Patent Publication Number: US-2019185877-A1

Title: Anti-microbial proteins

Description:
REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Application No. 62/280,597, filed Jan. 19, 2016, which is herein incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to the field of agricultural biotechnology. More specifically, the invention relates to nucleotide and polypeptide molecules, DNA constructs, and methods for producing plants with improved microbial pathogen or pest tolerance, as well as transgenic plants with improved pesticidal and/or fungicidal activity. The invention further relates to pesticidal and/or fungicidal compositions. 
     INCORPORATION OF SEQUENCE LISTING 
     A sequence listing contained in the file named “MONS388WO_ST25.txt” which is 592 KB (measured in MS-Windows®) and created on Jan. 18, 2017, is filed electronically herewith and incorporated by reference in its entirety. 
     BACKGROUND 
     Control of fungal pathogens and other disease-causing microbes that affect plants is one of the major difficulties facing the agricultural and horticultural industries. Although chemical agents have been successfully employed, a range of environmental and regulatory concerns is associated with the continued use of chemical approaches to control plant pests. Furthermore, with increased use of chemical pesticides comes increased resistance to these chemicals in pathogen and pest populations. Thus, further investigation to discover alternative mechanisms of providing resistance in plants to pathogens, such as insects, nematodes, microorganisms, fungi, bacteria and viruses, is needed. 
     SUMMARY 
     In one aspect, the invention provides a recombinant DNA construct comprising a nucleic acid sequence encoding a multi-domain defensin polypeptide comprising a first defensin region connected to a second defensin region by a linker region, the first defensin region and the second defensin region each comprising a gamma-thionin domain, wherein the nucleic acid sequence encoding the defensin polypeptide is operably linked to a promoter functional in a plant cell. 
     In another aspect, the invention provides a recombinant DNA construct comprising a nucleic acid sequence encoding a single domain defensin polypeptide. 
     For the multi-domain defensin, in some embodiments, the first defensin region is heterologous with respect to the second defensin region or the linker region, while in other embodiments the first defensin region or the second defensin region is heterologous with respect to the linker region. In certain embodiments, the first defensin region is identical to the second defensin region. In other embodiments, the first defensin region is different from the second defensin region. In certain embodiments, the promoter may be a heterologous promoter. In further embodiments, each defensin domain comprises at least 6 cysteine residues. The multi-domain defensin may be at least 90 amino acids in length. Each defensin domain may comprise: (a) between 5 and 10 cysteine residues; (b) a Pfam gamma-thionin domain (PF00304) with an E-value cutoff of 1e −3 ; or (c) a Cys-stabilized αβ (CSαβ) motif. 
     In certain embodiments, the first defensin region or the second defensin region of a multi-domain defensin comprises a polypeptide having at least 80% identity, at least 90% identity, at least 95% identity, or at least 97% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 559-662, or a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 559-662. In some embodiments, the linker region comprises a polypeptide having at least 80% identity, at least 90% identity, at least 95% identity, or at least 97% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 153-202, or a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 153-202. In other embodiments, the multi-domain defensin polypeptide comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 97% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 52-102, 329-454, and 1156, or a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-102, 329-454 and 1156. In further embodiments, the first defensin region or the second defensin region of a multi-domain defensin comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 97% identity to a defensin sequence portion of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1054-1152 and 1154, or an amino acid sequence selected from the group consisting of SEQ ID NOs: 1054-1152 and 1154. In further embodiments, the multi-domain defensin polypeptide encoded by nucleic acid sequence further comprises an N-terminal transit signal sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 97% identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 809-954. 
     In certain embodiments, the defensin polypeptide comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 97% identity to a defensin sequence portion of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1054-1152 and 1154, or an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1054-1152 and 1154. In further embodiments, the multi-domain defensin polypeptide encoded by nucleic acid sequence further comprises an N-terminal transit signal sequence having at least 80% identity, at least 90% identity, at least 95% identity, or at least 97% identity to an amino acid sequence selected form the group consisting of: SEQ ID NOs: 809-954. 
     In another aspect, the invention provides a synthetic promoter as set forth in SEQ ID NO: 1158 to express single domain defensin or multi-domain defensin in plant cells. 
     In another aspect, the invention provides a plant, seed, plant tissue, plant part, or cell comprising a recombinant DNA construct provided herein or comprising the multi-domain defensin polypeptide encoded by a recombinant DNA construct provided herein. The plant, seed, plant tissue, plant part, or cell may exhibit tolerance or activity against at least one plant fungal pathogen species within one or more of the following genera of fungi:  Fusarium, Collectotrichum, Stenocarpella , and/or  Phakopsora . The plant, seed, plant tissue, plant part, or cell may exhibit tolerance or activity against one or more of the following fungal species:  Fusarium graminearum, Fusarium verticilloides, Collectotrichum graminicola, Stenocarpella maydis , and/or  Phakopsora pachyrhizi.    
     In another aspect, the invention provides a microorganism comprising a recombinant DNA construct provided herein, or a DNA molecule or vector comprising a recombinant DNA construct provided herein. The DNA molecule or vector may comprise a polynucleotide sequence having at least 70% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 97% identity to a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 1-51, 203-328, 955-1053 and 1155, or a polynucleotide sequence having a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 1-51, 203-328, 955-1053, and 1155. 
     The invention further provides a method for conferring fungal pathogen tolerance or resistance to a plant, seed, cell, or plant part comprising expressing in said plant, seed, cell, or plant part the single domain defensin or the multi-domain defensin polypeptide encoded by the recombinant DNA construct disclosed herein. The invention further provides methods for expressing the recombinant DNA constructs of the invention in a plant cell or microorganism to produce a multi-domain defensin polypeptide, for example such that the multi-domain defensin polypeptide accumulates in a plant cell at a higher level relative to a single domain (1D) defensin control. The invention further provides a method for producing a transgenic plant with resistance or tolerance to a fungal pathogen comprising transforming a plant cell or tissue with the recombinant DNA molecule or vector provided herein, and regenerating a transgenic plant. 
     In another aspect, the invention provides a recombinant DNA construct comprising a nucleic acid sequence encoding a defensin having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1054-1152, wherein the nucleic acid sequence encoding the defensin polypeptide is operably linked to a promoter functional in a plant cell. In certain embodiments, the defensin polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1054-1152. In further embodiments, the promoter comprises a nucleotide sequence as set for in SEQ ID NO: 1158. 
     The invention further provides a plant, seed, plant tissue, plant part, or cell comprising a recombinant DNA construct described herein. In some embodiments, the plant, seed, plant tissue, plant part, or cell has tolerance or activity against at least one plant fungal pathogen selected from the group consisting of  Fusarium, Collectotrichum, Stenocarpella , and  Phakopsora , for example at least one fungal species selected from the group consisting of  Fusarium graminearum, Fusarium verticilloides, Collectotrichum graminicola, Stenocarpella maydis , and  Phakopsora pachyrhizi.    
     In a further aspect, the invention provides methods of producing a transgenic plant with tolerance to a fungal pathogen comprising transforming a plant cell or tissue with the recombinant DNA molecule or vector described herein, and regenerating a transgenic plant. 
     BRIEF DESCRIPTION OF THE SEQUENCES 
     SEQ ID NOs: 1-51: Nucleotide sequences of native multi-domain defensins.
 
SEQ ID NOs: 52-102: Polypeptide sequences of native multi-domain defensins.
 
SEQ ID NOs: 103-152: Nucleotide sequences of linker regions from multi-domain defensins.
 
SEQ ID NOs: 153-202: Polypeptide sequences of linker regions from multi-domain defensins.
 
SEQ ID NOs: 203-328: Nucleotide sequences of several synthetic multi-domain defensins.
 
SEQ ID NOs: 329-454: Polypeptide sequences of several synthetic multi-domain defensins.
 
SEQ ID NOs: 455-558: Nucleotide sequences of defensin regions from multi-domain defensins.
 
SEQ ID NOs: 559-662: Polypeptide sequences of defensin regions from multi-domain defensins.
 
SEQ ID NOs: 663-808: Nucleotide sequences of transit signals (TS) of multi-domain defensins.
 
SEQ ID NOs: 809-954: Polypeptide sequences of TS of multi-domain defensins.
 
SEQ ID NOs: 955-1053: Nucleotide sequences of 1D defensins.
 
SEQ ID NOs: 1054-1152: Polypeptide sequences of 1D defensins.
 
SEQ ID NO: 1153: Nucleotide sequence of MEDsa.AFPm1.
 
SEQ ID NO: 1154: Polypeptide sequence of MEDsa.AFPm1.
 
SEQ ID NO: 1155: Nucleotide sequence of a synthetic heterodimeric defensin.
 
SEQ ID NO: 1156: Polypeptide sequence of a synthetic heterodimeric defensin.
 
SEQ ID NO: 1157: Nucleotide sequence of codon optimized PINSY.AFP1.
 
SEQ ID NO: 1158: Nucleotide sequence of a synthetic regulatory element for controlling transgene expression.
 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows: (Top) a diagram depicting the domain configuration of two single domain (1D) defensins and four homodimeric synthetic two-domain (2D) defensins derived from the naturally occurring single domain defensins Coix22 and MtDef4. L1 and L2 represent different 2D linker regions described herein. The N-terminal transit signal (TS) sequences are also shown. (Bottom) a diagram depicting the domain configuration of a heterodimeric synthetic 2D defensin derived from Coix22 and AMAru.AFP10. L3 represent a 2D linker region described herein. 
         FIG. 2  shows: Relative RNA and protein expression levels of several homodimeric 2D defensins as compared to those levels of the corresponding 1D defensin. 
     
    
    
     DETAILED DESCRIPTION 
     Plant diseases caused by fungal or other microbial pathogens can severely impact yield in crop plants, resulting in millions of tons of grain loss annually. Fungal plant diseases can result from a combination of several pathogens, and nearly every field of crop plants may experience fungal disease pressure to some extent. The development of effective methods of fungal control has been hindered by a lack of available agents with activity against different fungal pathogens, and/or agents which can be effectively combined with existing methods for fungal control. Yield loss due to fungal disease in agricultural plants therefore remains a significant problem. 
     The invention provides novel anti-microbial peptides (AMPs) comprising defensin or defensin-like proteins, including multi-domain defensin proteins, capable of conferring pest resistance or tolerance and/or fungicidal activity to plants. Novel polynucleotide molecules and sequences encoding defensin or defensin-like proteins, as well as recombinant DNA constructs comprising these novel defensin-encoding polynucleotide sequences, are also provided. In addition, methods are provided for producing plants with increased pest control or pesticidal activity by expressing in a plant a polynucleotide of the invention encoding a defensin or defensin-like protein, such as by transforming a plant or plant cell with a recombinant DNA construct comprising a polynucleotide sequence encoding a defensin or defensin-like protein, and transgenic plants or plant cells produced by these methods and comprising a defensin-encoding DNA construct of the invention. Pesticidal and plant health compositions and methods are also contemplated for administering or applying a defensin or defensin-like protein(s) of the invention to a plant, a plant growth medium or soil associated with the plant, or a plant part or seed. 
     The invention provides novel polynucleotide and polypeptide sequences of defensins or defensin-like protein molecules including multi-domain defensins, and the use of these sequences and molecules for generating pest resistance or tolerance in plants. As used herein, “tolerance” or “improved tolerance” in a plant to a pest or pathogen is an indication that the plant is less affected by the pest or pathogen with respect to yield, survivability and/or other relevant agronomic measures, compared to a less resistant, more “susceptible” plant. “Resistance” or “improved resistance” in a plant to a pest or pathogen is an indication that the plant is more able to reduce the effect of the pest or pathogen than a non-resistant or less resistant plant. The defensins described herein may be introduced into various plant species to confer anti-fungal and/or anti-microbial activity, and thus resistance or tolerance to one or more plant pests or pathogens. The defensins and defensin-like molecules disclosed herein may be introduced and expressed in a plant or plant cell to confer the anti-fungal and/or anti-microbial activity to the plant or plant cell. According to embodiments of the invention, polynucleotides, constructs and proteins of the invention may confer resistance or tolerance to, and activity against, one or more fungal pathogens, including a  Fusarium, Colletotrichum, Stenocarpella , and/or  Phakopsora  species, such as  Fusarium graminearum, Fusarium verticilloides, Colletotrichum graminicola, Stenocarpella maydis , and/or  Phakopsora pachyrhizi . In addition to having anti-fungal activity, constructs and proteins of the invention may confer resistance or tolerance to, and activity against, other microbial plant pathogen(s) and/or plant pest(s), such as oomycetes, bacteria, insects, nematodes, etc. 
     Defensins or defensin-like proteins or polypeptides, hereinafter referred to jointly as defensins, are cysteine-rich cationic peptides, many of which exhibit inhibitory activity against a variety of microbial plant pathogens and agricultural pests. Single domain (1D) defensins are small globular proteins or peptide molecules, typically comprising approximately 50 amino acid (aa) residues that may be highly variable in sequence. Despite this variability, defensin peptides do share a gamma-thionin core consensus sequence (a gamma-thionin domain) and contain about 6-8 cysteine residues that may form disulfide bonds with protein folding. The three-dimensional structure of 1D defensins has been described as a Cys-stabilized αβ motif (CSαβ) having three anti-parallel β-sheets and one α-helix stabilized by multiple disulfide bridges formed by conserved cysteine residues (typically four disulfide bridges formed by eight conserved cysteine residues). See, e.g., Carvalho, A O et al.,  Peptides  30: 1007-1020 (2009); and Thomma, B et al.,  Planta  216: 193-202 (2002), the entire contents and disclosures of which are incorporated herein by reference. Defensins may further comprise an N-terminal transit signal (TS) sequence of variable length from only a few amino acids up to 20-30 amino acids, and/or a C-terminal extension sequence of variable length up to 35-30 amino acids when present. The N-terminal TS sequence on a defensin pro-protein may play a role in the targeting and/or export of the mature defensin protein into the apoplastic space or other sub-cellular compartment. However, the TS sequence may generally become cleaved and removed from the remainder of the defensin co/post-translationally to produce a mature defensin protein or peptide without the TS sequence. Some defensins may also have a C-terminal extension sequence that may also play a variety of roles in a plant cell and/or become cleaved from a mature defensin protein or peptide. 
     In one aspect, the invention provides several multi-domain (MD) defensins from plants, including two-domain (2D) and four-domain (4D) defensins, comprising two or more defensin regions or domains connected or bridged together by one or more linker regions, in addition to N-terminal TS sequences and/or C-terminal extension sequences. Each of the defensin regions of a 2D or MD defensin may also be referred to as a “defensin component” of the 2D or MD defensin. Thus, polypeptide sequences of the invention may comprise one of these multi-domain defensin proteins or polypeptides. Examples of multi-domain defensins including 2D defensins that may be used according to embodiments of the invention include those provided herein as SEQ ID NOs: 52-102. Further provided are polynucleotide molecules and constructs encoding one of these multi-domain defensins, such as those provided as SEQ ID NOs: 1-51. These MD defensin-encoding polynucleotides may be used according to embodiments of the invention to confer pesticidal and/or anti-fungal activity when expressed in a plant. Multi-domain defensin proteins of the invention may also optionally comprise an N-terminal transit signal (TS) sequence, such as one of SEQ ID NOs: 809-954, and/or a C-terminal sequence, such as one identified by annotation in Table 2 below for SEQ ID NOs: 101 and 102. 
     In one aspect, the invention provides several single domain (1D) defensins from plants that have pesticidal and/or anti-fungal activity, as well as polynucleotides encoding these 1D defensins. Thus, embodiments of the invention include polynucleotides and constructs encoding these 1D defensins for expression in a plant, or at least comprising a defensin sequence portion of these 1D defensins as identified herein. Examples of 1D defensins that are used according to embodiments of the invention include one or more of those provided herein as SEQ ID NOs: 1054-1127 and 1129-1152, and examples of polynucleotide sequences encoding these 1D defensins include one or more of those provided herein as SEQ ID NOs: 955-1028 and 1030-1053. 1D defensins of the invention may further include those comprising a defensin component of a 2D or MD defensin identified herein, such as one or more of those provided herein as SEQ ID NOs: 559-662, as well as polynucleotides sequences encoding these defensin components, such as one or more of those provided herein as SEQ ID NOs: 455-558. It is further contemplated that 1D defensins of the invention may comprise polypeptide and polynucleotide sequences having a relaxed sequence identity relative to the above identified sequences. For example, a 1D defensin may have a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to at least a defensin sequence portion of one of SEQ ID NOs: 1054-1127 or 1129-1152, and/or a 1D defensin may be encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to at least a defensin sequence portion of one of SEQ ID NOs: 955-1028 or 1030-1053. A 1D defensin may further comprise a fragment comprising at least 25, least 50, at least 75, or at least 100, contiguous amino acids of a defensin sequence portion of one of SEQ ID NOs: 1054-1127 or 1129-1152, and/or a 1D defensin may be encoded by a polynucleotide sequence that is fragment comprising at least 25, at least 50, at least 75, or at least 100 contiguous nucleotides of a defensin sequence portion of one of SEQ ID NOs: 955-1028 or 1030-1053. In some embodiments, a fragment has the activity of the full-length defensin sequence portion of one of SEQ ID NOs: 1054-1127 or 1129-1152, or encoded by one of SEQ ID NOs: 955-1028 or 1030-1053. The 1D defensin-encoding polynucleotides and constructs may be used according to embodiments of the invention to confer pesticidal and/or anti-fungal activity when introduced and expressed in a plant. 
     In addition to newly discovered single and multi-domain defensins from plants, methods and compositions are provided for the construction, synthesis and expression of synthetic multi-domain defensin proteins comprising heterologous or non-naturally occurring combinations of two or more defensin regions or domains and one or more linker region(s) connecting or bridging the two or more defensin regions together, as well as polynucleotides encoding these synthetic multi-domain defensins. For example, each of the defensin regions of a synthetic multi-domain defensin may comprise a defensin sequence portion of a 1D defensin or a defensin component of a 2D or MD defensin, as identified herein. These synthetic multi-domain defensin proteins may thus be referred to as chimeric multi-domain defensins comprising defensin region(s) and/or linker region(s) that are heterologous in their origin. In addition to their novel structure, synthetic multi-domain defensins may also have one or more novel properties or characteristics, such as increased accumulation when expressed in a plant cell and/or new, altered or enhanced anti-fungal and/or pesticidal activity, relative to existing or known defensins, such as 1D defensins comprising one of their defensin components. A synthetic multi-domain defensin of the invention may comprise a heterologous or non-naturally occurring combination of a first defensin domain or region, a second defensin domain or region, and a linker region, wherein the first defensin region and the second defensin region are linked or connected to each other by the linker region. For example, a synthetic 2D defensin of the invention can comprise these regions in the following order (in the N-terminal to C-terminal direction): (i) first defensin region, (ii) linker region, and (iii) second defensin region. Synthetic multi-domain defensins may further comprise additional defensin region(s) connected by additional linker region(s). Furthermore, a 3D defensin may have the following order: (i) first defensin region, (ii) first linker region, (iii) second defensin region, (iv) second linker region, and (v) third defensin region, whereas a 4D defensin may additionally comprise a third linker region and a fourth defensin region with the third linker region being between the third and fourth defensin regions, and so on. 
     Accordingly, synthetic multi-domain defensin proteins or polypeptides of the invention may comprise combinations of two or more defensin domains or regions, each of the defensin region(s) comprising of one of the sequences provided herein as SEQ ID NOs: 559-662 and/or a defensin sequence portion of one of SEQ ID NOs: 1054-1152 and/or 1154, wherein the two or more defensin regions are linked or joined together by linker region(s), such as one or more of those provided herein as SEQ ID NOs: 153-202. For example, the first defensin region of a synthetic 2D defensin may comprise one of SEQ ID NOs: 559-662, and/or a defensin sequence portion of one of SEQ ID NOs: 1054-1152 and/or 1154, the second defensin region may comprise one of SEQ ID NOs: 559-662 and/or a defensin sequence portion of one of SEQ ID NOs: 1054-1152 and/or 1154, which may be the same as, or different than, the first defensin region, and the linker region connecting the first and second defensin regions may be one of SEQ ID NOs: 153-202. 
     In one embodiment of the invention, a “defensin sequence portion” shall refer to the sequence portion of a 1D defensin or defensin-like protein that excludes the N-terminal TS sequence and the C-terminal extension sequence (if present), as well as a polynucleotide sequence encoding the defensin sequence portion of a defensin or defensin-like protein. See, e.g., Table 15 below providing annotation for the defensin sequence portion of 1D defensin sequences. However, according to some embodiments, a defensin region near the N-terminus of a synthetic 2D or MD protein may retain its native TS sequence, and/or a defensin region near the C-terminus of the synthetic 2D or MD protein may retain its native C-terminal extension. Synthetic multi-domain defensins of the invention may also optionally comprise an N-terminal transit signal (TS) sequence, such as one of SEQ ID NOs: 809-954, and/or a C-terminal sequence, such as one identified by annotation in Table 2 below for SEQ ID NOs: 101 and 102. For example, synthetic multi-domain defensins of the invention may comprise one or more of those provided herein as SEQ ID NOs: 329-454 and 1156. 
     Polynucleotides of the invention may include sequences encoding synthetic multi-domain defensins. These polynucleotides may comprise combinations of two or more polynucleotide sequences encoding defensin domains or regions, each of these polynucleotide sequences encoding a defensin region may comprise a polynucleotide sequence provided herein as SEQ ID NOs: 455-558 or a defensin sequence portion of one of the polynucleotide sequences provided herein as SEQ ID NOs: 955-1053 and/or 1153, linked or joined together by a polynucleotide sequence(s) encoding a linker region(s), such as one or more of those provided herein as SEQ ID NOs: 103-152. For example, the sequence encoding the first defensin region of a synthetic 2D defensin may comprise one of SEQ ID NOs: 455-558 or a defensin sequence portion of one of the polynucleotide sequences provided herein as SEQ ID NOs: 955-1053 and/or 1153, the sequence encoding the second defensin region of the synthetic 2D defensin may comprise one of SEQ ID NOs: 455-558 or a defensin sequence portion of one of the polynucleotide sequences provided herein as SEQ ID NOs: 955-1053 and/or 1153, which may be the same as or different than the sequence encoding the first defensin region, and the sequence encoding the linker region of the synthetic 2D defensin connecting the first and second defensin regions may be one of SEQ ID NOs: 103-152. Polynucleotides of the invention may also optionally comprise sequences encoding a N-terminal targeting signal (TS) sequence, such as one of SEQ ID NOs: 663-808, and/or a C-terminal sequence, such as one of the sequences identified by annotation in Table 1 herein for SEQ ID NOs: 50 and 51. For example, polynucleotides encoding synthetic multi-domain defensins of the invention may comprise one or more of those provided herein as SEQ ID NOs: 203-328 and 1155. 
     Multi-domain defensin proteins of the invention may further include variants and homologues of the native and synthetic defensin sequences provided herein. According to some embodiments, multi-domain defensin variants may comprise one or more mutations, deletions, insertions, etc., relative to a native or synthetic defensin or multi-domain defensin sequence, or other engineered defensin-like sequences. Multi-domain defensin proteins or polypeptides of the invention may comprise combinations of two or more defensin domains or regions, wherein one or more of those defensin regions has a relaxed sequence identity relative to one or identity to one or more of SEQ ID NOs: 559-662, and/or a defensin sequence portion of one or more of SEQ ID NOs: 1054-1152 and/or 1154. Each defensin region of a multi-domain defensin may be at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to one of SEQ ID NOs: 559-662 and/or a defensin sequence portion of one of SEQ ID NOs: 1054-1152 and/or 1154. A defensin region of a multi-domain defensin may further comprise a fragment comprising at least 25, at least 50, at least 75, or at least 100, contiguous amino acids of one of SEQ ID NOs: 1054-1152 and/or 1154. In some embodiments, a fragment has the activity of the full-length defensin region of one of SEQ ID NOs: 1054-1154. Each of the linker domain(s) or region(s) of a multi-domain defensin protein may also have a relaxed sequence identity relative to SEQ ID NOs: 153-202. Each linker region of a multi-domain defensin may be at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to one of SEQ ID NOs: 153-202. Indeed, multi-domain defensin proteins of the invention may comprise a polypeptide sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to any one of SEQ ID NOs: 52-102, 329-454, or 1156. A multi-domain defensin protein may further comprise a fragment comprising at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, or at least 200 contiguous amino acids of any one of SEQ ID NOs: 52-102, 329-454 or 1156. In some embodiments, a fragment has the activity of SEQ ID NOs: 52-102, 329-454, or 1156. However, a linker region of a multi-domain may also be highly variable in sequence and length with little or no sequence identity of similarity to SEQ ID NOs: 153-202. A linker region may also comprise two or more linker sequences arranged in tandem, wherein each of the linker sequences may comprise one of SEQ ID NOs: 153-202, a polypeptide sequence having a relaxed sequence identity relative to SEQ ID NOs: 153-202, or other sequence. 
     The N-terminal targeting signal (TS) and/or a C-terminal sequences of a multi-domain defensin (if present) may each also have relaxed sequence identity relative to the sequences provided herein. The N-terminal targeting signal (TS) sequence may be at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to one of SEQ ID NOs: 809-954. The C-terminal sequence may be at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to one of the C-terminal sequences identified by annotation in Table 2 for SEQ ID NOs: 101 and 102. According to some embodiments, however, the N-terminal and/or C-terminal sequences of a multi-domain defensin may instead be dissimilar or have a lower sequence identity relative to the sequences provided herein. 
     Polynucleotides of the invention may comprise sequences encoding multi-domain defensin proteins with relaxed sequence identity relative to the sequences provided herein. Polynucleotides encoding these multi-domain defensins may comprise combinations of two or more sequences encoding defensin domains or regions, wherein one or more of the polynucleotide sequences encoding these defensin regions have a relaxed sequence identity relative to one or more of SEQ ID NOs: 455-558 and/or a defensin sequence portion of one or more of SEQ ID NOs: 955-1053 and/or 1153. Accordingly, polynucleotide sequences encoding each defensin region of a multi-domain defensin may be at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to one of SEQ ID NOs: 455-558 and/or a defensin sequence portion of one of SEQ ID NOs: 955-1053 and/or 1153. A defensin region of a multi-domain defensin may further comprise a fragment comprising at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, or at least 200 contiguous nucleotides of any one of SEQ ID NOs: 455-558 and/or a defensin sequence portion of one of SEQ ID NOs: 955-1053 and/or 1153. In some embodiments, a fragment encodes a protein having the activity of a protein encoded by SEQ ID NOs: 455-558 and/or a defensin sequence portion of one of SEQ ID NOs: 955-1053 and/or 1153. The polynucleotide sequences encoding the linker domain(s) or region(s) of a multi-domain defensin protein may also have a relaxed sequence identity relative to SEQ ID NOs: 103-152. Accordingly, the polynucleotide sequence encoding a linker region of a multi-domain defensin may be at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to one of SEQ ID NOs: 103-152. Indeed, polynucleotides encoding multi-domain defensin proteins of the invention may comprise a polynucleotide sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to any one of SEQ ID NOs: 1-51, 203-328, or 1155. However, a polynucleotide sequence encoding the linker region(s) of a multi-domain may also be highly variable in sequence and length with little or no sequence identity of similarity to SEQ ID NOs: 103-152. As mentioned above, a linker region may also comprise two or more linker sequences arranged in tandem, wherein each of the linker sequences may comprise one of SEQ ID NOs: 103-152, a polynucleotide sequence having a relaxed sequence identity relative to SEQ ID NOs: 103-152, or other sequence. 
     If present, polynucleotide sequences encoding the N-terminal targeting signal (TS) sequence and/or the C-terminal sequence of a multi-domain defensin may also have a relaxed sequence identity relative to sequences provided herein. The polynucleotide sequence encoding the N-terminal targeting signal (TS) sequence of a multi-domain defensin may be at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to one SEQ ID NOs: 663-808. The polynucleotide sequence encoding the C-terminal sequence of a multi-domain defensin may be at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to one of the C-terminal sequences identified in Table 1 for SEQ ID NOs: 50 and 51. 
     According to some embodiments, however, the N-terminal and/or C-terminal sequences of a multi-domain defensin may instead be dissimilar or have a lower sequence identity relative to the sequences provided herein. 
     For purposes of the invention, the percent identity of two polynucleotide or polypeptide sequences may be determined by first optimally aligning the two sequences and then determining the percentage of nucleotide bases or amino acid residues that are the same between the two sequences over a comparison window, which may be over the full length of one of the two sequences. An optimal alignment is defined as a best fit or match of the two sequences with resistance or tolerance for any gaps in the alignment. A number of computerized programs and algorithms are known in the art for achieving an optimal alignment of two or more sequences (GAP, BESTFIT, FASTA, BLAST, Smith-Waterman). When optimally aligned, the percent identity of a subject sequence to a reference sequence is determined by taking the number of matched or identical bases or amino acids between the two sequences, dividing by the length of the reference sequence, and then multiplying the quotient by 100%. 
     Multi-domain defensins of the invention may further have characteristic structural features, which may be related to their pesticidal and anti-fungal activity. These features may allow for the identification of additional defensins that may be used in designing novel multi-domain defensins. For example, multi-domain defensin proteins of the invention may be defined as comprising the following features: (a) a Pfam gamma-thionin domain (PF00304), as determined by a search of the Pfam protein families database using an E-value cutoff of 1e −3  (Finn, et al.  Nucleic Acids Research,  2014, Database Issue 42:D222-D230); (b) polypeptides comprising more than 8 cysteine residues; and (c) polypeptides comprising two or more gamma-thionin domains (GXCX n C) separated by a polypeptide linker sequence, wherein n is 3-22 amino acids in length, and X is any amino acid. These putative MD defensins may further have a protein length of at least 90 amino acids or greater and an absence of any premature stop codons in its coding sequence. Furthermore, each defensin region of a multi-domain defensin may be defined as having at least 6 cysteine residues, or at least 8 cysteine residues, and a predicted Cys-stabilized αβ motif (CSαβ) structure when folded as described above. However, one or more of the defensin region(s) may each have fewer cysteines according to some embodiments, such as 4 or 5 cysteines in one or more of the defensin region(s). Thus, a multi-domain defensin may have at least 8 cysteine residues, or at least 10 cysteine residues, at least 12 cysteine residues, or at least 14 cysteine residues, and/or two or more predicted CSαβ structural motifs. Multi-domain defensins may also be defined functionally as having certain pesticidal or anti-fungal activity against one or more plant fungal pathogens. 
     As introduced above, the invention provides synthetic 2D or other MD defensins comprising at least a first defensin region linked to a second defensin region by a linker region. The defensin regions of a multi-domain defensin protein may be homomeric (i.e., the defensin regions are the same) or heteromeric (i.e., the defensin regions are different). A 2D or MD defensin of the invention may comprise a native combination of defensin regions (i.e., comprising the same combination of defensin regions present in a native multi-domain defensin) or a heterologous combination of defensin regions (i.e., comprising defensin regions that do not exist together in a native multi-domain defensin and/or derived from different defensins). One or more of the defensin region(s) may be heterologous with respect to a linker region(s) of the MD defensin. Indeed, a multi-domain defensin of the disclosure may comprise a native combination of defensin regions that may be homomeric or heteromeric relative to each other but heterologous with respect to a linker region. Without being bound by any theory, synthetic multi-domain defensins having a linker region that is heterologous with respect to at least one defensin region may tend to accumulate to higher levels when expressed in a plant or plant cell and/or confer new or altered anti-fungal or anti-microbial activities. 
     As further described above, embodiments of the disclosure provide nucleic acids and polynucleotides comprising one or more of SEQ ID NOs: 455-558 and/or a defensin sequence portion of one or more of 955-1053, such as SEQ ID NOs: 1-51, 203-328, or 1155 and defensin polypeptides or proteins encoded by these polynucleotide sequences. Defensin polypeptides or proteins may comprise one or more of SEQ ID NOs: 559-662 and/or a defensin sequence portion of one or more of 1054-1152, such as SEQ ID NOs: 52-102, 329-454, or 1156. Complements to any of the nucleic acid or polynucleotide sequences described herein are also provided. Polynucleotide molecules of the invention may encode a multi-domain defensin polypeptide comprising two or more defensin domains or regions, such as a 2D, 3D, 4D, or other MD defensin protein. Each defensin region of a multi-domain defensin is separated from all other defensin regions by one or more linker region(s) with at least one linker region being present between adjacent or neighboring defensin regions. In specific embodiments, a multi-domain defensin protein may comprise two or more defensin domains each comprising 4, 5, 6, 7, 8, 9, 10, or more cysteine residues. In certain embodiments as explained above, a polynucleotide molecule provided herein may be defined as comprising one or more nucleotide sequences, each having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the full length sequence of SEQ ID NOs: 1-51, 103-152, 203-328, 455-558, 663-808, 1155 and/or a defensin sequence portion of 955-1053 and/or 1153. Similarly, a polypeptide molecule provided herein may be defined as comprising one or more protein sequences, each having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the full length sequence of SEQ ID NOs: 52-102, 153-202, 329-454, 559-662, 809-954, 1156, and/or a defensin sequence portion of 1054-1152 and/or 1154. 
     Polynucleotide sequences of the disclosure may, when expressed in a plant, confer increased pesticidal or fungicidal activity and/or increased pest or fungal control to the plant. Indeed, polynucleotide constructs encoding multi-domain defensins of the disclosure may be used to generate transgenic plants with greater resistance or tolerance to one or more fungal pathogens. Such pesticidal or fungicidal activity may be effective against one or more of a  Fusarium, Colletotrichum, Stenocarpella , and/or  Phakopsora  species, such as one or more of  Fusarium graminearum, Fusarium verticilloides, Colletotrichum graminicola, Stenocarpella maydis , and/or  Phakopsora pachyrhizi . Such pesticidal activity may be further effective against other microbial plant pathogen(s) and/or plant pest(s), such as oomycetes, bacteria, insects, nematodes, etc. 
     The invention also provides recombinant DNA constructs comprising the polynucleotide sequences described herein, as well as plants, plant cells and seeds transformed therewith. In one embodiment, such DNA constructs may be used in expressing nucleotide sequences encoding 1D, 2D, or other MD defensins in plants for the purposes of protecting the plant from plant pests, such as one or more fungi. In another embodiment, such constructs may be of use in generating transgenic or recombinant plants with increased or enhanced resistance or tolerance to plant fungal pathogens. Thus, embodiments of the invention further comprise transformation vectors comprising a defensin-encoding DNA construct of the invention or a portion of such defensin-encoding DNA construct. According to some embodiments, defensin-encoding DNA constructs and vectors may be used for generating 1D, 2D, or MD defensin transcripts and/or proteins in microorganisms such as bacteria or yeast. 
     A recombinant DNA construct, molecule or vector of the invention may comprise a polynucleotide expression cassette comprising a coding sequence that encodes a 1D defensin or multi-domain defensin protein, wherein the coding sequence is operably linked to a promoter that is functional in a plant cell. Alternatively, the defensin-encoding polynucleotide sequence may be operably linked to a promoter suitable for expression of a defensin protein in a microorganism. Any suitable promoter known in the art may be used to express a defensin coding sequence of the invention in a plant, such as a constitutive, tissue-specific, tissue-enhanced or tissue-preferred, developmental, inducible, disease inducible, etc., promoter. Further, the plant promoter operably linked to the defensin coding sequence may be native, homologous or heterologous relative to the plant species to be transformed with the defensin-encoding DNA construct, or alternatively, the promoter may be chimeric or synthetic. A synthetic nucleotide sequence may be a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence. It is recognized that a gene-regulatory element of the invention comprises a synthetic nucleotide sequence. Preferably, the synthetic nucleotide sequence shares little or no extended homology to natural sequences. Extended homology in this context generally refers to 100% sequence identity extending beyond about 25 nucleotides of contiguous sequence. A synthetic gene-regulatory element of the invention comprises a synthetic nucleotide sequence. 
     The polynucleotide coding sequence for expressing the defensin protein may also be operatively linked to one or more additional regulatory element(s), such as an enhancer(s), leader, transcription start site (TSS), linker, 5′ and 3′ untranslated region(s), intron(s), polyadenylation signal, termination region or sequence, etc., that are suitable or necessary for regulating or allowing expression of the multi-domain defensin in a plant cell. Such additional regulatory element(s) may be optional and/or used to enhance or optimize expression of the defensin transgene or coding sequence. The term “operably linked” refers to a functional connection between the two sequences. A promoter or enhancer is “operably linked” to a transgene or coding sequence by affecting, causing, driving, promoting, etc., transcription and expression of the transgene or coding sequence. 
     According to embodiments of the invention, the term “recombinant” in reference to a DNA molecule, construct, vector, etc., refers to a DNA molecule or sequence that is not found in nature and/or is present in a context in which it is not found in nature, including a DNA molecule, construct, etc., comprising a combination of DNA sequences that would not naturally occur contiguously or in close proximity together without human intervention, and/or a DNA molecule, construct, etc., comprising at least two DNA sequences that are heterologous with respect to each other. A recombinant DNA molecule, construct, etc., may comprise DNA sequence(s) that is/are separated from other polynucleotide sequence(s) that exist in proximity to such DNA sequence(s) in nature, and/or a DNA sequence that is adjacent to (or contiguous with) other polynucleotide sequence(s) that are not naturally in proximity with each other. A recombinant DNA molecule, construct, etc., may also refer to a DNA molecule or sequence that has been genetically engineered and constructed outside of a cell. For example, a recombinant DNA molecule may comprise any suitable plasmid, vector, etc., and may include a linear or circular DNA molecule. Such plasmids, vectors, etc., may contain various maintenance elements including a prokaryotic origin of replication and selectable marker, as well as a defensin expressing transgene or cassette perhaps in addition to a plant selectable marker gene, etc. The term “recombinant” may also further refer to proteins expressed or encoded by these recombinant DNA molecules, constructs, etc., if they comprise non-native sequences. 
     As used herein, the term “isolated” in reference to a DNA molecule, construct, vector, etc., may refer to a DNA molecule or sequence that is not found in nature and/or is present in a context in which it is not found in nature. The term “isolated” may also refer to a nucleic acid molecule that has undergone at least one step towards being isolated or concentrated or enriched from a more complex solution or source. The term “isolated,” however, is in no way intended to limit the nucleic acid molecule to a particular location or state, and the invention extends to the nucleic acid molecule when introduced into the genome of a cell or when it is resident in progeny of cells into which the nucleic acid molecule has been introduced into its genome. 
     According to another broad aspect of the invention, methods are provided for transforming a plant cell, tissue or explant with a recombinant DNA molecule or construct comprising a defensin transgene or coding sequence to produce a defensin containing transgenic plant. Numerous methods for transforming chromosomes in a plant cell with a recombinant DNA molecule are known in the art, which may be used according to methods of the invention to produce a transgenic plant cell and plant. Any suitable method or technique for transformation of a plant cell known in the art may be used according to present methods. Effective methods for transformation of plants include bacterially mediated transformation, such as  Agrobacterium -mediated or Rhizhobium-mediated transformation, and microprojectile bombardment-mediated transformation. Other methods for plant transformation are also known in the art including, but not limited to, gene editing, site-directed integration, PEG-mediated transformation, protoplast transformation, electroporation, microinjection, agitation with silica/carbon fibers, virus-mediated or liposome-mediated transformation, etc. A variety of methods are known in the art for transforming explants with a transformation vector via bacterially mediated transformation or microprojectile bombardment and then subsequently culturing, etc, those explants to regenerate or develop transgenic plants. Methods are further provided for expressing a multi-domain defensin transgene of the invention in one or more plant cells or tissues under the control of a promoter operable in a plant cell. Such methods may be used to confer anti-fungal and pesticidal properties to a plant and combat a fungal infection. 
     Transformation of a target plant material or explant may be practiced in tissue culture on nutrient media, for example a mixture of nutrients that allow cells to grow in vitro. Recipient cell targets or explants may include, but are not limited to, meristems, shoot tips, protoplasts, hypocotyls, calli, immature or mature embryos, shoots, buds, nodal sections, leaves, gametic cells such as microspores, pollen, sperm and egg cells, etc., or any suitable portions thereof. It is contemplated that any transformable cell or tissue from which a fertile plant can be regenerated or grown/developed may be used as a target for transformation. Transformed explants, cells or tissues may be subjected to additional culturing steps, such as callus induction, selection, regeneration, etc., as known in the art. Transformed cells, tissues or explants containing a recombinant DNA insertion may be grown, developed or regenerated into transgenic plants in culture, plugs or soil according to methods known in the art. Transgenic plants may be further crossed to themselves or other plants to produce transgenic seeds and progeny. A transgenic plant may also be prepared by crossing a first plant comprising the recombinant DNA sequence or transformation event with a second plant lacking the insertion. For example, a recombinant DNA sequence may be introduced into a first plant line that is amenable to transformation, which may then be crossed with a second plant line to introgress the recombinant DNA sequence into the second plant line. Progeny of these crosses can be further back crossed into the more desirable line multiple times, such as through 6 to 8 generations or back crosses, to produce a progeny plant with substantially the same genotype as the original parental line but for the introduction of the recombinant DNA sequence. 
     A recombinant DNA molecule or construct of the invention may be included within a DNA transformation vector for use in transformation of a target plant cell, tissue or explant. Such a transformation vector of the invention may generally comprise sequences or elements necessary or beneficial for effective transformation in addition to the defensin expressing transgene or expression cassette. For  Agrobacterium -mediated transformation, the transformation vector may comprise an engineered transfer DNA (or T-DNA) segment or region having two border sequences, a left border (LB) and a right border (RB), flanking at least the defensin expressing transgene or cassette, such that insertion of the T-DNA into the plant genome will create a transformation event for the defensin transgene or cassette. In other words, the defensin transgene would be located between the left and right borders of the T-DNA, perhaps along with an additional transgene(s) or expression cassette(s), such as a plant selectable marker transgene and/or other gene(s) of agronomic interest that may confer a trait or phenotype of agronomic interest to a plant. In addition to protein encoding sequences, a gene of agronomic interest may further comprise a polynucleotide sequence encoding a RNA suppression element. According to alternative embodiments, the defensin-encoding transgene or cassette and the plant selectable marker transgene (or other gene of agronomic interest) may be present in separate T-DNA segments on the same or different recombinant DNA molecule(s), such as for co-transformation. A transformation vector or construct may further comprise prokaryotic maintenance elements, which for  Agrobacterium -mediated transformation may be located in the vector backbone outside of the T-DNA region(s). 
     A plant selectable marker transgene in a transformation vector or construct of the invention may be used to assist in the selection of transformed cells or tissue due to the presence of a selection agent, such as an antibiotic or herbicide, wherein the plant selectable marker transgene provides tolerance or resistance to the selection agent. Thus, the selection agent may bias or favor the survival, development, growth, proliferation, etc., of transformed cells expressing the plant selectable marker gene, such as to increase the proportion of transformed cells or tissues in the R 0  plant. Commonly used plant selectable marker genes include, for example, those conferring tolerance or resistance to antibiotics, such as kanamycin and paromomycin (nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aadA) and gentamycin (aac3 and aacC4), or those conferring tolerance or resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Plant screenable marker genes may also be used, which provide an ability to visually screen for transformants, such as luciferase or green fluorescent protein (GFP), or a gene expressing a beta glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. 
     Additionally provided herein are transgenic plants, plant parts, propagules and plant cells transformed with a recombinant DNA construct or vector of the invention having a polynucleotide sequence encoding a multi-domain defensin. Such a transgenic plant, plant part, or plant cell may comprise a transformation event or insertion of a defensin-encoding recombinant DNA construct or sequence of the invention into the genome of at least one plant cell thereof. A transgenic plant comprising the defensin-encoding DNA construct or sequence may be produced by any suitable transformation method, which may be followed by selection, culturing, regeneration, development, etc., as desired or needed to produce a transgenic R 0  plant, which may then be selfed or crossed to other plants to generate R1 seed and subsequent progeny generations and seed through additional crosses, etc. Similarly, embodiments of the invention further include a plant cell, tissue, explant, etc., comprising one or more transgenic cells having a transformation event or genomic insertion of a recombinant DNA or polynucleotide sequence comprising the defensin-encoding transgene, construct or sequence. Transgenic plants comprising a defensin-encoding transgene may have increased resistance or tolerance to one or more plant pests or fungi and/or increased anti-fungal properties or activities, relative to a wild type or control plant not having the defensin-encoding transgene. 
     For purposes of the invention, a “plant” may include an explant, embryo, seedling, plantlet or whole plant at any stage of regeneration or development. As used herein, a “transgenic plant” refers to a plant whose genome has been altered by the integration or insertion of a recombinant DNA molecule, construct or sequence. A transgenic plant includes an R 0  plant developed or regenerated from an originally transformed plant cell(s) as well as progeny transgenic plants in later generations or crosses from the R 0  transgenic plant. As used herein, a “plant part” may refer to any organ or intact tissue of a plant, such as a meristem, shoot organ/structure (e.g., leaf, stem and tuber), root, flower or floral organ/structure (e.g., bract, sepal, petal, stamen, carpel, anther, pollen and ovule), seed (e.g., embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), propagule, or other plant tissues (e.g., cuttings, vascular tissue, ground tissue, and the like), callus, protoplasts, or any portion thereof. Plant parts of the invention may be viable, nonviable, regenerable, and/or non-regenerable. A “propagule” may include any plant part that is capable of growing into an entire plant. As used herein, a “transgenic plant cell” simply refers to any plant cell that is transformed with a stably-integrated recombinant DNA molecule or sequence. A transgenic plant cell may include an originally-transformed plant cell, a transgenic plant cell of a regenerated or developed R 0  plant, or a transgenic plant cell from any progeny plant or offspring of the transformed R 0  plant, including cell(s) of a plant seed or embryo, or a cultured plant or callus cell, etc. 
     The transformed plants may be analyzed for the presence of the defensin-encoding sequence or transgene and/or its expression level and/or profile in a plant or plant cell or tissue. Those of skill in the art are aware of numerous methods available for the analysis of transformed plants. For example, methods for plant analysis include, but are not limited to, Southern blots or northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, and immunoblotting assays. The expression of a transcribable DNA molecule or transgene can be measured, for example, using TaqMan® (Applied Biosystems, Foster City, Calif.) reagents and methods as described by the manufacturer and PCR cycle times determined using the TaqMan® Testing Matrix. As an alternative example, the Invader® (Third Wave Technologies, Madison, Wis.) reagents and methods as described by the manufacturer can be used to evaluate transgene expression. 
     The transgenic plants of the invention comprising a defensin-encoding polynucleotide sequence or construct can be any agricultural crop species. The species may be a monocotyledonous or dicotyledonous plant. Particularly useful plants may include but are not limited to wheat, carrot, sorghum rice, barley, soybean, potato, corn,  Brassica , canola, tomato, alfalfa, peanut, sugarcane and cotton. The plant can be an R 0  transgenic plant (i.e., a plant derived from the original transformed tissue). The transgenic plant can also be any generation of progeny plants derived from the original R 0  transgenic plant, such as by any known method of crossing, introgressing, converting or propagating plants. 
     The invention also provides methods for producing a transgenic plant with increased pest resistance or tolerance and/or pesticidal activity comprising introducing or transforming into the plant a recombinant DNA construct encoding a defensin protein as described herein. Such a method may further comprise: growing said plant to produce a further generation; and selecting at least one plant from said further generation comprising the recombinant DNA construct, wherein said plant has increased pest resistance or tolerance and/or anti-fungal activity relative to a control plant that does not comprise the recombinant DNA construct. Further provided is a method for producing plants with increased pest resistance or tolerance and/or pesticidal activity comprising crossing a transgenic plant of the invention with itself or a second plant to produce at least a first progeny plant, wherein said progeny plant comprises increased pest resistance or tolerance. Seed from plants comprising the recombinant DNA construct and having increased pesticidal activity and/or pest resistance or tolerance may be obtained from any number of sources. 
     According to another aspect of the invention, pesticidal compositions are provided comprising a defensin polypeptide or protein of the invention having pesticidal or anti-fungal activity. Such pesticidal compositions may further comprise other compounds or active pesticidal molecules that may be effective against one or more insects, nematodes, microbes, fungi, nematodes, or viruses. These pesticidal compositions comprising one or more recombinant defensin(s) of the invention may be formulated as a solid or liquid and/or applied as a topical, foliar, soil, or granular application or treatment to prevent or inhibit fungal infections and/or other plant pest infestations. Examples of other types of plant pests include insects, nematodes, weeds, microbes, such as bacteria, fungi, and viruses, etc. Methods for formulating a pesticidal composition of the invention may be similar to methods known in the art for other pesticidal formulations. Ingredients or components for pesticidal composition of the invention may include one or more carriers, diluents, surfactants, or other formulation ingredients known in the art. 
     EXAMPLES 
     Example 1. Identification of Defensins with Two or More Defensin Regions 
     Plant defensins are small polypeptides comprising an N-terminal signal peptide and a defensin region comprising approximately 50 amino acids and usually having 6 to 8 cysteines. The invention provides new multi-domain defensins comprising two defensin regions (2D defensins) or multiple defensin regions (MD defensins) connected by a short linker region. Many of these 2D or MD defensins were further found to comprise an N-terminal transit signal (TS) sequence or region that may generally become cleaved from the remainder of the protein to produce a mature defensin protein. Some of the 2D or MD defensins were also found to comprise a C-terminal extension sequence. 
     To identify novel multi-domain defensins, genomic and transciptome sequences from 19 different plant species were mined and analyzed to identify longer polypeptide sequences having one or more structural or functional features characteristic of defensins. Polypeptides meeting the following criteria were identified as multi-domain defensins and subjected to further analysis: (a) Polypeptides comprising a Pfam gamma-thionin domain (PF00304), as determined by a search of the Pfam protein families database using an E-value cutoff of 1e −3  (Finn, et al.  Nucleic Acids Research,  2014, Database Issue 42:D222-D230); (b) Polypeptides comprising at least 100 amino acids; (c) Polypeptides comprising at least 8 cysteine residues; and (d) Polypeptides comprising two or more extended gamma-thionin domains (GXCX n C) separated by a short polypeptide linker sequence, wherein the n is 3-22 amino acids in length, and X is any amino acid. These putative multi-domain defensins were further identified by the absence of a premature stop codon in their coding sequence. 
     A total of 51 multi-domain defensin proteins including mostly 2D defensins were identified (see below) by mining the genomes of the following 19 plant genomes:  Arabidopsis lyrata, Arabidopsis thaliana, Brassica rapa, Brassica napus, Carica papaya, Citrus clementina, Citrus sinensis, Cucumis melo, Cucumis sativus, Glycine max, Kochia scoparia, Malus domestica, Medicago truncatula, Portulaca oleracea, Prunus persica, Raphanus raphanistrum, Rosa blanda, Trifolium repens , and  Zea mays . The detection of mRNA transcripts for these multi-domain defensins and their discovery in many plant species supports the conclusion that these multi-domain defensins are expressed in plants. 
     Example 2. Annotation of Multi-Domain Defensins 
     Full-length multi-domain protein sequences identified by the above criteria were further analyzed using a combination of manual calls, multiple sequence alignments, and N-terminal cleavage site (SignalP) predictions to identify and locate the N-terminal TS or signal peptide sequences, defensin regions, intervening linker regions, and C-terminal extensions. Linker regions were identified as being between the last cysteine residue of the defensin region on the N-terminal side and one or two amino acids upstream of the first cysteine residue in the defensin region on the C-terminal side. Determining the position of the C-terminal end of the linker region of an identified 2D or MD defensin is based on the expected position of the predicted cleavage site of the downstream defensin region on the C-terminal side of the linker region, although this site may generally not become cleaved, unlike the cleavage site between the N-terminal TS sequence and the adjacent defensin region. 
     The 51 multi-domain defensin sequences identified from the 19 plant genomes are shown in Tables 1 and 2. SEQ ID NOs: 1-51 represent nucleotide coding sequences of the newly identified 2D or MD defensins, and SEQ ID NOs: 52-102 represent the corresponding polypeptide sequences of these identified 2D or MD defensins. For each of the identified 2D and MD defensins, the sequence boundaries (start and stop positions) for each of the defensin regions (D1, D2, etc.), linker region(s) (L1, L2, etc.), N-terminal transit signal (TS) sequence, and C-terminal (CT) extension sequence (if present; “NA” if not present) are shown for the DNA and protein sequences in Tables 1 and 2, respectively. The full coding sequence (CDS) is also provided in Table 1. The defensin components of these 2D or MD defensins correspond to each of the defensin regions annotated in Tables 1 and 2. For illustration, the defensin components of “ARAly_AFP26” on a nucleotide level correspond to nucleotides positions 82-207 (D1) and 229-372 (D2) of SEQ ID NO: 1, whereas the defensin components of “ARAly_AFP26” on a protein level correspond to amino acid positions 28-69 (D1) and 77-124 (D2) of SEQ ID NO: 52. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Nucleotide start and end positions for each region within the identified MD or 2D defensin sequences. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 TS 
                 CDS 
                 CT 
                 D1 
                 D2 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 DNA 
                 Protein 
                 TS 
                 TS 
                 CDS 
                 CDS 
                 CT 
                 CT 
                 D1 
                 D1 
                 D2 
                 D2 
               
               
                 Locus ID 
                 SEQ ID NO. 
                 SEQ ID NO. 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
               
               
                   
               
               
                 ARAly_AFP26 
                 1 
                 52 
                 1 
                 81 
                 82 
                 372 
                 NA 
                 NA 
                 82 
                 207 
                 229 
                 372 
               
               
                 At_AFP10 
                 2 
                 53 
                 1 
                 63 
                 64 
                 387 
                 NA 
                 NA 
                 64 
                 234 
                 259 
                 387 
               
               
                 At_AFP11 
                 3 
                 54 
                 1 
                 78 
                 79 
                 381 
                 NA 
                 NA 
                 79 
                 210 
                 241 
                 381 
               
               
                 At_AFP12 
                 4 
                 55 
                 1 
                 81 
                 82 
                 360 
                 NA 
                 NA 
                 82 
                 195 
                 220 
                 360 
               
               
                 At_AFP8 
                 5 
                 56 
                 1 
                 78 
                 79 
                 378 
                 NA 
                 NA 
                 79 
                 204 
                 259 
                 378 
               
               
                 At_AFP9 
                 6 
                 57 
                 1 
                 78 
                 79 
                 366 
                 NA 
                 NA 
                 79 
                 210 
                 226 
                 366 
               
               
                 BASsc_AFP21 
                 7 
                 58 
                 1 
                 21 
                 22 
                 354 
                 NA 
                 NA 
                 22 
                 162 
                 193 
                 354 
               
               
                 Bn_AFP35 
                 8 
                 59 
                 1 
                 99 
                 100 
                 402 
                 NA 
                 NA 
                 100 
                 228 
                 259 
                 402 
               
               
                 Bn_AFP36 
                 9 
                 60 
                 1 
                 18 
                 19 
                 330 
                 NA 
                 NA 
                 19 
                 150 
                 187 
                 330 
               
               
                 Bn_AFP47 
                 10 
                 61 
                 1 
                 78 
                 79 
                 366 
                 NA 
                 NA 
                 79 
                 204 
                 238 
                 366 
               
               
                 Bn_AFP74 
                 11 
                 62 
                 1 
                 78 
                 79 
                 381 
                 NA 
                 NA 
                 79 
                 210 
                 238 
                 381 
               
               
                 Bn_AFP75 
                 12 
                 63 
                 1 
                 45 
                 46 
                 357 
                 NA 
                 NA 
                 46 
                 180 
                 214 
                 357 
               
               
                 Bn_AFP76 
                 13 
                 64 
                 1 
                 78 
                 79 
                 381 
                 NA 
                 NA 
                 79 
                 210 
                 238 
                 381 
               
               
                 Bn_AFP79 
                 14 
                 65 
                 1 
                 78 
                 79 
                 366 
                 NA 
                 NA 
                 79 
                 192 
                 223 
                 366 
               
               
                 Bn_AFP80 
                 15 
                 66 
                 1 
                 78 
                 79 
                 366 
                 NA 
                 NA 
                 79 
                 192 
                 223 
                 366 
               
               
                 Br_AFP1 
                 16 
                 67 
                 1 
                 42 
                 43 
                 333 
                 NA 
                 NA 
                 43 
                 177 
                 241 
                 333 
               
               
                 Br_AFP2 
                 17 
                 68 
                 1 
                 78 
                 79 
                 360 
                 NA 
                 NA 
                 79 
                 198 
                 220 
                 360 
               
               
                 CARpa_AFP1 
                 18 
                 69 
                 1 
                 45 
                 46 
                 402 
                 NA 
                 NA 
                 46 
                 192 
                 217 
                 402 
               
               
                 CARpa_AFP2 
                 19 
                 70 
                 1 
                 63 
                 64 
                 420 
                 NA 
                 NA 
                 64 
                 207 
                 232 
                 420 
               
               
                 CARpa_AFP3 
                 20 
                 71 
                 1 
                 69 
                 70 
                 384 
                 NA 
                 NA 
                 70 
                 192 
                 202 
                 384 
               
               
                 CITcl_AFP10 
                 21 
                 72 
                 1 
                 81 
                 82 
                 393 
                 NA 
                 NA 
                 82 
                 198 
                 208 
                 393 
               
               
                 CITcl_AFP11 
                 22 
                 73 
                 1 
                 66 
                 67 
                 402 
                 NA 
                 NA 
                 67 
                 195 
                 220 
                 402 
               
               
                 CITcl_AFP2 
                 23 
                 74 
                 1 
                 90 
                 91 
                 408 
                 NA 
                 NA 
                 91 
                 225 
                 247 
                 408 
               
               
                 CITcl_AFP3 
                 24 
                 75 
                 1 
                 75 
                 76 
                 390 
                 NA 
                 NA 
                 76 
                 216 
                 250 
                 390 
               
               
                 CITsi_AFP1 
                 25 
                 76 
                 1 
                 93 
                 94 
                 411 
                 NA 
                 NA 
                 94 
                 216 
                 226 
                 411 
               
               
                 CITsi_AFP2 
                 26 
                 77 
                 1 
                 54 
                 55 
                 369 
                 NA 
                 NA 
                 55 
                 195 
                 226 
                 369 
               
               
                 CUCme_AFP1 
                 27 
                 78 
                 1 
                 24 
                 25 
                 345 
                 NA 
                 NA 
                 25 
                 168 
                 205 
                 345 
               
               
                 CUCsa_AFP3 
                 28 
                 79 
                 1 
                 24 
                 25 
                 345 
                 NA 
                 NA 
                 25 
                 168 
                 205 
                 345 
               
               
                 Gm_AFP1 
                 29 
                 80 
                 1 
                 81 
                 82 
                 411 
                 NA 
                 NA 
                 82 
                 207 
                 217 
                 411 
               
               
                 Gm_AFP2 
                 30 
                 81 
                 1 
                 81 
                 82 
                 387 
                 NA 
                 NA 
                 82 
                 207 
                 244 
                 387 
               
               
                 MALdo_AFP11 
                 31 
                 82 
                 1 
                 87 
                 88 
                 408 
                 NA 
                 NA 
                 88 
                 219 
                 250 
                 408 
               
               
                 Mt_AFP14 
                 32 
                 83 
                 1 
                 87 
                 88 
                 408 
                 NA 
                 NA 
                 88 
                 237 
                 259 
                 408 
               
               
                 Mt_AFP60 
                 33 
                 84 
                 1 
                 84 
                 85 
                 390 
                 NA 
                 NA 
                 85 
                 204 
                 244 
                 390 
               
               
                 Mt_AFP65 
                 34 
                 85 
                 1 
                 78 
                 79 
                 384 
                 NA 
                 NA 
                 79 
                 204 
                 235 
                 384 
               
               
                 Mt_AFP66 
                 35 
                 86 
                 1 
                 69 
                 70 
                 285 
                 NA 
                 NA 
                 70 
                 105 
                 142 
                 285 
               
               
                 Mt_AFP67 
                 36 
                 87 
                 1 
                 81 
                 82 
                 387 
                 NA 
                 NA 
                 79 
                 207 
                 235 
                 387 
               
               
                 Mt_AFP77 
                 37 
                 88 
                 1 
                 81 
                 82 
                 390 
                 NA 
                 NA 
                 82 
                 189 
                 232 
                 390 
               
               
                 Mt_AFP78 
                 38 
                 89 
                 1 
                 78 
                 79 
                 384 
                 NA 
                 NA 
                 79 
                 207 
                 238 
                 384 
               
               
                 PORol_AFP1 
                 39 
                 90 
                 1 
                 63 
                 64 
                 360 
                 NA 
                 NA 
                 64 
                 228 
                 256 
                 360 
               
               
                 PORol_AFP2 
                 40 
                 91 
                 NA 
                 NA 
                 1 
                 297 
                 NA 
                 NA 
                 1 
                 165 
                 193 
                 297 
               
               
                 PORol_AFP27 
                 41 
                 92 
                 1 
                 87 
                 88 
                 390 
                 NA 
                 NA 
                 88 
                 219 
                 241 
                 390 
               
               
                 PRUpe_AFP1 
                 42 
                 93 
                 1 
                 18 
                 19 
                 402 
                 NA 
                 NA 
                 19 
                 201 
                 211 
                 402 
               
               
                 RAPra_AFP23 
                 43 
                 94 
                 1 
                 96 
                 97 
                 411 
                 NA 
                 NA 
                 97 
                 228 
                 265 
                 411 
               
               
                 RAPra_AFP24 
                 44 
                 95 
                 1 
                 78 
                 79 
                 384 
                 NA 
                 NA 
                 79 
                 213 
                 238 
                 384 
               
               
                 RAPra_AFP25 
                 45 
                 96 
                 1 
                 27 
                 28 
                 345 
                 NA 
                 NA 
                 28 
                 165 
                 199 
                 345 
               
               
                 RAPra_AFP26 
                 46 
                 97 
                 1 
                 9 
                 10 
                 294 
                 NA 
                 NA 
                 10 
                 132 
                 163 
                 294 
               
               
                 RAPra_AFP27 
                 47 
                 98 
                 1 
                 78 
                 79 
                 363 
                 NA 
                 NA 
                 79 
                 201 
                 232 
                 363 
               
               
                 ROSbl_AFP11 
                 48 
                 99 
                 1 
                 78 
                 79 
                 360 
                 NA 
                 NA 
                 79 
                 204 
                 214 
                 360 
               
               
                 TRIre_AFP5 
                 49 
                 100 
                 1 
                 84 
                 85 
                 387 
                 NA 
                 NA 
                 85 
                 204 
                 241 
                 387 
               
               
                 Zm_AFP101 
                 50 
                 101 
                 1 
                 24 
                 25 
                 420 
                 421 
                 474 
                 25 
                 120 
                 283 
                 420 
               
               
                 Zm_AFP100 
                 51 
                 102 
                 1 
                 162 
                 163 
                 966 
                 967 
                 1002 
                 163 
                 312 
                 397 
                 534 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 D3 
                 D4 
                 L1 
                 L2 
                 L3 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 D3 
                 D3 
                 D4 
                 D4 
                 Linker1 
                 Linker1 
                 Linker2 
                 Linker2 
                 Linker3 
                 Linker3 
               
               
                   
                 Locus ID 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
               
               
                   
                   
               
               
                   
                 ARAly_AFP26 
                   
                   
                   
                   
                 208 
                 228 
               
               
                   
                 At_AFP10 
                   
                   
                   
                   
                 235 
                 258 
               
               
                   
                 At_AFP11 
                   
                   
                   
                   
                 211 
                 240 
               
               
                   
                 At_AFP12 
                   
                   
                   
                   
                 196 
                 219 
               
               
                   
                 At_AFP8 
                   
                   
                   
                   
                 205 
                 258 
               
               
                   
                 At_AFP9 
                   
                   
                   
                   
                 211 
                 225 
               
               
                   
                 BASsc_AFP21 
                   
                   
                   
                   
                 163 
                 192 
               
               
                   
                 Bn_AFP35 
                   
                   
                   
                   
                 229 
                 258 
               
               
                   
                 Bn_AFP36 
                   
                   
                   
                   
                 151 
                 186 
               
               
                   
                 Bn_AFP47 
                   
                   
                   
                   
                 205 
                 237 
               
               
                   
                 Bn_AFP74 
                   
                   
                   
                   
                 211 
                 237 
               
               
                   
                 Bn_AFP75 
                   
                   
                   
                   
                 181 
                 213 
               
               
                   
                 Bn_AFP76 
                   
                   
                   
                   
                 211 
                 237 
               
               
                   
                 Bn_AFP79 
                   
                   
                   
                   
                 193 
                 222 
               
               
                   
                 Bn_AFP80 
                   
                   
                   
                   
                 193 
                 222 
               
               
                   
                 Br_AFP1 
                   
                   
                   
                   
                 178 
                 240 
               
               
                   
                 Br_AFP2 
                   
                   
                   
                   
                 199 
                 219 
               
               
                   
                 CARpa_AFP1 
                   
                   
                   
                   
                 193 
                 216 
               
               
                   
                 CARpa_AFP2 
                   
                   
                   
                   
                 208 
                 231 
               
               
                   
                 CARpa_AFP3 
                   
                   
                   
                   
                 193 
                 201 
               
               
                   
                 CITcl_AFP10 
                   
                   
                   
                   
                 199 
                 207 
               
               
                   
                 CITcl_AFP11 
                   
                   
                   
                   
                 196 
                 219 
               
               
                   
                 CITcl_AFP2 
                   
                   
                   
                   
                 226 
                 246 
               
               
                   
                 CITcl_AFP3 
                   
                   
                   
                   
                 217 
                 249 
               
               
                   
                 CITsi_AFP1 
                   
                   
                   
                   
                 217 
                 225 
               
               
                   
                 CITsi_AFP2 
                   
                   
                   
                   
                 196 
                 225 
               
               
                   
                 CUCme_AFP1 
                   
                   
                   
                   
                 169 
                 204 
               
               
                   
                 CUCsa_AFP3 
                   
                   
                   
                   
                 169 
                 204 
               
               
                   
                 Gm_AFP1 
                   
                   
                   
                   
                 208 
                 216 
               
               
                   
                 Gm_AFP2 
                   
                   
                   
                   
                 208 
                 243 
               
               
                   
                 MALdo_AFP11 
                   
                   
                   
                   
                 220 
                 249 
               
               
                   
                 Mt_AFP14 
                   
                   
                   
                   
                 238 
                 258 
               
               
                   
                 Mt_AFP60 
                   
                   
                   
                   
                 205 
                 243 
               
               
                   
                 Mt_AFP65 
                   
                   
                   
                   
                 205 
                 234 
               
               
                   
                 Mt_AFP66 
                   
                   
                   
                   
                 106 
                 141 
               
               
                   
                 Mt_AFP67 
                   
                   
                   
                   
                 208 
                 234 
               
               
                   
                 Mt_AFP77 
                   
                   
                   
                   
                 190 
                 231 
               
               
                   
                 Mt_AFP78 
                   
                   
                   
                   
                 208 
                 237 
               
               
                   
                 PORol_AFP1 
                   
                   
                   
                   
                 229 
                 255 
               
               
                   
                 PORol_AFP2 
                   
                   
                   
                   
                 166 
                 192 
               
               
                   
                 PORol_AFP27 
                   
                   
                   
                   
                 220 
                 240 
               
               
                   
                 PRUpe_AFP1 
                   
                   
                   
                   
                 202 
                 210 
               
               
                   
                 RAPra_AFP23 
                   
                   
                   
                   
                 229 
                 264 
               
               
                   
                 RAPra_AFP24 
                   
                   
                   
                   
                 214 
                 237 
               
               
                   
                 RAPra_AFP25 
                   
                   
                   
                   
                 166 
                 198 
               
               
                   
                 RAPra_AFP26 
                   
                   
                   
                   
                 133 
                 162 
               
               
                   
                 RAPra_AFP27 
                   
                   
                   
                   
                 202 
                 231 
               
               
                   
                 ROSbl_AFP11 
                   
                   
                   
                   
                 205 
                 213 
               
               
                   
                 TRIre_AFP5 
                   
                   
                   
                   
                 205 
                 240 
               
               
                   
                 Zm_AFP101 
                   
                   
                   
                   
                 121 
                 282 
               
               
                   
                 Zm_AFP100 
                 613 
                 750 
                 829 
                 966 
                 313 
                 396 
                 535 
                 612 
                 751 
                 828 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Protein start and end positions for each region within the identified MD or 2D defensin sequences. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 TS 
                 CT 
                 D1 
                 D2 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 DNA 
                 Protein 
                 TS 
                 TS 
                 CT 
                 CT 
                 D1 
                 D1 
                 D2 
                 D2 
               
               
                 Locus ID 
                 SEQ ID NO. 
                 SEQ ID NO. 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
               
               
                   
               
               
                 ARAly_AFP26 
                 1 
                 52 
                 1 
                 27 
                 NA 
                 NA 
                 28 
                 69 
                 77 
                 124 
               
               
                 At_AFP10 
                 2 
                 53 
                 1 
                 21 
                 NA 
                 NA 
                 22 
                 78 
                 87 
                 129 
               
               
                 At_AFP11 
                 3 
                 54 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 70 
                 81 
                 127 
               
               
                 At_AFP12 
                 4 
                 55 
                 1 
                 27 
                 NA 
                 NA 
                 28 
                 65 
                 74 
                 120 
               
               
                 At_AFP8 
                 5 
                 56 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 68 
                 87 
                 126 
               
               
                 At_AFP9 
                 6 
                 57 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 70 
                 76 
                 122 
               
               
                 BASsc_AFP21 
                 7 
                 58 
                 1 
                 7 
                 NA 
                 NA 
                 8 
                 54 
                 65 
                 118 
               
               
                 Bn_AFP35 
                 8 
                 59 
                 1 
                 33 
                 NA 
                 NA 
                 34 
                 76 
                 87 
                 134 
               
               
                 Bn_AFP36 
                 9 
                 60 
                 1 
                 6 
                 NA 
                 NA 
                 7 
                 50 
                 63 
                 110 
               
               
                 Bn_AFP47 
                 10 
                 61 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 68 
                 80 
                 122 
               
               
                 Bn_AFP74 
                 11 
                 62 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 70 
                 80 
                 127 
               
               
                 Bn_AFP75 
                 12 
                 63 
                 1 
                 15 
                 NA 
                 NA 
                 16 
                 60 
                 72 
                 119 
               
               
                 Bn_AFP76 
                 13 
                 64 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 70 
                 80 
                 127 
               
               
                 Bn_AFP79 
                 14 
                 65 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 64 
                 75 
                 122 
               
               
                 Bn_AFP80 
                 15 
                 66 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 64 
                 75 
                 122 
               
               
                 Br_AFP1 
                 16 
                 67 
                 1 
                 14 
                 NA 
                 NA 
                 15 
                 59 
                 81 
                 111 
               
               
                 Br_AFP2 
                 17 
                 68 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 66 
                 74 
                 120 
               
               
                 CARpa_AFP1 
                 18 
                 69 
                 1 
                 15 
                 NA 
                 NA 
                 16 
                 64 
                 73 
                 134 
               
               
                 CARpa_AFP2 
                 19 
                 70 
                 1 
                 21 
                 NA 
                 NA 
                 22 
                 69 
                 78 
                 140 
               
               
                 CARpa_AFP3 
                 20 
                 71 
                 1 
                 23 
                 NA 
                 NA 
                 24 
                 64 
                 68 
                 128 
               
               
                 CITcl_AFP10 
                 21 
                 72 
                 1 
                 27 
                 NA 
                 NA 
                 28 
                 66 
                 70 
                 131 
               
               
                 CITcl_AFP11 
                 22 
                 73 
                 1 
                 22 
                 NA 
                 NA 
                 23 
                 65 
                 74 
                 134 
               
               
                 CITcl_AFP2 
                 23 
                 74 
                 1 
                 30 
                 NA 
                 NA 
                 31 
                 75 
                 83 
                 136 
               
               
                 CITcl_AFP3 
                 24 
                 75 
                 1 
                 25 
                 NA 
                 NA 
                 26 
                 72 
                 84 
                 130 
               
               
                 CITsi_AFP1 
                 25 
                 76 
                 1 
                 31 
                 NA 
                 NA 
                 32 
                 72 
                 76 
                 137 
               
               
                 CITsi_AFP2 
                 26 
                 77 
                 1 
                 18 
                 NA 
                 NA 
                 19 
                 65 
                 76 
                 123 
               
               
                 CUCme_AFP1 
                 27 
                 78 
                 1 
                 8 
                 NA 
                 NA 
                 9 
                 56 
                 69 
                 115 
               
               
                 CUCsa_AFP3 
                 28 
                 79 
                 1 
                 8 
                 NA 
                 NA 
                 9 
                 56 
                 69 
                 115 
               
               
                 Gm_AFP1 
                 29 
                 80 
                 1 
                 27 
                 NA 
                 NA 
                 28 
                 69 
                 73 
                 137 
               
               
                 Gm_AFP2 
                 30 
                 81 
                 1 
                 27 
                 NA 
                 NA 
                 28 
                 69 
                 82 
                 129 
               
               
                 MALdo_AFP11 
                 31 
                 82 
                 1 
                 29 
                 NA 
                 NA 
                 30 
                 73 
                 84 
                 136 
               
               
                 Mt_AFP14 
                 32 
                 83 
                 1 
                 29 
                 NA 
                 NA 
                 30 
                 79 
                 87 
                 136 
               
               
                 Mt_AFP60 
                 33 
                 84 
                 1 
                 28 
                 NA 
                 NA 
                 29 
                 68 
                 82 
                 130 
               
               
                 Mt_AFP65 
                 34 
                 85 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 68 
                 79 
                 128 
               
               
                 Mt_AFP66 
                 35 
                 86 
                 1 
                 23 
                 NA 
                 NA 
                 24 
                 35 
                 48 
                 95 
               
               
                 Mt_AFP67 
                 36 
                 87 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 69 
                 79 
                 129 
               
               
                 Mt_AFP77 
                 37 
                 88 
                 1 
                 27 
                 NA 
                 NA 
                 28 
                 63 
                 78 
                 130 
               
               
                 Mt_AFP78 
                 38 
                 89 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 69 
                 80 
                 128 
               
               
                 PORol_AFP1 
                 39 
                 90 
                 1 
                 21 
                 NA 
                 NA 
                 22 
                 76 
                 86 
                 120 
               
               
                 PORol_AFP2 
                 40 
                 91 
                 NA 
                 NA 
                 NA 
                 NA 
                 1 
                 55 
                 65 
                 99 
               
               
                 PORol_AFP27 
                 41 
                 92 
                 1 
                 29 
                 NA 
                 NA 
                 30 
                 73 
                 81 
                 130 
               
               
                 PRUpe_AFP1 
                 42 
                 93 
                 1 
                 6 
                 NA 
                 NA 
                 7 
                 67 
                 71 
                 134 
               
               
                 RAPra_AFP23 
                 43 
                 94 
                 1 
                 32 
                 NA 
                 NA 
                 33 
                 76 
                 89 
                 137 
               
               
                 RAPra_AFP24 
                 44 
                 95 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 71 
                 80 
                 128 
               
               
                 RAPra_AFP25 
                 45 
                 96 
                 1 
                 9 
                 NA 
                 NA 
                 10 
                 55 
                 67 
                 115 
               
               
                 RAPra_AFP26 
                 46 
                 97 
                 1 
                 3 
                 NA 
                 NA 
                 4 
                 44 
                 55 
                 98 
               
               
                 RAPra_AFP27 
                 47 
                 98 
                 1 
                 26 
                 NA 
                 NA 
                 27 
                 67 
                 78 
                 121 
               
               
                 ROSbl_AFP11 
                 48 
                 99 
                 1 
                 26 
                 NA 
                 NA 
                 11 
                 68 
                 72 
                 120 
               
               
                 TRIre_AFP5 
                 49 
                 100 
                 1 
                 28 
                 NA 
                 NA 
                 29 
                 68 
                 81 
                 129 
               
               
                 Zm_AFP101 
                 50 
                 101 
                 1 
                 8 
                 967 
                 1002 
                 9 
                 40 
                 95 
                 140 
               
               
                 Zm_AFP100 
                 51 
                 102 
                 1 
                 54 
                 421 
                 474 
                 55 
                 104 
                 133 
                 178 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 D3 
                 D4 
                 L1 
                 L2 
                 L3 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 D3 
                 D3 
                 D4 
                 D4 
                 Linker1 
                 Linker1 
                 Linker2 
                 Linker2 
                 Linker3 
                 Linker3 
               
               
                 Locus ID 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
               
               
                   
               
               
                 ARAly_AFP26 
                   
                   
                   
                   
                 70 
                 76 
               
               
                 At_AFP10 
                   
                   
                   
                   
                 79 
                 86 
               
               
                 At_AFP11 
                   
                   
                   
                   
                 71 
                 80 
               
               
                 At_AFP12 
                   
                   
                   
                   
                 66 
                 73 
               
               
                 At_AFP8 
                   
                   
                   
                   
                 69 
                 86 
               
               
                 At_AFP9 
                   
                   
                   
                   
                 71 
                 75 
               
               
                 BASsc_AFP21 
                   
                   
                   
                   
                 55 
                 64 
               
               
                 Bn_AFP35 
                   
                   
                   
                   
                 77 
                 86 
               
               
                 Bn_AFP36 
                   
                   
                   
                   
                 51 
                 62 
               
               
                 Bn_AFP47 
                   
                   
                   
                   
                 69 
                 79 
               
               
                 Bn_AFP74 
                   
                   
                   
                   
                 71 
                 79 
               
               
                 Bn_AFP75 
                   
                   
                   
                   
                 61 
                 71 
               
               
                 Bn_AFP76 
                   
                   
                   
                   
                 71 
                 79 
               
               
                 Bn_AFP79 
                   
                   
                   
                   
                 65 
                 74 
               
               
                 Bn_AFP80 
                   
                   
                   
                   
                 65 
                 74 
               
               
                 Br_AFP1 
                   
                   
                   
                   
                 60 
                 80 
               
               
                 Br_AFP2 
                   
                   
                   
                   
                 67 
                 73 
               
               
                 CARpa_AFP1 
                   
                   
                   
                   
                 65 
                 72 
               
               
                 CARpa_AFP2 
                   
                   
                   
                   
                 70 
                 77 
               
               
                 CARpa_AFP3 
                   
                   
                   
                   
                 65 
                 67 
               
               
                 CITcl_AFP10 
                   
                   
                   
                   
                 67 
                 69 
               
               
                 CITcl_AFP11 
                   
                   
                   
                   
                 66 
                 73 
               
               
                 CITcl_AFP2 
                   
                   
                   
                   
                 76 
                 82 
               
               
                 CITcl_AFP3 
                   
                   
                   
                   
                 73 
                 83 
               
               
                 CITsi_AFP1 
                   
                   
                   
                   
                 73 
                 75 
               
               
                 CITsi_AFP2 
                   
                   
                   
                   
                 66 
                 75 
               
               
                 CUCme_AFP1 
                   
                   
                   
                   
                 57 
                 68 
               
               
                 CUCsa_AFP3 
                   
                   
                   
                   
                 57 
                 68 
               
               
                 Gm_AFP1 
                   
                   
                   
                   
                 70 
                 72 
               
               
                 Gm_AFP2 
                   
                   
                   
                   
                 70 
                 81 
               
               
                 MALdo_AFP11 
                   
                   
                   
                   
                 74 
                 83 
               
               
                 Mt_AFP14 
                   
                   
                   
                   
                 80 
                 86 
               
               
                 Mt_AFP60 
                   
                   
                   
                   
                 69 
                 81 
               
               
                 Mt_AFP65 
                   
                   
                   
                   
                 69 
                 78 
               
               
                 Mt_AFP66 
                   
                   
                   
                   
                 36 
                 47 
               
               
                 Mt_AFP67 
                   
                   
                   
                   
                 70 
                 78 
               
               
                 Mt_AFP77 
                   
                   
                   
                   
                 64 
                 77 
               
               
                 Mt_AFP78 
                   
                   
                   
                   
                 70 
                 79 
               
               
                 PORol_AFP1 
                   
                   
                   
                   
                 77 
                 85 
               
               
                 PORol_AFP2 
                   
                   
                   
                   
                 56 
                 64 
               
               
                 PORol_AFP27 
                   
                   
                   
                   
                 74 
                 80 
               
               
                 PRUpe_AFP1 
                   
                   
                   
                   
                 68 
                 70 
               
               
                 RAPra_AFP23 
                   
                   
                   
                   
                 77 
                 88 
               
               
                 RAPra_AFP24 
                   
                   
                   
                   
                 72 
                 79 
               
               
                 RAPra_AFP25 
                   
                   
                   
                   
                 56 
                 66 
               
               
                 RAPra_AFP26 
                   
                   
                   
                   
                 45 
                 54 
               
               
                 RAPra_AFP27 
                   
                   
                   
                   
                 68 
                 77 
               
               
                 ROSbl_AFP11 
                   
                   
                   
                   
                 69 
                 71 
               
               
                 TRIre_AFP5 
                   
                   
                   
                   
                 69 
                 80 
               
               
                 Zm_AFP101 
                   
                   
                   
                   
                 41 
                 94 
               
               
                 Zm_AFP100 
                 205 
                 250 
                 277 
                 322 
                 105 
                 132 
                 179 
                 204 
                 251 
                 276 
               
               
                   
               
            
           
         
       
     
     Example 3. Identification of Linker Regions from Multi-Domain Defensins 
     Upon examination of the discovered multi-domain plant defensins, linker regions were identified between the defensin regions. The unique linker regions identified from the 2D and MD defensins are shown in Table 3. SEQ ID NOs: 103-152 represent nucleotide sequences encoding these identified defensin linker regions, and SEQ ID NOs: 153-202 represent the corresponding polypeptide sequences of these defensin linker regions. As can be seen in Table 3, there were 50 unique linker region DNA and protein sequences identified from these MD defensins since two of the linker regions for the 4D defensin (Zm_AFP100) are the same (SEQ ID NOs: 152 and 202). N-terminal TS sequences and C-terminal extension sequences (when present) for each of these identified MD defensins are further shown by sequence position annotations in Tables 1 and 2 above. The lengths of the TS sequences (if present) were observed to vary from a few amino acids to over 30 amino acids, and the C-terminal extension sequence was generally absent from the identified 2D defensins but was present in a 2D defensin (Zm_AFP101) and a 4D defensin (Zm_AFP100) at a variable length of about 35-50 amino acids. Most of the linkers were found to be proline-rich, while others were enriched with glycine or charged amino acids. Many of the linkers were approximately 10-20 amino acids in length, but a small subset of linkers had either very short linkers of only a few amino acids or longer linker sequence lengths of 20-65 amino acids. 
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 D and MD Defensin Linker Regions. 
               
            
           
           
               
               
               
               
            
               
                 Locus ID 
                 Linker ID 
                 DNA SEQ ID NO 
                 Protein SEQ ID NO 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 ARAly_AFP26 
                 ARAly26L10 
                 103 
                 153 
               
               
                 At_AFP10 
                 At10L18 
                 104 
                 154 
               
               
                 At_AFP11 
                 At11L19 
                 105 
                 155 
               
               
                 At_AFP12 
                 At12L20 
                 106 
                 156 
               
               
                 At_AFP8 
                 At8L21 
                 107 
                 157 
               
               
                 At_AFP9 
                 At9L22 
                 108 
                 158 
               
               
                 BASsc_AFP21 
                 BASsc21L23 
                 109 
                 159 
               
               
                 Bn_AFP35 
                 Bn35L11 
                 110 
                 160 
               
               
                 Bn_AFP36 
                 Bn36L1 
                 111 
                 161 
               
               
                 Bn_AFP47 
                 Bn47L2 
                 112 
                 162 
               
               
                 Bn_AFP74 
                 Bn74L3 
                 113 
                 163 
               
               
                 Bn_AFP75 
                 Bn75L13 
                 114 
                 164 
               
               
                 Bn_AFP76 
                 Bn76L3 
                 113 
                 163 
               
               
                 Bn_AFP79 
                 Bn79L7 
                 115 
                 165 
               
               
                 Bn_AFP80 
                 Bn80L7 
                 115 
                 165 
               
               
                 Br_AFP1 
                 Br1L24 
                 116 
                 166 
               
               
                 Br_AFP2 
                 Br2L25 
                 117 
                 167 
               
               
                 CARpa_AFP1 
                 CARpa1L26 
                 118 
                 168 
               
               
                 CARpa_AFP2 
                 CARpa2L27 
                 119 
                 169 
               
               
                 CARpa_AFP3 
                 CARpa3L28 
                 120 
                 170 
               
               
                 CITcl_AFP10 
                 CITcl10L29 
                 121 
                 171 
               
               
                 CITcl_AFP11 
                 CITcl11L30 
                 122 
                 172 
               
               
                 CITcl_AFP2 
                 CITcl2L17 
                 123 
                 173 
               
               
                 CITcl_AFP3 
                 CITcl3L14 
                 124 
                 174 
               
               
                 CITsi_AFP1 
                 CITsi1L31 
                 125 
                 175 
               
               
                 CITsi_AFP2 
                 CITsi2L32 
                 126 
                 176 
               
               
                 CUCme_AFP1 
                 CUCme1L33 
                 127 
                 177 
               
               
                 CUCsa_AFP3 
                 CUCsa3L34 
                 128 
                 178 
               
               
                 Gm_AFP1 
                 Gm1L35 
                 129 
                 179 
               
               
                 Gm_AFP2 
                 Gm2L36 
                 130 
                 180 
               
               
                 MALdo_AFP11 
                 MALdo11L12 
                 131 
                 181 
               
               
                 Mt_AFP14 
                 Mt14L6 
                 132 
                 182 
               
               
                 Mt_AFP60 
                 Mt60L4 
                 133 
                 183 
               
               
                 Mt_AFP65 
                 Mt65L8 
                 134 
                 184 
               
               
                 Mt_AFP66 
                 Mt66L15 
                 135 
                 185 
               
               
                 Mt_AFP67 
                 Mt67L16 
                 136 
                 186 
               
               
                 Mt_AFP77 
                 Mt77L5 
                 137 
                 187 
               
               
                 Mt_AFP78 
                 Mt78L9 
                 138 
                 188 
               
               
                 PORol_AFP1 
                 PORol1L37 
                 139 
                 189 
               
               
                 PORol_AFP2 
                 PORol2L38 
                 140 
                 190 
               
               
                 PORol_AFP27 
                 PORol27L39 
                 141 
                 191 
               
               
                 PRUpe_AFP1 
                 PRUpe1L40 
                 142 
                 192 
               
               
                 RAPra_AFP23 
                 RAPra23L41 
                 143 
                 193 
               
               
                 RAPra_AFP24 
                 RAPra24L42 
                 144 
                 194 
               
               
                 RAPra_AFP25 
                 RAPra25L43 
                 145 
                 195 
               
               
                 RAPra_AFP26 
                 RAPra26L44 
                 146 
                 196 
               
               
                 RAPra_AFP27 
                 RAPra27L45 
                 147 
                 197 
               
               
                 ROSbl_AFP11 
                 ROSbl11L46 
                 148 
                 198 
               
               
                 TRIre_AFP5 
                 TRIre5L47 
                 149 
                 199 
               
               
                 Zm_AFP101 
                 Zm101L49 
                 150 
                 200 
               
               
                 Zm_AFP100 L1 
                 Zm100L48 
                 151 
                 201 
               
               
                 Zm_AFP100 L2 
                 Zm100L50 
                 152 
                 202 
               
               
                 Zm_AFP100 L3 
                 Zm100L50 
                 152 
                 202 
               
               
                   
               
            
           
         
       
     
     Example 4. Design of Synthetic Multi-Domain Defensins 
     In this example, synthetic homodimeric 2D defensins were designed and constructed to comprise two copies of a cognate single domain (1D) defensin, for example MtDef4 (nucleotide SEQ ID NO: 1029; polypeptide SEQ ID NO: 1128) or Coix22 (nucleotide SEQ ID NO: 990; polypeptide SEQ ID NO: 1089), connected by various linker regions derived from native 2D or MD defensins described herein. Synthetic multi-domain defensins comprising Coix22 defensin regions and having one of the nucleic acid sequences of SEQ ID NOs: 203-219 (encoding the protein sequences of SEQ ID NOs: 329-345, respectively) linked or connected by 17 different linker regions (identified in Table 4) were assessed in corn protoplasts. Synthetic multi-domain defensins comprising MtDef4 defensin regions and having nucleic acid sequences of SEQ ID NOs: 220-236 (encoding the protein sequences of SEQ ID NOs: 346-362, respectively) with the same linker regions as identified in Table 4 were also assessed in corn and soy protoplasts. Further reference is made to Tables 3 and 5 for the sequence identifiers corresponding to these 17 linker regions. 
     Also described in this example is the design of a synthetic heterodimeric defensin as set forth in nucleotide SEQ ID NO: 1155 and polypeptide SEQ ID NO: 1156 using Coix 22, linker Mt.AFP65 (nucleotide SEQ ID NO: 134; polypeptide SEQ ID NO: 184) and AMAru.AFP10 (nucleotide SEQ ID NO: 963; polypeptide SEQ ID NO: 1062). Stop codons were removed from the N-terminal defensin region of these synthetic multi-domain 2D defensins. The defensin N-terminal transit signal (TS) domain was maintained at the N-terminus of the N-terminal defensin region, but removed from the N-terminus of the C-terminal defensin region. Diagrammatic examples of these synthetic defensins are shown in  FIG. 1  (L1, L2 and L3 refer to linkers derived from different 2D defensins identified herein). 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Homodimeric synthetic 2D using MtDef4 or Coix22 defensins 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Nucle- 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 otide 
                 Protein 
                   
                   
                 Protein 
                 Protein 
                 Protein 
                 Protein 
                 Protein 
                 Protein 
               
               
                 SEQ 
                 SEQ 
                   
                   
                 TS 
                 TS 
                 MP 
                 MP 
                 Linker 
                 Linker 
               
               
                 ID NO 
                 ID NO 
                 Template ID 
                 Linker ID 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
               
               
                   
               
               
                 203 
                 329 
                 Cl22FL_ARAly26L10_Cl22D1 
                 ARAly26L10 
                 1 
                 31 
                 32 
                 80 
                 81 
                 87 
               
               
                 204 
                 330 
                 Cl22FL_Bn35L11_Cl22D1 
                 Bn35L11 
                 1 
                 31 
                 32 
                 80 
                 81 
                 90 
               
               
                 205 
                 331 
                 Cl22FL_Bn36L1_Cl22D1 
                 Bn36L1 
                 1 
                 31 
                 32 
                 80 
                 81 
                 92 
               
               
                 206 
                 332 
                 Cl22FL_Bn47L2_Cl22D1 
                 Bn47L2 
                 1 
                 31 
                 32 
                 80 
                 81 
                 91 
               
               
                 207 
                 333 
                 Cl22FL_Bn74L3_Cl22D1 
                 Bn74L3 
                 1 
                 31 
                 32 
                 80 
                 81 
                 89 
               
               
                 208 
                 334 
                 Cl22FL_Bn75L1_Cl22D1 
                 Bn75L13 
                 1 
                 31 
                 32 
                 80 
                 81 
                 91 
               
               
                 209 
                 335 
                 Cl22FL_Bn79L7_Cl22D1 
                 Bn79L7 
                 1 
                 31 
                 32 
                 80 
                 81 
                 90 
               
               
                 210 
                 336 
                 Cl22FL_CITcl2L17_Cl22D1 
                 CITcl2L17 
                 1 
                 31 
                 32 
                 80 
                 81 
                 87 
               
               
                 211 
                 337 
                 Cl22FL_CITcl3L14_Cl22D1 
                 CITcl3L14 
                 1 
                 31 
                 32 
                 80 
                 81 
                 91 
               
               
                 212 
                 338 
                 Cl22FL_MALdo11L12_Cl22D1 
                 MALdo11L12 
                 1 
                 31 
                 32 
                 80 
                 81 
                 90 
               
               
                 213 
                 339 
                 Cl22FL_Mt14L6_Cl22D1 
                 Mt14L6 
                 1 
                 31 
                 32 
                 80 
                 81 
                 87 
               
               
                 214 
                 340 
                 Cl22FL_Mt60L4_Cl22D1 
                 Mt60L4 
                 1 
                 31 
                 32 
                 80 
                 81 
                 93 
               
               
                 215 
                 341 
                 Cl22FL_Mt65L8_Cl22D1 
                 Mt65L8 
                 1 
                 31 
                 32 
                 80 
                 81 
                 90 
               
               
                 216 
                 342 
                 Cl22FL_Mt66L15_Cl22D1 
                 Mt66L15 
                 1 
                 31 
                 32 
                 80 
                 81 
                 92 
               
               
                 217 
                 343 
                 Cl22FL_Mt67L16_Cl22D1 
                 Mt67L16 
                 1 
                 31 
                 32 
                 80 
                 81 
                 89 
               
               
                 218 
                 344 
                 Cl22FL_Mt77L5_Cl22D1 
                 Mt77L5 
                 1 
                 31 
                 32 
                 80 
                 81 
                 94 
               
               
                 219 
                 345 
                 Cl22FL_Mt78L9_Cl22D1 
                 Mt78L9 
                 1 
                 31 
                 32 
                 80 
                 81 
                 90 
               
               
                 220 
                 346 
                 MtDef4FL_ARAly26L10_MtDef4D1 
                 ARAly26L10 
                 1 
                 29 
                 30 
                 76 
                 77 
                 83 
               
               
                 222 
                 348 
                 MtDef4FL_Bn36L1_MtDef4D1 
                 Bn36L1 
                 1 
                 29 
                 30 
                 76 
                 77 
                 88 
               
               
                 223 
                 349 
                 MtDef4FL_Bn47L2_MtDef4D1 
                 Bn47L2 
                 1 
                 29 
                 30 
                 76 
                 77 
                 87 
               
               
                 224 
                 350 
                 MtDef4FL_Bn74L3_MtDef4D1 
                 Bn74L3 
                 1 
                 29 
                 30 
                 76 
                 77 
                 85 
               
               
                 225 
                 351 
                 MtDef4FL_Bn75L13_MtDef4D1 
                 Bn75L13 
                 1 
                 29 
                 30 
                 76 
                 77 
                 87 
               
               
                 226 
                 352 
                 MtDef4FL_Bn79L7_MtDef4D1 
                 Bn79L7 
                 1 
                 29 
                 30 
                 76 
                 77 
                 86 
               
               
                 227 
                 353 
                 MtDef4FL_CITcl2L17_MtDef4D1 
                 CITcl2L17 
                 1 
                 29 
                 30 
                 76 
                 77 
                 83 
               
               
                 228 
                 354 
                 MtDef4FL_CITcl3L14_MtDef4D1 
                 CITcl3L14 
                 1 
                 29 
                 30 
                 76 
                 77 
                 87 
               
               
                 229 
                 355 
                 MtDef4FL_MALdo11L12_MtDef4D1 
                 MALdo11L12 
                 1 
                 29 
                 30 
                 76 
                 77 
                 86 
               
               
                 230 
                 356 
                 MtDef4FL_Mt14L6_MtDef4D1 
                 Mt14L6 
                 1 
                 29 
                 30 
                 76 
                 77 
                 83 
               
               
                 231 
                 357 
                 MtDef4FL_Mt60L4_MtDef4D1 
                 Mt60L4 
                 1 
                 29 
                 30 
                 76 
                 77 
                 89 
               
               
                 232 
                 358 
                 MtDef4FL_Mt65L8_MtDef4D1 
                 Mt65L8 
                 1 
                 29 
                 30 
                 76 
                 77 
                 86 
               
               
                 233 
                 359 
                 MtDef4FL_Mt66L15_MtDef4D1 
                 Mt66L15 
                 1 
                 29 
                 30 
                 76 
                 77 
                 88 
               
               
                 234 
                 360 
                 MtDef4FL_Mt67L16_MtDef4D1 
                 Mt67L16 
                 1 
                 29 
                 30 
                 76 
                 77 
                 85 
               
               
                 235 
                 361 
                 MtDef4FL_Mt77L5_MtDef4D1 
                 Mt77L5 
                 1 
                 29 
                 30 
                 76 
                 77 
                 90 
               
               
                 236 
                 362 
                 MtDef4FL_Mt78L9_MtDef4D1 
                 Mt78L9 
                 1 
                 29 
                 30 
                 76 
                 77 
                 86 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Nucle- 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 otide 
                 Protein 
                 Protein 
                 DNA 
                 DNA 
                 DNA 
                 DNA 
                 DNA 
                 DNA 
                 DNA 
                 DNA 
               
               
                   
                 SEQ 
                 MP2 
                 MP2 
                 TS 
                 TS 
                 MP 
                 MP 
                 Linker 
                 Linker 
                 MP2 
                 MP2 
               
               
                   
                 ID NO 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
                 Start 
                 End 
               
               
                   
                   
               
               
                   
                 203 
                 88 
                 136 
                 1 
                 93 
                 94 
                 240 
                 241 
                 261 
                 262 
                 408 
               
               
                   
                 204 
                 91 
                 139 
                 1 
                 93 
                 94 
                 240 
                 241 
                 270 
                 271 
                 417 
               
               
                   
                 205 
                 93 
                 141 
                 1 
                 93 
                 94 
                 240 
                 241 
                 276 
                 277 
                 423 
               
               
                   
                 206 
                 92 
                 140 
                 1 
                 93 
                 94 
                 240 
                 241 
                 273 
                 274 
                 420 
               
               
                   
                 207 
                 90 
                 138 
                 1 
                 93 
                 94 
                 240 
                 241 
                 267 
                 268 
                 414 
               
               
                   
                 208 
                 92 
                 140 
                 1 
                 93 
                 94 
                 240 
                 241 
                 273 
                 274 
                 420 
               
               
                   
                 209 
                 91 
                 139 
                 1 
                 93 
                 94 
                 240 
                 241 
                 270 
                 271 
                 417 
               
               
                   
                 210 
                 88 
                 136 
                 1 
                 93 
                 94 
                 240 
                 241 
                 261 
                 262 
                 408 
               
               
                   
                 211 
                 92 
                 140 
                 1 
                 93 
                 94 
                 240 
                 241 
                 273 
                 274 
                 420 
               
               
                   
                 212 
                 91 
                 139 
                 1 
                 93 
                 94 
                 240 
                 241 
                 270 
                 271 
                 417 
               
               
                   
                 213 
                 88 
                 136 
                 1 
                 93 
                 94 
                 240 
                 241 
                 261 
                 262 
                 408 
               
               
                   
                 214 
                 94 
                 142 
                 1 
                 93 
                 94 
                 240 
                 241 
                 279 
                 280 
                 426 
               
               
                   
                 215 
                 91 
                 139 
                 1 
                 93 
                 94 
                 240 
                 241 
                 270 
                 271 
                 417 
               
               
                   
                 216 
                 93 
                 141 
                 1 
                 93 
                 94 
                 240 
                 241 
                 276 
                 277 
                 423 
               
               
                   
                 217 
                 90 
                 138 
                 1 
                 93 
                 94 
                 240 
                 241 
                 267 
                 268 
                 414 
               
               
                   
                 218 
                 95 
                 143 
                 1 
                 93 
                 94 
                 240 
                 241 
                 282 
                 283 
                 429 
               
               
                   
                 219 
                 91 
                 139 
                 1 
                 93 
                 94 
                 240 
                 241 
                 270 
                 271 
                 417 
               
               
                   
                 220 
                 84 
                 130 
                 1 
                 87 
                 88 
                 228 
                 229 
                 249 
                 250 
                 390 
               
               
                   
                 222 
                 89 
                 135 
                 1 
                 87 
                 88 
                 228 
                 229 
                 264 
                 265 
                 405 
               
               
                   
                 223 
                 88 
                 134 
                 1 
                 87 
                 88 
                 228 
                 229 
                 261 
                 262 
                 402 
               
               
                   
                 224 
                 86 
                 132 
                 1 
                 87 
                 88 
                 228 
                 229 
                 255 
                 256 
                 396 
               
               
                   
                 225 
                 88 
                 134 
                 1 
                 87 
                 88 
                 228 
                 229 
                 261 
                 262 
                 402 
               
               
                   
                 226 
                 87 
                 133 
                 1 
                 87 
                 88 
                 228 
                 229 
                 258 
                 259 
                 399 
               
               
                   
                 227 
                 84 
                 130 
                 1 
                 87 
                 88 
                 228 
                 229 
                 249 
                 250 
                 390 
               
               
                   
                 228 
                 88 
                 134 
                 1 
                 87 
                 88 
                 228 
                 229 
                 261 
                 262 
                 402 
               
               
                   
                 229 
                 87 
                 133 
                 1 
                 87 
                 88 
                 228 
                 229 
                 258 
                 259 
                 399 
               
               
                   
                 230 
                 84 
                 130 
                 1 
                 87 
                 88 
                 228 
                 229 
                 249 
                 250 
                 390 
               
               
                   
                 231 
                 90 
                 136 
                 1 
                 87 
                 88 
                 228 
                 229 
                 267 
                 268 
                 408 
               
               
                   
                 232 
                 87 
                 133 
                 1 
                 87 
                 88 
                 228 
                 229 
                 258 
                 259 
                 399 
               
               
                   
                 233 
                 89 
                 135 
                 1 
                 87 
                 88 
                 228 
                 229 
                 264 
                 265 
                 405 
               
               
                   
                 234 
                 86 
                 132 
                 1 
                 87 
                 88 
                 228 
                 229 
                 255 
                 256 
                 396 
               
               
                   
                 235 
                 91 
                 137 
                 1 
                 87 
                 88 
                 228 
                 229 
                 270 
                 271 
                 411 
               
               
                   
                 236 
                 87 
                 133 
                 1 
                 87 
                 88 
                 228 
                 229 
                 258 
                 259 
                 399 
               
               
                   
                   
               
            
           
         
       
     
     Example 5. Protein Accumulation of 1D Versus 2D Defensins in Corn and Soy Plant Cells 
     A. Protein Accumulation in Corn Protoplasts 
     Select synthetic homodimeric 2D defensins described in Example 4 were synthesized and delivered in a pUC57 cloning vector. Polymerase chain reaction (PCR) amplification and restriction digestion were used to amplify sequences encoding the synthetic 2D defensins from the pUC57 vectors, which were then ligated into a protoplast expression vector comprising a 35S promoter linked to the synthetic 2D defensin coding sequences. Protoplasts isolated from corn leaf mesophyll tissues were transformed with the protoplast expression plasmid vectors expressing native 1D Coix22 or synthetic 2D defensins comprising Coix22 defensin domains/regions connected by a linker region. For these synthetic 2D constructs, the homodimeric Coix22 defensin regions were heterologous with respect to the linker region. Following transformation, the protoplasts were lysed and the total proteins were subjected to standard Western immunoblotting using rabbit polyclonal antibodies raised against the native 1D Coix22. A goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP) was used as secondary antibody and the blots were visualized with the Super Signal West Femto kit (Thermo Fisher Scientific, Waltham, Mass.). Following Western blotting, the membranes were subjected to Coomassie staining to confirm equal sample loading. Dilutions of a purified full length CI_AFP22 were western blotted and the resulting bands were subject to densitometry imaging (Imager Lab 5.1) and used to create a standard curve. The standard curve was then used as a basis for comparison to quantify the 1D and 2D CI_AFP22 proteins that were expressed in corn protoplasts. 
     Protein accumulation of homodimeric Coix22 domains in combination with various heterologous linker sequences (see Table 4) were compared with protein accumulation for Coix22 1D defensin components. In these experiments, many of the synthetic 2D defensin proteins appear to accumulate to greater levels than their 1D defensin counterparts. Although one of the synthetic 2D defensins tested, homodimeric Coix22 2D linked by Mt77L5 (nucleic acid SEQ ID NO: 218; protein SEQ ID NO: 344), was cleaved in both soy and corn protoplasts, no cleavage product was observed for the remainder of the synthetic 2D Coix22 defensins tested. Accordingly, it is proposed that linker regions may be leveraged to enhance accumulation of 2D and MD defensins in plants to enable more effective control of plant pathogens and/or pests. 

 
     B. Protein Accumulation in Transgenic Soybean Plants 
     Transgenic soybean plants expressing Coix22 and Six Coix22 homodimeric 2D defensins with different heterologous linkers were established, and samples were collected at V2 and lyophilized prior to RNA and protein quantification. Protein quantification was performed using an indirect ELISA with a rabbit IgG primary antibody raised against the protein of interest and an anti-rabbit goat secondary antibody. Two concentrations of standard protein were used to calculate protein detected which is then normalized by total protein extracted. The QuantiGene technology measures RNA directly via a nucleic acid hybridization platform in which target RNAs are captured through cooperative binding of multiple oligonucleotide probes that are conjugated to magnetic microbeads. Cooperative binding of the multiple oligonucleotide probes with specificity for the target sequence results in exceptionally high assay specificity. Detection of this oligonucleotide complex occurs through amplification of a branched DNA amplifier and fluorescent signal, which is counted digitally by high-throughput flow cytometry sorting of the microbead. The results of RNA and Protein levels are shown in  FIG. 2 . While the variance in RNA is not significant between the 1D coix22 and the homodimeric Coix22 2D defensins, three of the five 2D proteins expressed at higher levels than the single domain, even after normalizing for the difference in protein size (2.25-fold increase for the 2D defensin using linker Bn36L1, 2-fold for the 2D defensin using linker Bn35L1, and 1.6-fold for the 2D defensin using linker CITcl2L17). These results demonstrate that multiple domains may help in protein accumulation through increased stability in transgenic soybean plants. 
     Example 6. Methods for Assessing Activity of Multi-Domain Defensins 
     In this example, synthetic 2D defensins and their 1D defensin counterparts were tested for their ability to inhibit growth of several fungal species. The defensins were expressed in  Pichia  yeast cells and purified for use in fungal plate assays as described below. 
     Defensin Production Using  Pichia  Cells 
     Constructs designed for  Pichia  and containing a  Pichia  signal peptide were synthesized by Bio Basic Inc. For clarity, this “ Pichia  signal peptide” used for these experiments is different than the N-terminal TS sequence of defensin proteins and may be present in addition to the TS sequence. The  Pichia  signal peptide is used to enable secretion of the synthesized protein from the  Pichia  cells to facilitate collection and protein purification. Approximately 5 μg of DNA was linearized, purified, and transformed into electrocompetent PichiaPink™ cells (Thermo Scientific) by electroporation. After recovery, the cells were plated onto  Pichia  Adenine Dropout (PAD) selection plates and incubated at 29° C. for 3 days. One colony from each transformation was cultured for 3 days for outgrowth. The cell pellets were then collected and induced with media containing methanol. The cells were cultured and supplemented with methanol daily for 3 additional days. The cell pellets were collected and the media used for the purification. Methods for purification of the  Pichia -expressed defensin proteins depended on their expected isoelectric point (pI). 
     Purification of Defensins Having pI&gt;7 Using Cation Exchange. 
     Media was diluted at a ratio of 8:1 with 50 mM sodium acetate at pH 5.0 and passed over SP sepharose resin (GE Healthcare #17-0729-01) using a vacuum manifold. The resin was washed with 50 mM sodium acetate at pH 5.0 and eluted in twice the resin volume with 50 mM sodium acetate at pH 5.0 containing 1M NaCl. 
     Purification of Defensins Having pI&lt;7 Using Anion Exchange. 
     Media was diluted at a ratio of 8:1 with 50 mM CHES at pH 9.0 and passed over Q sepharose resin (GE Healthcare #17-0510-01) using a vacuum manifold. The resin was washed with 50 mM CHES at pH 9.0 and eluted in twice the resin volume with 50 mM CHES at pH 9.0 containing 1M NaCl. 
     Buffer Exchange and Concentration. 
     The purified protein was dialyzed overnight into 8 mM Tris HCl at pH 8 using Pura-A-Lyzer Maxi 3500 Dialysis tubes (Sigma #PURX35050). The resulting solution was concentrated by drying in the Pura-A-Lyzer tubes until a final volume of 400-450 μl was achieved. 
     Fungal Growth Conditions 
     Fungi were first grown according to the conditions in Table 5 for 3-4 weeks or less depending on fungal species as indicated. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Fungal Growth Conditions. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Lighting and Temperature 
                   
               
               
                 Fungus 
                 Media 
                 Conditions 
                 Time before use 
               
               
                   
               
               
                 
                   Fusarium 
                 
                 0.25x Potato 
                 12 hour diurnal cycle at 19° C. 
                 21-28 days 
               
               
                 
                   graminearum 
                 
                 Dextrose Agar 
                 in the dark and 23° C. with 
                   
               
               
                 (Gibberella stalk rot) 
                   
                 100 μmol light 
                   
               
               
                 
                   Fusarium 
                 
                 0.25x Potato 
                 12 hour diurnal cycle at 19° C. 
                 21-28 days 
               
               
                 
                   verticilloides 
                 
                 Dextrose Agar 
                 in the dark and 23° C. with 
                   
               
               
                 (Fusarium stalk rot) 
                   
                 100 μmol light 
                   
               
               
                 
                   Colletotrichum 
                 
                 1x oatmeal 
                 12 hour diurnal cycle at 19° C. 
                 21-28 days 
               
               
                 
                   graminicola 
                 
                 agar 
                 in the dark and 23° C. with 
                   
               
               
                 (Anthracnose stalk 
                   
                 100 μmol light 
                   
               
               
                 rot) 
                   
                   
                   
               
               
                 
                   Stenocarpella maydis 
                 
                 1X PDA + 
                 12 hour diurnal cycle at 26° C. 
                 21-28 days 
               
               
                 (Diplodia ear rot) 
                 Cefotaxime 
                 with 100 μmol of light 
                   
               
               
                   
                   
                 during the daytime period 
                   
               
               
                 
                   Phakopsora 
                 
                 Culture in 
                 14 hour diurnal light cycle 
                 At least 12 days after 
               
               
                   pachyrhizi  (Soy Asian 
                 planta 
                 with 19° C. in the dark and  
                 plant inoculation 
               
               
                 rust) 
                   
                 22° C. with 500 μmol light 
                   
               
               
                   
                   
                 during the daytime period, 
                   
               
               
                   
                   
                 75% relative humidity 
               
               
                   
               
            
           
         
       
     
     Plate Functional Assay for Fungal Growth Inhibition 
     Following expression and purification of the 1D and 2D defensin proteins and growth of the fungal species in culture, the ability of the individual defensins to inhibit fungal growth was tested in plate assays. On the day of the growth test assay, fungi were collected in test media, filtered, and resuspended to the appropriate titer as shown in Table 7. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Conditions for Fungal Growth Inhibition Assay. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Test  
                 Filter  
                   
                   
               
               
                 Fungus 
                 media 
                 condition 
                 Test media 
                 Titer 
               
               
                   
               
               
                 
                   Fusarium  
                 
                 0.05%  
                 100 μm  
                 0.5x POTATO 
                   4e 4    
               
               
                 
                   graminearum 
                 
                 Triton 
                 Steriflip  
                 DEXTROSE 
                 spores/ml 
               
               
                 (Gibberella stalk rot) 
                 X100 
                 filter 
                 BROTH 
                   
               
               
                 
                   Fusarium verticilloides 
                 
                 0.05%  
                 100 μm  
                 0.5x POTATO 
                 6.5e 4   
               
               
                 (Fusarium stalk rot) 
                 Triton 
                 Steriflip  
                 DEXTROSE 
                 spores/ml 
               
               
                   
                 X100 
                 filter 
                 BROTH 
                   
               
               
                 
                   Colletotrichum 
                 
                 0.05%  
                 100 μm  
                   2x POTATO 
                   1e 5    
               
               
                 
                   graminicola 
                 
                 Triton 
                 Steriflip  
                 DEXTROSE 
                 spores/ml 
               
               
                 (Anthracnose stalk rot) 
                 X100 
                 filter 
                 BROTH 
                   
               
               
                 
                   Stenocarpella maydis 
                 
                 0.05%  
                 100 μm  
                   2x POTATO 
                 6.5e 4   
               
               
                 (Diplodia ear rot) 
                 Triton 
                 Steriflip  
                 DEXTROSE 
                 spores/ml 
               
               
                   
                 X100 
                 filter 
                 BROTH 
                   
               
               
                 
                   Phakopsora pachyrhizi 
                 
                 0.05%  
                 20 μm and 60 
                 0.5x POTATO 
                   5e 4    
               
               
                 (Soy Asian rust) 
                 Triton 
                 μm Steriflip 
                 DEXTROSE 
                 spores/ml 
               
               
                   
                 X100 
                 filter 
                 BROTH 
               
               
                   
               
            
           
         
       
     
     20 μl of each fungal solution according to Table 7 was added to 20 μl of the purified defensin protein to be tested in 20 mM Tris HCL, pH 8.0, in 96 well view plates (Perkin Elmer). Plates were sealed with parafilm and incubated in a clear humidity box for a predetermined period of time, temperature and conditions according to Table 8, before being imaged for fungal growth in the presence of the defensin. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Conditions and Times for Incubation of Fungi with Defensins. 
               
            
           
           
               
               
               
            
               
                   
                 Lighting and  
                   
               
               
                 Fungus 
                 Temperature Conditions 
                 Time 
               
               
                   
               
               
                 
                   Fusarium  
                 
                 12 hour diurnal cycle at 23° C.  
                 24 hours 
               
               
                 
                   graminearum 
                 
                 with 100 μmol of light during  
                   
               
               
                 (Gibberella stalk rot) 
                 the daytime period 
                   
               
               
                 
                   Fusarium  
                 
                 12 hour diurnal cycle at 23° C.  
                 24 hours 
               
               
                 
                   verticilloides 
                 
                 with 100 μmol of light during 
                   
               
               
                 (Fusarium stalk rot) 
                 the daytime period 
                   
               
               
                 
                   Colletotrichum  
                 
                 12 hour diurnal cycle at 26° C.  
                 24 hours 
               
               
                 
                   graminicola 
                 
                 with 100 μmol of light during  
                   
               
               
                 (Anthracnose stalk rot) 
                 the daytime period 
                   
               
               
                 
                   Stenocarpella maydis 
                 
                 12 hour diurnal cycle at 26° C.  
                 24 hours 
               
               
                 (Diplodia ear rot) 
                 with 100 μmol of light during 
                   
               
               
                   
                 the daytime period 
                   
               
               
                 
                   Phakopsora  
                 
                 12 hour diurnal cycle at 23° C.  
                 16 hours 
               
               
                 
                   pachyrhizi 
                 
                 with 100 μmol of light during  
                   
               
               
                 (Soy Asian rust) 
                 the daytime period 
               
               
                   
               
            
           
         
       
     
     Imaging Fungal Growth 
     The plates were visually imaged using the Perkin Elmer Operetta High Content Imager, using brightfield settings and a 10× objective lens. Depending on the particular fungal species being tested, the stored images were scored according to the ratings or criteria provided in Table 9. Two replicates and up to 4 fields per replicates were averaged for each of the visual inhibition scores. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Scoring of Inhibition of Fungal Growth. 
               
            
           
           
               
               
            
               
                 Fungus 
                 Scoring 
               
               
                   
               
               
                 
                   Fusarium graminearum 
                 
                 0-Germinated, hyphal elongation and no activity 
               
               
                 (Gibberella stalk rot) 
                 1-Germinated and hyphal elongation of ≥250 μm 
               
               
                   
                 2-Germinated and hyphal elongation of 100-250 μm 
               
               
                   
                 3-germinated and hyphal elongation of 50-100 μm 
               
               
                   
                 4-no germination &gt;80% of spores; hyperbranching “starburst” phenotype 
               
               
                   
                 with hyphal elongation &lt;50 μm 
               
               
                 
                   Fusarium verticilloides 
                 
                 0-Germinated, hyphal elongation and no activity 
               
               
                 (Fusarium stalk rot) 
                 1-Germinated and hyphal elongation of ≥400 μm 
               
               
                   
                 2-Germinated and hyphal elongation of 200-400 μm 
               
               
                   
                 3-germinated and hyphal elongation of 100-200 μm 
               
               
                   
                 4-no germination &gt;50% of spores; hyperbranching “starburst” phenotype 
               
               
                   
                 with hyphal elongation &lt;100 μm 
               
               
                 
                   Colletotrichum 
                 
                 0-Germinated and hyphal elongation ≥ 400 μm 
               
               
                 
                   graminicola 
                 
                 1-Germinated and hyphal elongation of 200-400 μm 
               
               
                 (Anthracnose stalk rot) 
                 2-Germinated and hyphal elongation of 125-200 μm 
               
               
                   
                 3-germinated and hyphal elongation of &lt;125 μm 
               
               
                   
                 4-no germination 
               
               
                 
                   Stenocarpella maydis 
                 
                 0-Germinated and hyphal elongation ≥ 400 μm 
               
               
                 (Diplodia ear rot) 
                 1-Germinated and hyphal elongation of 200-400 μm 
               
               
                   
                 2-Germinated and hyphal elongation of 125-200 μm 
               
               
                   
                 3-germinated and hyphal elongation of &lt;125 μm 
               
               
                   
                 4-activity with no germination &gt;25% of spores; 
               
               
                   
                 hyphal elongation &lt; 25 μm 
               
               
                 
                   Phakopsora pachyrhizi 
                 
                 0-0% Inhabitation ≥ 330 μm 
               
               
                 (Soy Asian rust) 
                 1-25% Inhabitation 330-250 um 
               
               
                   
                 2-20-75% Inhabitation 100-250 μm 
               
               
                   
                 3-75-100% Inhabitation 0-100 μm 
               
               
                   
               
            
           
         
       
     
     Example 7. Fungal Inhibition Activity of Multi-Domain Defensins 
     A. 2D Defensins Inhibit Fungal Growth in Plate Assay 
     Eighteen native 2D defensins of the invention were tested in the above in vitro plate assay and compared with their 1D defensin counterparts to determine whether the 2D defensins are active against the fungal pathogens listed in Table 9. These 19 2D defensins are identified in Table 10, and their sequences are provided above in Tables 1 and 2 (their linker sequences are also provided in Table 3; and corresponding protein sequences may be determined from the nucleotide sequences listed). The anti-fungal activities of the 2D defensins were also compared to their cognate or component 1D defensins to determine if their potency and spectrum of activity were enhanced relative to their 1D components. The 1D or 2D defensins tested were expressed in  Pichia  and purified as described above. The fungal inhibition scores for each defensin were calculated as an average of the reps performed. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Fungal Inhibition Activity of 2D Defensins. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 NUC 
                   
                   
                   
                   
                   Colletot - 
                   
                 
                   Fusarium 
                 
                   
                   
                   
               
               
                 SEQ 
                 protein 
                   
                   
                   
                 
                   richum 
                 
                 
                   Fusarium 
                 
                   verticil - 
                 
                   Phakopsora 
                 
                 
                   Stenocarpella 
                 
               
               
                 ID NO 
                 name 
                 locus ID 
                 Domain 
                 Reps 
                 
                   graminicola 
                 
                 
                   graminearum 
                 
                 
                   lioides 
                 
                 
                   pachyrhizi 
                 
                 
                   maydis 
                 
                 &gt;20 ppm 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 PHT001688 
                 ARAly_AFP26 
                 D1D2 
                 2 
                 0 
                 1 
                 0 
                 0 
                 1 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 8 
                 PHT002159 
                 Bn_AFP35 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 9 
                 PHT002230 
                 Bn_AFP36 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                   
                   
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 10 
                 PHT002187 
                 Bn_AFP47 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0.5 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 11 
                 PHT002172 
                 Bn_AFP74 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 12 
                 PHT002235 
                 Bn_AFP75 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                   
                 0 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 13 
                 PHT002236 
                 Bn_AFP76 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 14 
                 PHT002182 
                 Bn_AFP79 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0.5 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 15 
                 PHT002219 
                 Bn_AFP80 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 24 
                 PHT002366 
                 CITcl_AFP3 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0.5 
                 1.5 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 31 
                 PHT002131 
                 MAFdo_AFP11 
                 D1D2 
                 2 
                 0 
                 1.5 
                 0.5 
                 1.5 
                 1 
                 yes 
               
               
                 32 
                 PHT002270 
                 Mt_AFP14 
                 D1D2 
                 2 
                 0 
                 4 
                 2.5 
                   
                 2 
                 yes 
               
               
                 33 
                 PHT002295 
                 Mt_AFP60 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 34 
                 PHT002294 
                 Mt_AFP65 
                 D1D2 
                 2 
                 0 
                 0.5 
                 0 
                 0 
                 0 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 35 
                 PHT002293 
                 Mt_AFP66 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 37 
                 PHT002296 
                 Mt_AFP77 
                 D1D2 
                 2 
                 0 
                 1 
                 0 
                 0 
                 0.5 
                 yes 
               
               
                 38 
                 PHT002297 
                 Mt_AFP78 
                 D1D2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 1 
                 not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 known 
               
               
                 — 
                 Empty Vector 
                 negative control 
                 N/A 
                 4 
                 0 
                 0.25 
                 0 
                 0 
                 1.5 
                 no 
               
               
                 1153 
                 MEDsa.AFPm1 
                 positive control 
                 N/A 
                 4 
                 2.75 
                 3.7 
                 2.3 
                 1.25 
                 2.7 
                 yes 
               
               
                   
               
            
           
         
       
     
     As shown in Table 10, some of these native 2D defensins were able to inhibit growth of fungal pathogens tested in this plate functional assay. Protein concentrations (ppm) of some of the defensin preparations used in the fungal growth assay were measured and compared. In this experiment, at least one of the different 2D defensins was found to have activity against each of the fungal species tested. In cases where the protein concentration was measured and determined to be greater than 20 ppm, inhibitory activity against one or more pathogens was observed for that defensin protein. 
     B. Transgenic Corn Plants Expressing MALdo_AFP11 have Enhanced Disease Resistance 
     Transgenic corn plants expressing MALdo_AFP11 (nucleotide SEQ ID NO: 31 and polypeptide SEQ ID NO: 82) were generated and grown in the greenhouse. At the VT growth stage, two nodes were infected by wounding the stalk and injecting a suspension of  Colletotrichum graminicola  inoculum into the wound. Corn plants were evaluated ˜2-3 weeks after inoculation. Stalks were harvested, leaves were removed and the stalks were split longitudinally. Disease severity was reported as the percent necrosis of the cut surface and is compared to the percent necrosis of non-transformed plants. Five transgenic corn events out of 9 transgenic corn events tested were shown to have significant disease reduction at p-value &lt;0.05 as compared to controls. 
     Example 8. Fungal Inhibition by Synthetic Multi-Domain Defensins 
     Synthetic 2D defensins comprising two identical defensin regions connected or bridged together by a linker region were compared with 1D defensins. As shown in Example 5 above, many of these longer 2D defensins exhibited increased accumulation relative to their 1D counterparts when expressed in the corn and soy protoplast system, perhaps due to their increased size. The synthetic 2D defensins that accumulated to higher levels in protoplasts also tended to have enhanced or altered activity against fungal pathogens in the plate assays. 
     Coix22 (PHT000006; nucleotide SEQ ID NO: 990; polypeptide SEQ ID NO: 1089) and MtDef4 (PHT000025; nucleotide SEQ ID NO: 1029; polypeptide SEQ ID NO: 1128) are 1D defensins shown to be active against fungal pathogens. These Coix22 and MtDef4 domains were fused with heterologous defensin linker regions to create novel synthetic 2D defensins similar to those expressed in Examples 4 and 5 above but modified for expression in  Pichia . Seventeen unique defensin linkers from 2D defensins identified from different plant species were tested for their ability to create active 2D defensins. Each synthetic defensin was expressed from a  Pichia  expression vector, and proteins were purified and tested in vitro for activity as described above. The 1D Coix22 and MtDef4 defensins were used as controls. The fungal inhibition activities or scores for these synthetic 2D defensins are shown in Tables 11, 12, and 13. Tables 12 and 13 provide these fungal inhibition activities or scores standardized to 1 μM or 0.5 μM protein concentration. Standardization of these fungal inhibition scores allows for a more direct comparison of the intrinsic anti-fungal activities of these 2D and 1D defensins, apart from the effects of their variable protein accumulation levels. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Fungal Inhibition Activity of Synthetic 2D defensins. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Defensin 
                   
                   
                 final 
                 estimate 
                   
                   
                   
                   
                   
               
               
                 PRT SEQ 
                 Protein 
                   
                 assay 
                 of Pichia 
                   C . 
                   F . 
                   F . 
                   P . 
                   S . 
               
               
                 ID NO 
                 name 
                 linker 
                 cone (μM) 
                 processing 
                 
                   graminicola 
                 
                 
                   graminearum 
                 
                 
                   verticilloides 
                 
                 
                   pachyrhizi 
                 
                 
                   maydis 
                 
               
               
                   
               
               
                 — 
                 Empty Vector 
                 (Empty) 
                 5 
                 (Empty) 
                 0 
                 1.5 
                 0.5 
                 0 
                 1 
               
               
                   
               
               
                 1154 
                 MEDsa.AFPm1 
                 (Empty) 
                 3.72 
                 (Empty) 
                 4 
                 3 
                 3 
                 3 
                 3 
               
               
                   
               
               
                 1089 
                 PHT000006 
                 none 
                 5 
                 N/A 
                 3 
                 1 
                 0 
                 2 
                 2.5 
               
               
                   
               
               
                  365 
                 PHT002411 
                 PPTPPSPFrRP 
                 5 
                 not cleaved 
                 3 
                 2.5 
                 2.5 
                 2 
                 1.5 
               
               
                   
               
               
                  366 
                 PHT002412 
                 GPPSPTPPHHK 
                 5 
                 cleaved 
                 3.5 
                 1 
                 2 
                 1 
                 2.5 
               
               
                   
               
               
                  373 
                 PHT002416 
                 APKKVEP 
                 3.96 
                 not cleaved 
                 3 
                 2 
                 1 
                 2 
                 0.5 
               
               
                   
               
               
                  369 
                 PHT002417 
                 NNESASPASK 
                 1.75 
                 not cleaved 
                 2.5 
                 2 
                 1 
                 1 
                 1 
               
               
                   
               
               
                  375 
                 PHT002418 
                 GGKAGKKAPK 
                 5 
                 not cleaved 
                 2.5 
                 4 
                 3.5 
                 2 
                 1 
               
               
                   
               
               
                  363 
                 PHT002420 
                 ASIKPAK 
                 2.86 
                 cleaved 
                 2 
                 1 
                 2 
                 0.5 
                 2 
               
               
                   
               
               
                  364 
                 PHT002529 
                 ATPPTPTPPK 
                 5 
                 not cleaved 
                 3 
                 3 
                 2.5 
                 2 
                 0.5 
               
               
                   
               
               
                  372 
                 PHT002530 
                 GPPSPPPYSK 
                 5 
                 cleaved 
                 2 
                 1.5 
                 0.5 
                 2 
                 3 
               
               
                   
               
               
                  368 
                 PHT002531 
                 EPPSLTSTPLN 
                 5 
                 not cleaved 
                 2 
                 2.5 
                 2 
                 2 
                 0.5 
               
               
                   
               
               
                  377 
                 PHT002534 
                 GGKPGKKAP 
                 1.1 
                 not cleaved 
                 1 
                 1.5 
                 1 
                 0 
                 2.5 
               
               
                   
               
               
                  370 
                 PHT002535 
                 AGRGDKK 
                 5 
                 not cleaved 
                 2 
                 1 
                 1 
                 0.5 
                 2.5 
               
               
                   
               
               
                 — 
                 Tris 
                 (Empty) 
                 (Empty) 
                 (Empty) 
                 0.9 
                 0.7 
                 0 
                 0.3 
                 0.4 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Fungal Inhibition Activity of Synthetic 2D Defensins Standardized to a Protein Concentration of 1 μM. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Defensin 
                   
                   
                 final 
                 estimate 
                   
                   
                   
                   
                   
               
               
                 PRT SEQ 
                 Protein 
                   
                 assay 
                 of Pichia 
                   C . 
                   F . 
                   F . 
                   P . 
                   S . 
               
               
                 ID NO 
                 name 
                 linker 
                 cone (μM) 
                 processing 
                 
                   graminicola 
                 
                 
                   graminearum 
                 
                 
                   verticilloides 
                 
                 
                   pachyrhizi 
                 
                 
                   maydis 
                 
               
               
                   
               
               
                 — 
                 Empty Vector 
                 (Empty) 
                 1 
                 (Empty) 
                 1.5 
                 1 
                 0 
                 0 
                 1 
               
               
                   
               
               
                 1154 
                 MEDsa.AEPm1 
                 (Empty) 
                 1 
                 (Empty) 
                 2.5 
                 3 
                 2.5 
                 0.5 
                 3 
               
               
                   
               
               
                 1089 
                 PHT000006 
                 none 
                 1 
                 N/A 
                 0.5 
                 0 
                 1 
                 0.5 
                 2 
               
               
                   
               
               
                  365 
                 PHT002411 
                 PPTPPSPPTRP 
                 1 
                 not cleaved 
                 1.5 
                 0 
                 1 
                 1 
                 3 
               
               
                   
               
               
                  366 
                 PHT002412 
                 GPPSPTPPHHK 
                 1 
                 cleaved 
                 1.5 
                 0 
                 1 
                 1 
                 2.5 
               
               
                   
               
               
                  373 
                 PHT002416 
                 APKKVEP 
                 1 
                 not cleaved 
                 1 
                 1 
                 2 
                 0.5 
                 3 
               
               
                   
               
               
                  369 
                 PHT002417 
                 NNESASPASK 
                 1 
                 not cleaved 
                 1.5 
                 1 
                 1 
                 0 
                 1.5 
               
               
                   
               
               
                  375 
                 PHT002418 
                 GGKAGKKAPK 
                 1 
                 not cleaved 
                 2 
                 1 
                 1.5 
                 0 
                 2 
               
               
                   
               
               
                  363 
                 PHT002420 
                 ASIKPAK 
                 1 
                 cleaved 
                 2 
                 1 
                 1.5 
                 0.5 
                 1.5 
               
               
                   
               
               
                  364 
                 PHT002529 
                 ATPFrrrppK 
                 1 
                 not cleaved 
                 2.5 
                 0 
                 1 
                 1 
                 2 
               
               
                   
               
               
                  372 
                 PHT002530 
                 GPPSPPPYSK 
                 1 
                 cleaved 
                 1.5 
                 1 
                 0.5 
                 0.5 
                 1.5 
               
               
                   
               
               
                  368 
                 PHT002531 
                 EPPSLTSTPLN 
                 1 
                 not cleaved 
                 1.5 
                 0 
                 1 
                 1 
                 1 
               
               
                   
               
               
                  377 
                 PHT002534 
                 GGKPGKKAP 
                 1 
                 not cleaved 
                 0 
                 1 
                 0.5 
                 0 
                 0 
               
               
                   
               
               
                  370 
                 PHT002535 
                 AGRGDKK 
                 1 
                 not cleaved 
                 1 
                 0 
                 0 
                 0 
                 1.5 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Fungal Inhibition Activity of Synthetic 2D Defensins Standardized to a Protein Concentration of 0.5 μM. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Defensin 
                   
                   
                 final 
                 estimate 
                   
                   
                   
                   
                   
               
               
                 PRT SEQ 
                 Protein 
                   
                 assay 
                 of Pichia 
                   C . 
                   F . 
                   F . 
                   P . 
                   S . 
               
               
                 ID NO 
                 name 
                 linker 
                 cone (μM) 
                 processing 
                 
                   graminicola 
                 
                 
                   graminearum 
                 
                 
                   verticilloides 
                 
                 
                   pachyrhizi 
                 
                 
                   maydis 
                 
               
               
                   
               
               
                 — 
                 Empty Vector 
                 (Empty) 
                 0.5 
                 (Empty) 
                 1 
                 0 
                 0 
                 0 
                 1.5 
               
               
                   
               
               
                 1154 
                 MEDsa.AFPm1 
                 (Empty) 
                 0.5 
                 (Empty) 
                 1 
                 3 
                 1.5 
                 1 
                 2.5 
               
               
                   
               
               
                 1089 
                 PHT000006 
                 none 
                 0.5 
                 N/A 
                 2.5 
                 0.5 
                 1 
                 1 
                 1 
               
               
                   
               
               
                  365 
                 PHT002411 
                 PPTPPSPPTRP 
                 0.5 
                 not cleaved 
                 2.5 
                 0 
                 1 
                 1 
                 2 
               
               
                   
               
               
                  366 
                 PHT002412 
                 GPPSPTPPHHK 
                 0.5 
                 cleaved 
                 2.5 
                 0.5 
                 1 
                 0.5 
                 2 
               
               
                   
               
               
                  373 
                 PHT002416 
                 APKKVEP 
                 0.5 
                 not cleaved 
                 0.5 
                 0 
                 1.5 
                 1 
                 1 
               
               
                   
               
               
                  369 
                 PHT002417 
                 NNESASPASK 
                 0.5 
                 not cleaved 
                 2.5 
                 0 
                 1 
                 1.5 
                 2.5 
               
               
                   
               
               
                  375 
                 PHT002418 
                 GGKAGKKAPK 
                 0.5 
                 not cleaved 
                 1 
                 0.5 
                 1 
                 1 
                 2 
               
               
                   
               
               
                  363 
                 PHT002420 
                 ASFKPAK 
                 0.5 
                 cleaved 
                 1 
                 1 
                 1.5 
                 0.5 
                 2.5 
               
               
                   
               
               
                  364 
                 PHT002529 
                 ATPPTPTPPK 
                 0.5 
                 not cleaved 
                 2 
                 1 
                 0.5 
                 1.5 
                 2.5 
               
               
                   
               
               
                  372 
                 PHT002530 
                 GPPSPPPYSK 
                 0.5 
                 cleaved 
                 0.5 
                 0 
                 1 
                 1 
                 2 
               
               
                   
               
               
                  368 
                 PHT002531 
                 EPPSLTSTPLN 
                 0.5 
                 not cleaved 
                 0 
                 0 
                 1 
                 0 
                 2 
               
               
                   
               
               
                  377 
                 PHT002534 
                 GGKPGKKAP 
                 0.5 
                 not cleaved 
                 0.5 
                 1 
                 1 
                 0 
                 1 
               
               
                   
               
               
                  370 
                 PHT002535 
                 AGRGDKK 
                 0.5 
                 not cleaved 
                 1 
                 1 
                 0 
                 0 
                 2.5 
               
               
                   
               
            
           
         
       
     
     Example 9. Fungal Inhibition by Synthetic Multi-Domain Defensins 
     As demonstrated in Example 8, defensin regions can be connected by a linker region sequence to create a functionally active synthetic or chimeric 2D defensin and tested for anti-fungal activity. Seventeen linker regions from different 2D defensins were tested in combination with five native 2D defensin regions by replacing the native linker region with one of the 16 heterologous linkers (one of the 17 linkers was the native linker for that 2D defensin). The five native 2D defensins were chosen from a set of 17 2D defensins based on the cysteines in the 2 defensin regions. Two defensins, Mt_AFP14 and Bn_AFP79, represented the most common 8:8 class of 2D defensins. Bn_AFP79 and Mt_AFP65 represented the 8:10 class, and Mt_AFP60 represented the 7:9 class of defensins. See Tables 1 and 2 providing sequence identifiers for these native 2D proteins. For 2D defensins, this x:y nomenclature refers to the relative number of cysteines in the two defensin regions. For example, the 8:10 class refers to a 2D defensin having 8 cysteines in the first defensin region and 10 cysteines in the second defensin region. A total of 80 constructs (16 heterologous linker regions in 5 native 2D defensins) and three controls were tested for fungal inhibition activity in the plate assay. Data available for 57 of these constructs is shown in Table 14 below. Each of the five native 2D defensins were also tested (i.e., without the linker swap). The results are shown in Table 14. Proteins were expressed in  Pichia  and purified as described above. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Fungal Inhibition Activity of Synthetic Chimeric 2D Defensins. 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 NUC 
                 PRT 
                   
                   Colletot - 
                   
                 
                   Fusarium 
                 
                   
                   
                   
               
               
                 SEQ 
                 SEQ 
                   
                 
                   richum 
                 
                 
                   Fusarium 
                 
                   verticil - 
                 
                   Stenocarpella 
                 
                 
                   Phakopsora 
                 
                 protein 
               
               
                 ID NO 
                 ID NO 
                 template_id 
                 
                   graminicola 
                 
                 
                   graminearum 
                 
                 
                   lioides 
                 
                 
                   maydis 
                 
                 
                   pachyrhizi 
                 
                 expression 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 14 
                 65 
                 BRANA.G1156000011.1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 N/A 
               
               
                 10 
                 61 
                 BRANA.G181000052.1 
                 0 
                 0 
                 0 
                 0.5 
                 0 
                 N/A 
               
               
                 32 
                 83 
                 contig_21756_1.1 
                 0 
                 2.5 
                 0 
                 0 
                 0 
                 N/A 
               
               
                 33 
                 84 
                 Medtr2g037250.1 
                 0 
                 1.5 
                 0 
                 0 
                 0 
                 N/A 
               
               
                 34 
                 85 
                 Medtr2g037190.1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 N/A 
               
               
                 271 
                 397 
                 Bn47D1_Bn35L11_Bn47D2 
                   
                 0 
                 0 
                   
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 272 
                 398 
                 Bn47D1_Bn36L1_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 273 
                 399 
                 Bn47D1_MALdo11L12_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 274 
                 400 
                 Bn47D1_Bn74L3_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 275 
                 401 
                 Bn47D1_Bn75L13_Bn47D2 
                 0 
                 0 
                 0 
                 2 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 276 
                 402 
                 Bn47D1_Mt60L4_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 277 
                 403 
                 Bn47D1_Mt77L5_Bn47D2 
                   
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 278 
                 404 
                 Bn47D1_CITcl3L14_Bn47D2 
                   
                 0 
                 0 
                   
                 0 
                 + 
               
               
                 279 
                 405 
                 Bn47D1_Mt14L6_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 280 
                 406 
                 Bn47D1_Mt66L15_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 281 
                 407 
                 Bn47D1_Mt65L8_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 282 
                 408 
                 Bn47D1_Mt67L16_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 283 
                 409 
                 Bn47D1_Mt78L9_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 284 
                 410 
                 Bn47D1_CITcl2L17_Bn47D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 285 
                 411 
                 Mt60D1_Bn36L1_Mt60D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 286 
                 412 
                 Mt60D1_Bn47L2_Mt60D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 287 
                 413 
                 Mt60D1_MALdo11L12_Mt60D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 288 
                 414 
                 Mt60D1_Bn75L13_Mt60D2 
                 0 
                 0 
                 0 
                 3 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 289 
                 415 
                 Mt60D1_Mt77L5_Mt60D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 ++ 
               
               
                 290 
                 416 
                 Mt60D14_CITcl3L14_Mt60D2 
                 0 
                 2 
                 0 
                 0 
                 0 
                 ++ 
               
               
                 291 
                 417 
                 Mt60D1_Mt14L6_Mt60D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 292 
                 418 
                 Mt60D1_Bn79L7_Mt60D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 293 
                 419 
                 Mt60D1_Mt66L15_Mt60D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 ++ 
               
               
                 294 
                 420 
                 Mt60D1_Mt65L8_Mt60D2 
                 0 
                 0.5 
                 0 
                 0 
                 0 
                 ++ 
               
               
                 295 
                 421 
                 Mt60D1_Mt78L9_Mt60D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 296 
                 422 
                 Mt60D1_CITcl2L17_Mt60D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 ++ 
               
               
                 297 
                 423 
                 Mt14D1_Bn47L2_Mt14D2 
                 0 
                 2.5 
                 1 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 298 
                 424 
                 Mt14D1 _MALdo11L12_Mt14D2 
                 0 
                 1.5 
                 1 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 299 
                 425 
                 Mt14D1_Bn74L3_Mt14D2 
                 0 
                 4 
                 1.5 
                 1.5 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 300 
                 426 
                 Mt14D1_Bn75L13_Mt14D2 
                 0 
                 2 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 301 
                 427 
                 Mt14D1_Mt60L4_Mt14D2 
                 0 
                 3 
                 1 
                 1 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 302 
                 428 
                 Mt14D1_Mt77L5_Mt14D2 
                 0 
                 1 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 303 
                 429 
                 Mt14D1_CITcl3L14_Mt14D2 
                 0 
                 2.5 
                 1.5 
                 0 
                 0 
                 ++ 
               
               
                 304 
                 430 
                 Mt14D1_Mt78L9_Mt14D2 
                 0 
                 3.5 
                 1.5 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 305 
                 431 
                 Mt14D1_CITcl2L17_Mt14D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 306 
                 432 
                 Bn79D1_Bn35L11_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 307 
                 433 
                 Bn79D1_Bn47L2_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 308 
                 434 
                 Bn79D1_Bn74L3_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 309 
                 435 
                 Bn79D1_Bn75L13_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 310 
                 436 
                 Bn79D1_Mt60L4_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 311 
                 437 
                 Bn79D1_Mt77L5_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 312 
                 438 
                 Bn79D1_Mt14L6_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 313 
                 439 
                 Bn79D1_Mt66L15_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 ++ 
               
               
                 314 
                 440 
                 Bn79D1_Mt65L8_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 315 
                 441 
                 Bn79D1_Mt78L9_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 316 
                 442 
                 Bn79D1_CITcl2L17_Bn79D2 
                 0 
                 0 
                 0 
                 2.5 
                 0 
                 + 
               
               
                 317 
                 443 
                 Bn79D1_ARAly26L10_Bn79D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 failed 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 transformation 
               
               
                 318 
                 444 
                 Mt65D1_Bn35L11_Mt65D2 
                 0 
                 2 
                 0 
                 0 
                 0 
                 + 
               
               
                 319 
                 445 
                 Mt65D1_Bn36L1_Mt65D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 320 
                 446 
                 Mt65D1_MALdo11L12_Mt65D2 
                 0 
                 2 
                 1 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 321 
                 447 
                 Mt65D1_Bn75L13_Mt65D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 322 
                 448 
                 Mt65D1_Mt77L5_Mt65D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 323 
                 449 
                 Mt65D1_CITcl3L14_Mt65D2 
                 0 
                 0.5 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 324 
                 450 
                 Mt65D1_Bn79L7_Mt65D2 
                 0 
                 0 
                 0 
                   
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 325 
                 451 
                 Mt65D1_Mt66L15_Mt65D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 + 
               
               
                 326 
                 452 
                 Mt65D1_Mt78L9_Mt65D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 327 
                 453 
                 Mt65D1_CITcl2L17_Mt65D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                 328 
                 454 
                 Mt65D1_ARAly26L10_Mt65D2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 band not 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 visible 
               
               
                   
                   
                 MEDsa.AFPm1 
                 3 
                 3 
                 2.25 
                 2.6 
                 2 
                 N/A 
               
               
                   
                   
                 Empty Vector 
                 0 
                 0 
                 0 
                 0.5 
                 0 
                 N/A 
               
               
                   
                   
                 20 mM Tris-HCl pH 8.0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 N/A 
               
               
                   
               
            
           
         
       
     
     This data demonstrates that linkers have a broad capacity for linking two defensin regions together to create synthetic 2D defensin that are active against one or more fungal pathogens. Eight of the seventeen linkers tested (Bn35L11, Bn47L2, Bn74L3, Bn75L13, CITcl2L17, CITcl3L14, MALdo11L12, and Mt65L8) showed promise in working across different defensin backgrounds. Many of the synthetic multi-domain or 2D defensins had higher levels of protein accumulation in plant protoplasts with the potential for new or altered spectrums of activity against different fungal pathogens. 
     Example 10. Disease Reduction by Synthetic Heterodimeric 2D Defensins in Transgenic Soybean Plants 
     Transgenic soybean plants expressing heterodimeric 2D defensin Cl.AFP22/linker Mt.AFP65/AMAru.AFP10 (nucleotide SEQ ID NO: 1155 and polypeptide SEQ ID NO: 1156) were established and grown in soil in growth chambers and inoculated with a pathogen suspension containing  Phakopsora pachyrhizi  (pathogen for Asian soy rust, ASR), at the V2 stage. The plants were scored 12-14 days after inoculation for percent leaf infection. The score was compared to that of plants transformed with an empty vector. The transgenic soybean plants expressing the heterodimeric 2D defensin showed significant disease reduction as compared to the control plants. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Transgenic soybean plants expressing heterodimeric 2D defensin. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Numbers 
                   
                   
               
               
                   
                 NUC SEQ ID NO/ 
                 of Sample 
                 Mean of percent leaf 
                 Std 
               
               
                 Sample ID 
                 PRT SEQ ID NO 
                 tested 
                 infection * 
                 Error 
               
               
                   
               
               
                 Empty Vector Control 
                 / 
                 33 
                 41.8182 
                 1.7029 
               
               
                 Cl.AFP22/linker 
                 1155/1156 
                 30 
                 31.3333 
                 1.7860 
               
               
                 Mt.AFP65/AMAru.AFP10 
               
               
                   
               
               
                 * Means are statistically different at p &lt; 0.05 
               
            
           
         
       
     
     Example 11. 1D Defensin Capable of Inhibiting Fungal Growth 
     Several native 1D defensins were also identified that may be useful either alone or as defensin regions/domains when designing synthetic 2D or other MD defensins. Table 16 provides a list of native 1D defensins with nucleotide and protein sequence identifiers and annotated to identify the boundaries between different sequence regions or domains. The ninety-nine (99) defensins listed in Table 16 were selected based on their activity in the fungal plate assay described above in Example 6. Of the 413 1D defensin proteins tested, the 99 1D defensins listed in Table 16 had an average fungal growth inhibition score of 2 or greater over at least two repeated experiments against at least one of the five fungi listed in the Tables above. 
     As similarly described above, the polynucleotide sequence positions defining the boundaries of the polynucleotide sequences encoding the N-terminal TS sequence (TS_Start, TS_End), the defensin sequence portion (Def_Start, Def_End), and the C-terminal extension sequence (CT_Start, Ct_End; if present) are identified for each of the 1D defensins listed. Based on the information provided in Table 16, one can discern the corresponding protein sequence boundaries of the N-terminal TS sequence, defensin sequence portion, and C-terminal extension sequence (if present) of a 1D defensin encoded by the polynucleotide sequences in Table 15 by dividing the nucleotide positions by three (an amino acid corresponding to one codon of three nucleotides). As an exemplary illustration, the 1D defensin protein “ARAly_AFP15” has an amino acid length of 83 amino acids with its TS sequence corresponding to amino acids 1-19, its defensin sequence portion corresponding to amino acids 20-75, and its C-terminal extension sequence corresponding to amino acids 76-83 of SEQ ID NO: 1054. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Identification of 1D defensins. 
               
            
           
           
               
               
               
            
               
                   
                 Targeting Signal (TS) Sequence 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 DNA 
                 PRT 
                 DNA 
                 PRT 
                   
                   
                   
                   
                   
                   
               
               
                   
                 SEQ 
                 SEQ 
                 SEQ 
                 SEQ 
               
               
                 Locus_ID 
                 ID NO 
                 ID NO 
                 ID NO 
                 ID NO 
                 TS_Start 
                 TS_End 
                 Def_Start 
                 Def_End 
                 CT_Start 
                 CT_End 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 ARAly_AFP15 
                 955 
                 1054 
                 713 
                 859 
                 1 
                 57 
                 58 
                 225 
                 226 
                 249 
               
               
                 Zm_AFP82 
                 956 
                 1055 
                 714 
                 860 
                 1 
                 81 
                 82 
                 237 
                 238 
                 321 
               
               
                 IPOtr_AFP2 
                 957 
                 1056 
                 715 
                 861 
                 1 
                 96 
                 97 
                 237 
                 No CT 
                 No CT 
               
               
                 ABUth_AFP1 
                 958 
                 1057 
                 716 
                 862 
                 1 
                 81 
                 82 
                 222 
                 No CT 
                 No CT 
               
               
                 ABUth_AFP6 
                 959 
                 1058 
                 717 
                 863 
                 1 
                 24 
                 25 
                 180 
                 No CT 
                 No CT 
               
               
                 AMBar_AFP1 
                 960 
                 1059 
                 718 
                 864 
                 1 
                 93 
                 94 
                 234 
                 No CT 
                 No CT 
               
               
                 AMBar_AFP7 
                 961 
                 1060 
                 719 
                 865 
                 1 
                 117 
                 118 
                 249 
                 No CT 
                 No CT 
               
               
                 AMAru_AFP16 
                 962 
                 1061 
                 720 
                 866 
                 1 
                 90 
                 91 
                 240 
                 No CT 
                 No CT 
               
               
                 AMAru_AFP10 
                 963 
                 1062 
                 721 
                 867 
                 1 
                 81 
                 82 
                 240 
                 No CT 
                 No CT 
               
               
                 AMBtr_AFP6 
                 964 
                 1063 
                 722 
                 868 
                 1 
                 213 
                 214 
                 360 
                 No CT 
                 No CT 
               
               
                 AMBtr_AFP7 
                 965 
                 1064 
                 723 
                 869 
                 1 
                 93 
                 94 
                 234 
                 No CT 
                 No CT 
               
               
                 BRAdi_AFP13 
                 966 
                 1065 
                 724 
                 870 
                 1 
                 96 
                 97 
                 240 
                 No CT 
                 No CT 
               
               
                 Bn_AFP43 
                 967 
                 1066 
                 725 
                 871 
                 1 
                 78 
                 79 
                 240 
                 241 
                 255 
               
               
                 Bn_AFP1 
                 968 
                 1067 
                 726 
                 872 
                 1 
                 75 
                 76 
                 231 
                 232 
                 240 
               
               
                 Bn_AFP51 
                 969 
                 1068 
                 727 
                 873 
                 1 
                 90 
                 91 
                 267 
                 268 
                 282 
               
               
                 Bn_AFP98 
                 970 
                 1069 
                 728 
                 874 
                 1 
                 87 
                 88 
                 252 
                 253 
                 264 
               
               
                 Bn_AFP52 
                 971 
                 1070 
                 729 
                 875 
                 1 
                 231 
                 232 
                 402 
                 403 
                 417 
               
               
                 Bn_AFP103 
                 972 
                 1071 
                 730 
                 876 
                 1 
                 81 
                 82 
                 228 
                 229 
                 273 
               
               
                 Sb_AFP1 
                 973 
                 1072 
                 731 
                 877 
                 1 
                 78 
                 79 
                 231 
                 232 
                 384 
               
               
                 CANro_AFP2 
                 974 
                 1073 
                 732 
                 878 
                 1 
                 78 
                 79 
                 219 
                 No CT 
                 No CT 
               
               
                 CANro_AFP1 
                 975 
                 1074 
                 733 
                 879 
                 1 
                 93 
                 94 
                 234 
                 No CT 
                 No CT 
               
               
                 CANro_AFP13 
                 976 
                 1075 
                 734 
                 880 
                 1 
                 84 
                 85 
                 213 
                 214 
                 273 
               
               
                 CHEal_AFP7 
                 977 
                 1076 
                 735 
                 881 
                 1 
                 108 
                 109 
                 249 
                 No CT 
                 No CT 
               
               
                 CHEal_AFP1 
                 978 
                 1077 
                 736 
                 882 
                 1 
                 78 
                 79 
                 222 
                 No CT 
                 No CT 
               
               
                 CONca_AFP1 
                 979 
                 1078 
                 737 
                 883 
                 1 
                 96 
                 97 
                 237 
                 No CT 
                 No CT 
               
               
                 STEme_AFP1 
                 980 
                 1079 
                 738 
                 884 
                 1 
                 96 
                 97 
                 231 
                 No CT 
                 No CT 
               
               
                 ERAte_AFP11 
                 981 
                 1080 
                 739 
                 885 
                 1 
                 87 
                 88 
                 234 
                 No CT 
                 No CT 
               
               
                 Mt_AFP52 
                 982 
                 1081 
                 740 
                 886 
                 1 
                 9 
                 10 
                 171 
                 No CT 
                 No CT 
               
               
                 ERAte_AFP14 
                 983 
                 1082 
                 741 
                 887 
                 1 
                 90 
                 91 
                 276 
                 No CT 
                 No CT 
               
               
                 ERAte_AFP23 
                 984 
                 1083 
                 742 
                 888 
                 1 
                 90 
                 91 
                 255 
                 256 
                 303 
               
               
                 ERAte_AFP26 
                 985 
                 1084 
                 743 
                 889 
                 1 
                 87 
                 88 
                 258 
                 No CT 
                 No CT 
               
               
                 ERAte_AFP29 
                 986 
                 1085 
                 744 
                 890 
                 1 
                 87 
                 88 
                 249 
                 250 
                 291 
               
               
                 ERAte_AFP30 
                 987 
                 1086 
                 745 
                 891 
                 1 
                 66 
                 67 
                 207 
                 208 
                 303 
               
               
                 ERAte_AFP36 
                 988 
                 1087 
                 746 
                 892 
                 1 
                 72 
                 73 
                 216 
                 217 
                 324 
               
               
                 ERAte_AFP37 
                 989 
                 1088 
                 747 
                 893 
                 1 
                 99 
                 100 
                 258 
                 259 
                 303 
               
               
                 Cl_AFP22 
                 990 
                 1089 
                 748 
                 894 
                 1 
                 93 
                 94 
                 240 
                 No CT 
                 No CT 
               
               
                 Cl_AFP24 
                 991 
                 1090 
                 749 
                 895 
                 1 
                 69 
                 70 
                 210 
                 211 
                 312 
               
               
                 ERAte_AFP56 
                 992 
                 1091 
                 750 
                 896 
                 1 
                 69 
                 70 
                 213 
                 214 
                 333 
               
               
                 ERAte_AFP57 
                 993 
                 1092 
                 751 
                 897 
                 1 
                 87 
                 88 
                 249 
                 No CT 
                 No CT 
               
               
                 Cl_AFP39 
                 994 
                 1093 
                 752 
                 898 
                 1 
                 12 
                 13 
                 186 
                 187 
                 228 
               
               
                 ERAte_AFP58 
                 995 
                 1094 
                 753 
                 899 
                 1 
                 60 
                 61 
                 219 
                 220 
                 354 
               
               
                 Cl_AFP44 
                 996 
                 1095 
                 754 
                 900 
                 1 
                 96 
                 97 
                 240 
                 No CT 
                 No CT 
               
               
                 ERAte_AFP65 
                 997 
                 1096 
                 755 
                 901 
                 1 
                 81 
                 82 
                 249 
                 No CT 
                 No CT 
               
               
                 STEme_AFP2 
                 998 
                 1097 
                 756 
                 902 
                 1 
                 30 
                 31 
                 162 
                 No CT 
                 No CT 
               
               
                 STEme_AFP6 
                 999 
                 1098 
                 757 
                 903 
                 1 
                 138 
                 139 
                 282 
                 No CT 
                 No CT 
               
               
                 ERAte_AFP87 
                 1000 
                 1099 
                 758 
                 904 
                 1 
                 87 
                 88 
                 234 
                 No CT 
                 No CT 
               
               
                 ERAte_AFP92 
                 1001 
                 1100 
                 759 
                 905 
                 1 
                 144 
                 145 
                 351 
                 No CT 
                 No CT 
               
               
                 STEme_AFP4 
                 1002 
                 1101 
                 760 
                 906 
                 1 
                 96 
                 97 
                 246 
                 No CT 
                 No CT 
               
               
                 ERAte_AFP94 
                 1003 
                 1102 
                 761 
                 907 
                 1 
                 90 
                 91 
                 276 
                 No CT 
                 No CT 
               
               
                 ERAte_AFP95 
                 1004 
                 1103 
                 762 
                 908 
                 1 
                 81 
                 82 
                 246 
                 247 
                 312 
               
               
                 ARAly_AFP1 
                 1005 
                 1104 
                 763 
                 909 
                 1 
                 87 
                 88 
                 231 
                 232 
                 246 
               
               
                 At_AFP1 
                 1006 
                 1105 
                 764 
                 910 
                 1 
                 90 
                 91 
                 231 
                 No CT 
                 No CT 
               
               
                 Ps_AFP1 
                 1007 
                 1106 
                 — 
                 — 
                 — 
                 — 
                 1 
                 138 
                 No CT 
                 No CT 
               
               
                 PINsy_AFP1 
                 1008 
                 1107 
                 765 
                 911 
                 1 
                 99 
                 100 
                 246 
                 247 
                 252 
               
               
                 Ta_AFP1 
                 1009 
                 1108 
                 — 
                 — 
                 — 
                 — 
                 1 
                 141 
                 No CT 
                 No CT 
               
               
                 Ph_AFP2 
                 1010 
                 1109 
                 766 
                 912 
                 1 
                 75 
                 76 
                 222 
                 223 
                 306 
               
               
                 DIGsa_AFP1 
                 1011 
                 1110 
                 767 
                 913 
                 1 
                 192 
                 193 
                 348 
                 349 
                 399 
               
               
                 SACra_AFP18 
                 1012 
                 1111 
                 768 
                 914 
                 1 
                 75 
                 76 
                 195 
                 196 
                 282 
               
               
                 SACra_AFP20 
                 1013 
                 1112 
                 769 
                 915 
                 1 
                 15 
                 16 
                 171 
                 172 
                 207 
               
               
                 SACra_AFP23 
                 1014 
                 1113 
                 770 
                 916 
                 1 
                 81 
                 82 
                 228 
                 229 
                 324 
               
               
                 SACra_AFP27 
                 1015 
                 1114 
                 771 
                 917 
                 1 
                 78 
                 79 
                 231 
                 232 
                 330 
               
               
                 EUPma_AFP6 
                 1016 
                 1115 
                 772 
                 918 
                 1 
                 87 
                 88 
                 237 
                 No CT 
                 No CT 
               
               
                 EUPma_AFP7 
                 1017 
                 1116 
                 773 
                 919 
                 1 
                 87 
                 88 
                 237 
                 No CT 
                 No CT 
               
               
                 EUPma_AFP2 
                 1018 
                 1117 
                 774 
                 920 
                 1 
                 81 
                 82 
                 222 
                 No CT 
                 No CT 
               
               
                 FOEvu_AFP4 
                 1019 
                 1118 
                 775 
                 921 
                 1 
                 102 
                 103 
                 273 
                 No CT 
                 No CT 
               
               
                 BASsc_AFP18 
                 1020 
                 1119 
                 776 
                 922 
                 1 
                 54 
                 55 
                 207 
                 208 
                 261 
               
               
                 BASsc_AFP20 
                 1021 
                 1120 
                 777 
                 923 
                 1 
                 90 
                 91 
                 246 
                 No CT 
                 No CT 
               
               
                 BASsc_AFP3 
                 1022 
                 1121 
                 778 
                 924 
                 1 
                 81 
                 82 
                 225 
                 No CT 
                 No CT 
               
               
                 BASsc_AFP25 
                 1023 
                 1122 
                 779 
                 925 
                 1 
                 36 
                 37 
                 351 
                 No CT 
                 No CT 
               
               
                 BASsc_AFP11 
                 1024 
                 1123 
                 780 
                 926 
                 1 
                 24 
                 25 
                 174 
                 No CT 
                 No CT 
               
               
                 Os_AFP12 
                 1025 
                 1124 
                 781 
                 927 
                 1 
                 69 
                 70 
                 213 
                 214 
                 321 
               
               
                 Os_AFP9 
                 1026 
                 1125 
                 782 
                 928 
                 1 
                 72 
                 73 
                 213 
                 214 
                 258 
               
               
                 Mt_AFP36 
                 1027 
                 1126 
                 783 
                 929 
                 1 
                 87 
                 88 
                 213 
                 214 
                 222 
               
               
                 Ta_AFP4 
                 1028 
                 1127 
                 — 
                 — 
                 — 
                 — 
                 1 
                 375 
                 No CT 
                 No CT 
               
               
                 Mt_Def4 
                 1029 
                 1128 
                 784 
                 930 
                 1 
                 87 
                 88 
                 228 
                 No CT 
                 No CT 
               
               
                 PANha_AFP7 
                 1030 
                 1129 
                 785 
                 931 
                 1 
                 96 
                 97 
                 237 
                 No CT 
                 No CT 
               
               
                 PANvi_AFP55 
                 1031 
                 1130 
                 786 
                 932 
                 1 
                 75 
                 76 
                 303 
                 No CT 
                 No CT 
               
               
                 PANvi_AFP17 
                 1032 
                 1131 
                 787 
                 933 
                 1 
                 69 
                 70 
                 216 
                 217 
                 369 
               
               
                 PLAma_AFP7 
                 1033 
                 1132 
                 788 
                 934 
                 1 
                 15 
                 16 
                 156 
                 157 
                 330 
               
               
                 PORol_AFP12 
                 1034 
                 1133 
                 789 
                 935 
                 1 
                 81 
                 82 
                 225 
                 No CT 
                 No CT 
               
               
                 PORol_AFP15 
                 1035 
                 1134 
                 790 
                 936 
                 1 
                 87 
                 88 
                 231 
                 No CT 
                 No CT 
               
               
                 PORol_AFP4 
                 1036 
                 1135 
                 791 
                 937 
                 1 
                 90 
                 91 
                 231 
                 No CT 
                 No CT 
               
               
                 PORol_AFP6 
                 1037 
                 1136 
                 792 
                 938 
                 1 
                 24 
                 25 
                 165 
                 No CT 
                 No CT 
               
               
                 RAPra_AFP6 
                 1038 
                 1137 
                 793 
                 939 
                 1 
                 81 
                 82 
                 219 
                 No CT 
                 No CT 
               
               
                 RAPra_AFP8 
                 1039 
                 1138 
                 794 
                 940 
                 1 
                 87 
                 88 
                 240 
                 No CT 
                 No CT 
               
               
                 RAPra_AFP9 
                 1040 
                 1139 
                 795 
                 941 
                 1 
                 87 
                 88 
                 240 
                 No CT 
                 No CT 
               
               
                 RAPra_AFP11 
                 1041 
                 1140 
                 796 
                 942 
                 1 
                 87 
                 88 
                 237 
                 No CT 
                 No CT 
               
               
                 RAPra_AFP20 
                 1042 
                 1141 
                 797 
                 943 
                 1 
                 87 
                 88 
                 237 
                 No CT 
                 No CT 
               
               
                 RAPra_AFP12 
                 1043 
                 1142 
                 798 
                 944 
                 1 
                 87 
                 88 
                 240 
                 No CT 
                 No CT 
               
               
                 ROSbl_AFP13 
                 1044 
                 1143 
                 799 
                 945 
                 1 
                 30 
                 31 
                 198 
                 No CT 
                 No CT 
               
               
                 ROSbl_AFP7 
                 1045 
                 1144 
                 800 
                 946 
                 1 
                 81 
                 82 
                 225 
                 No CT 
                 No CT 
               
               
                 ROSbl_AFP8 
                 1046 
                 1145 
                 801 
                 947 
                 1 
                 81 
                 82 
                 222 
                 No CT 
                 No CT 
               
               
                 Sb_AFP6 
                 1047 
                 1146 
                 802 
                 948 
                 1 
                 96 
                 97 
                 237 
                 No CT 
                 No CT 
               
               
                 SPIol_AFP13 
                 1048 
                 1147 
                 803 
                 949 
                 1 
                 93 
                 94 
                 249 
                 No CT 
                 No CT 
               
               
                 SPIol_AFP15 
                 1049 
                 1148 
                 804 
                 950 
                 1 
                 84 
                 85 
                 231 
                 No CT 
                 No CT 
               
               
                 TARof_AFP4 
                 1050 
                 1149 
                 805 
                 951 
                 1 
                 84 
                 85 
                 231 
                 232 
                 414 
               
               
                 TARof_AFP6 
                 1051 
                 1150 
                 806 
                 952 
                 1 
                 39 
                 40 
                 189 
                 190 
                 345 
               
               
                 Zm_AFP73 
                 1052 
                 1151 
                 807 
                 953 
                 1 
                 90 
                 91 
                 246 
                 247 
                 314 
               
               
                 Zm_AFP10 
                 1053 
                 1152 
                 808 
                 954 
                 1 
                 99 
                 100 
                 246 
                 247 
                 318 
               
               
                   
               
            
           
         
       
     
     Example 12. Transgenic Soybean Plants Expressing 1D Defensin have Enhanced Disease Resistance to  Phakopsora Pachyrhizi    
     Transgenic soybean plants expressing 1D defensins listed in Table 17 were established and grown in soil in growth chambers and inoculated at the V2 stage with a pathogen suspension containing soybean rust pathogen,  Phakopsora pachyrhizi . The plants were scored 12-14 days after inoculation for percent leaf infection. Disease reduction is reported as percent disease relative to the negative control (plants transformed with an empty vector) in Table 17. These results show that the transgenic soybean plants expressing 1D defensin, Cl.AFP44, ERAte_AFP29, or ERAte_AFP30 have enhanced resistance to  Phakopsora pachyrhizi . 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Disease testing for trangenic soybean plants expressing 1D defensins 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 % disease 
                   
               
               
                   
                   
                   
                 relative to 
                 Statistically 
               
               
                   
                 NUC SEQ ID NO/ 
                   
                 negative 
                 Significant 
               
               
                 Tests 
                 PRT SEQ ID NO 
                 Locus ID 
                 control 
                 Activity? 
               
               
                   
               
               
                 1 
                 991/1090 
                 C1.AFP44 
                 31 
                 yes 
               
               
                 2 
                 991/1090 
                 C1.AFP44 
                 38 
                 yes 
               
               
                 3 
                 986/1085 
                 ERAte_AFP29 
                 71 
                 yes 
               
               
                 4 
                 987/1086 
                 ERAte_AFP30 
                 65 
                 yes 
               
               
                   
               
            
           
         
       
     
     Example 13. Transgenic Soybean Plants Expressing PINSY.AFP1 have Enhanced Disease Resistance to  Phakopsora Pachyrhizi    
     Transgenic soybean plants expressing a codon optimized PINSY.AFP1 gene (SEQ ID NO: 1157) under the control of a synthetic promoter as set forth in SEQ ID NO: 1158 were generated. Field trials of transgenic plants expressing PINSY.AFP1 (polypeptide SEQ ID NO: 1107) were conducted to assess plant resistance to  Phakopsora pachyrhizi  utilizing multiple planting dates. The field trials were conducted using a randomized complete block design for each of two planting dates. Efficacy parameters were scored throughout the field trial season and at the end of the season yield was determined. Trait efficacy was measured by scoring disease prevalence 3 times at 14 day intervals once ASR was observed in the wildtype plots. Disease Ratings were taken as a 1-9 rating of overall plot infection (RST) and as a percent disease infection in the plots (DPPF). Efficacy data was utilized to calculate area under the disease progress curve (AUDPC) to determine overall disease resistance of each event. Data are presented in Table 18. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Field Trial Efficacy Results as Rated disease infection (RST) 
               
               
                 and Percent disease infection reduction (DPPF) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Area under disease 
                 Area under disease 
                 RST 
                 DPPF 
               
               
                 Event 
                 progress curve RST 
                 progress curve DPPF 
                 LSD(.10) 
                 LSD(.10) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Non-transgenic control 
                 31.9 
                 319.4 
                 7.4 total 
                 99.7 total 
               
               
                 Transgenic Event 1 
                 26.3 
                 117.7** 
                 7.4 total 
                 99.7 total 
               
               
                 Transgenic Event 2 
                 21.0** 
                 95.4** 
                 7.4 total 
                 99.7 total 
               
               
                 Transgenic Event 3 
                 26.3 
                 207.8* 
                 7.4 total 
                 99.7 total 
               
               
                 Transgenic Event 4 
                 22.8** 
                 163.6** 
                 7.4 total 
                 99.7 total 
               
               
                   
               
               
                 *Significant reduction in disease at p ≤ 0.1 
               
               
                 **Significant reduction in disease at p ≤ 0.05 
               
            
           
         
       
     
     The average rated disease for the control plants was 31.9. The average rated crop injury by disease for transgenic PINSy.AFLP1 soybean Event 1, Event 2, Event 3, and Event 4 was 26.3, 21.0, 26.3, and 22.8 respectively. The rated disease reduction least significant difference (LSD) at 0.10 was 7.4 for all events tested. The average percent disease for the non-transgenic control utilizing Area Under the Disease Progress Curve (AUDPC) 319.4. The average AUDPC for percent crop injury by disease for transgenic PINSy.AFP1 soybean Event 1, Event 2, Event 3, and Event 4 was 117.7, 95.4, 207.8 and 163.6 respectively. The AUDPC percent disease reduction least significant difference (LSD) at 0.10 was 99.7 for all transgenic events tested. These results indicate that all transgenic soybean events expressing PINSY.AFP1 have enhanced disease resistance to  Phakopsora pachyrhizi.    
     Example 14. Transgenic Corn Plants Expressing 1D Defensins have Enhanced Disease Resistance 
     Transgenic corn plants expressing 1D defensin were generated and grown in the greenhouse. At the VT growth stage, two nodes were infected by wounding the stalk and injecting a suspension of  Colletotrichum graminicola  inoculum into the wound. Corn plants were evaluated ˜2-3 weeks after inoculation. Stalks were harvested, leaves were removed and the stalks were split longitudinally. Disease severity was reported as the percent necrosis of the cut surface and is compared to the percent necrosis of non-transformed plants as shown in Table 19. Transgenic corn plants expressing defensin AMBtr_AFP7, Cl_AFP22, Cl_AFP44, ERAte_AFP95, Ph_AFP2, or PINsy_AFP1 as shown in Table 19 have enhanced disease resistance to  Colletotrichum graminicola . Transgenic corn plants expressing defensin Ph_AFP2 were further tested for resistance to other pathogens and were shown to have enhanced broad-spectrum disease resistance to  S. maydis F. verticiliodes, F. graminearum . 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Transgenic corn plants expressing 1D defensins have enhanced disease resistance 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Number of events with significant 
               
               
                   
                   
                   
                 disease reduction/number of 
               
               
                 Defensin 
                 NUC SEQ ID NO 
                 PRT SEQ ID NO 
                 events tested (at p-value &lt; 0.2) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 AMBtr_AFP7 
                 961 
                 1060 
                 5 of 7 
               
               
                 Cl_AFP22 
                 990 
                 1089 
                 3 of 7 
               
               
                 Cl_AFP44 
                 996 
                 1095 
                  3 of 20 
               
               
                 ERAte_AFP95 
                 1004 
                 1103 
                 6 of 9 
               
               
                 Ph_AFP2 
                 1010 
                 1109 
                  9 of 17 
               
               
                 PINsy_AFP1 
                 1108 
                 1107 
                 4 of 9 
               
               
                   
               
            
           
         
       
     
     As various modifications could be made in the constructions and methods herein described and illustrated without departing from the scope of the invention, it is intended that all matter contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative rather than limiting. The breadth and scope of the invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims appended hereto and their equivalents. All patent and non-patent documents cited in this specification are incorporated herein by reference in their entireties.