# Patent

## Title
AtMIN7 mediated disease resistance to Pseudomonas syringae in Arabidopsis

## Abstract

The present invention relates to compositions and methods for enhancing plant defenses against pathogens. More particularly, the invention relates to enhancing plant immunity against bacterial pathogens, wherein AtMIN7 mediated protection is enhanced and/or there is a decrease in activity of an AtMIN7 associated virulence protein such as a Pseudomonas syringae pv. tomato DC3000 HopM1. Reagents of the present invention provide a means of studying cellular trafficking while formulations of the present inventions provide increased pathogen resistance in plants.

## Background
GOVERNMENT SUPPORT
The invention was made in part with Government support from the Department of Energy and National Institutes of Health, National Institute Of Allergy And Infectious Diseases, grants 1R01AI060761-01A2 and 1R21AI060761-01. As such, the Government has certain rights in the invention.


SEQUENCE LISTING
A Sequence Listing has been submitted on a compact disc, the entire content of which is herein incorporated by reference. The compact disc and its duplicate are labeled Copy 1 and Copy 2, respectively. Each disk contains a file named “13600seq.txt” created on Jul. 7, 2008 that is 294,912 bytes, and each disk is identical to the other.
FIELD OF THE INVENTION
The present invention relates to compositions and methods for enhancing plant defenses against pathogens. More particularly, the invention relates to enhancing plant immunity against bacterial pathogens, wherein AtMIN mediated protection is enhanced and/or there is a decrease in activity of an AtMIN7 associated virulence protein such as a Pseudomonas syringae pv. tomato DC3000 HopM1. Reagents of the present invention provide a means of studying cellular trafficking while formulations of the present inventions provide increased pathogen resistance in plants.
BACKGROUND OF THE INVENTION
Plants have a powerful immune system to defend against colonization by most microbial organisms. However, virulent plant pathogens, such as Pseudomonas syringae, have developed countermeasures and inject virulence proteins into the host cell of a susceptible plant to overcome plant immunity and cause disease. Host plants include tomato plants and collard plants, such as cabbage and kale. In particular, Pseudomonas syringae pv. tomato causes an economically devastating disease called bacterial speck of tomato plants.
Bacteria control strategies are based on a combination of practices such as use of pathogen-free seed and transplants, elimination of volunteer tomato plants, resistant cultivars, and frequent application of a copper and mancozeb mixture (Jones, et al. 1986, Phytopathology 76:430-434; Jones, et al. 1991, Phytopathology 81:714-719; Sherf, et al. 1986, In: Vegetable Diseases and Their Control. John Wiley and Sons, New York; all of which are herein incorporated by reference). Chemical control has been used extensively for controlling bacterial spot. In the 1950s, streptomycin was used, but resistant bacterial strains developed and rendered antibiotics ineffective (Stall, R. E., and Thayer, P. L. 1962, Plant Dis. Rep. 46:389-392; herein incorporated by reference). However, these strategies are of limited use, especially in the tropics and subtropics where weather conditions favor infection (Kucharek, T. 1994, Plant pathology fact sheet, PP-3, University of Florida, Gainesville; herein incorporated by reference).
One method of treatment is a biopesticide product containing as active ingredients bacteriophages of Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato “AgriPhage” EPA Registration #67986-1.
However, Pseudomonas quickly develops resistance to these treatment methods. Further, despite intensive research efforts, the molecular targets of bacterial virulence proteins important for plant disease development have remained obscure.
Therefore, there is a need for effective and economical bacterial pathogen treatments, and further, for enhancing plant immunity to virulent plant pathogens.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for enhancing plant defenses against pathogens. More particularly, the invention relates to enhancing plant immunity against bacterial pathogens, wherein AtMIN7 mediated protection is enhanced and/or there is a decrease in activity of an AtMIN7 associated virulence protein such as a Pseudomonas syringae pv. tomato DC3000 HopM1. Reagents of the present invention provide a means of studying cellular trafficking while formulations of the present inventions provide increased pathogen resistance in plants.
In one embodiment, the invention provides an expression vector construct comprising a nucleic acid molecule at least 57% identical to SEQ ID NO:02. Accordingly, in other embodiments, the present invention provides an expression vector construct comprising a nucleic acid molecule at least 57%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:02. In one embodiment, the nucleic acid molecule encodes a polypeptide that alters pathogen resistance in a plant. The present invention is not limited to any particular source of said nucleic acid molecule. Indeed, a variety of sources are contemplated, including but not limited to Brassicaceae, Solanaceae, and Poaceae. In some embodiments, the nucleic acid molecule encodes a polypeptide that is at least 38% identical to SEQ ID NO:01, wherein said polypeptide alters pathogen resistance in a plant. Accordingly, in other embodiments, the present invention provides an expression vector construct comprising a polypeptide at least 38%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:01. In one embodiment, the polypeptide alters pathogen resistance in a plant. The present invention is not limited to any particular type of polypeptide for altering pathogen resistance in a plant. In a preferred embodiment, the polypeptide increases pathogen resistance in a plant. In some embodiments, the nucleic acid is operably linked to an exogenous promoter. The present invention is not limited to any particular type of promoter. Indeed, the use of a variety of promoters is contemplated. In some embodiments, the promoter is a eukaryotic promoter. In some embodiments, the eukaryotic promoter is active in cell. In some embodiments, the eukaryotic promoter is active in a yeast cell. In some embodiments, the eukaryotic promoter is active in a plant cell. The present invention is not limited to any particular type of vector construct. Indeed, the use of a variety of vectors is contemplated. In some embodiments, the vector is an expression vector. In some embodiments, the expression vector is a eukaryotic expression vector. In other embodiments, said eukaryotic expression vector is a plant expression vector. In other embodiments, said plant expression vector comprises a T-DNA vector. In other embodiments, said expression vector is a prokaryotic expression vector.
In one embodiment, the invention provides a plant comprising a heterologous nucleic acid molecule encoding a polypeptide that is at least 38% identical to SEQ ID NO:01, wherein said polypeptide provides pathogen resistance. Accordingly, in other embodiments, the present invention provides an expression vector construct comprising a polypeptide at least 38%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:01, wherein said polypeptide provides pathogen resistance. The present invention is not limited to any particular source of said nucleic acid molecule. Indeed, a variety of sources are contemplated, including but not limited to Brassicaceae, Solanaceae, and Poaceae. In a preferred embodiment, the plant is a transgenic plant. In some embodiments, the pathogen resistance is increased pathogen resistance. In some embodiments, the increased pathogen resistance is increasing the resistance of a plant to a pathogen-induced symptom. The present invention is not limited to any particular type of pathogen-induced symptom. Indeed, a variety of symptoms are contemplated, including but not limited to a canker, a leaf canker, a stem canker, flower blast, dieback, brown spot, a necrotic leaf spot, a blister, and the like. The present invention is not limited to any particular type of pathogen. In some embodiments, the pathogen is a microbial pathogen. Indeed, a variety of microbial pathogens are contemplated, including but not limited to a bacterium, a virus and a fungus. The present invention is not limited to any particular type of bacterium. Indeed, a variety of bacteria are contemplated, including but not limited to a Pseudomonas species. The present invention is not limited to any particular type of fungi. Indeed, a variety of fungi are contemplated, including but not limited to a Cytospora species and Nectria species. The present invention is not limited to any particular type of plant. Indeed a variety of plants are contemplated, including but not limited to a fruit plant, a vegetable plant, a grass plant, a crop plant, a woody plant, and an ornamental plant. In some embodiments, the plant is an Arabidopsis plant. In some embodiments, the plant is a tomato plant. In some embodiments, the plant is a rice plant. In some embodiments, the plant is an oil seed rape plant. In some embodiments, the plant is a soybean plant. In some embodiments, the plant is a plant part. The present invention is not limited to any particular type of plant part. Indeed, a variety of plant parts are contemplated, including but not limited to a tiller, a seed, a leaf, and the like.
In one embodiment, the invention provides a method for providing pathogen resistance in a plant, comprising, a) providing, i) a plant, and ii) a nucleic acid molecule, wherein said nucleic acid molecule encodes a polypeptide that is at least 38% identical to SEQ ID NO:01, and b) introducing said nucleic acid molecule into said plant under conditions such that said nucleic acid is expressed and provides pathogen resistance in the plant. In some embodiments, the expressed nucleic acid is overexpressed. In some embodiments, the providing pathogen resistance in the plant is increasing expression of the nucleic acid molecule. In some embodiments, the plant is a transgenic plant. The present invention is not limited to any particular type of pathogen. In some embodiments, the pathogen is a microbial pathogen. Indeed, a variety of microbial pathogens are contemplated, including but not limited to a bacterium, a virus and a fungus. The present invention is not limited to any particular type of bacterium. Indeed, a variety of bacteria are contemplated, including but not limited to a Pseudomonas species. The present invention is not limited to any particular type of fungi. Indeed, a variety of fungi are contemplated, including but not limited to a Cytospora species and Nectria species. The present invention is not limited to any particular type of introducing. In some embodiments, the introducing said nucleic acid molecule into said plant is by transfection. In some embodiments, the introducing said nucleic acid molecule into said plant is by introgression of the nucleic acid molecule into a plant. In some embodiments, the plant is a transgenic plant. In some embodiments, the introducing said nucleic acid molecule into said plant is by transfection or by traditional breeding methods. In some embodiments, the providing pathogen resistance in the plant is by overexpression of the nucleic acid molecule.
In one embodiment, the invention provides an expression vector, comprising a nucleic acid sequence encoding a double stranded RNA sequence, one strand of which is complementary to a sequence at least 75% identical to SEQ ID NO:35. Accordingly, in other embodiments, the present invention provides a sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NOs:35. Accordingly, in some embodiments, the nucleic acid at least 75% identical to SEQ ID NO:35 comprises a full-length nucleic acid or a nucleic acid encoding a C-terminal fragment. The present invention is not limited to any particular C-terminal fragment. Indeed, a variety of C-terminal fragments are provided, including but not limited to nucleic acids comprising 333-2136 nucleic acids of the C-terminus. Accordingly, in other embodiments, the present invention provides a nucleic acid comprising at least 333, 433, 533, 633, 733, 833, 933, 1033, 1133, 1233, (or more) contiguous C-terminal nucleic acids. In some embodiments, the nucleic acid at least 75% identical to SEQ ID NO:35 ranges in size from 10-2136 contiguous C-terminal nucleic acids. In some embodiments, the double stranded RNA sequence mediates RNA interference. The present invention is not limited to any particular type of interference. In some embodiments, the nucleic acid product that interferes is an antisense sequence. In some embodiments, the interference is decreased translation of the polypeptide encoded by SEQ ID NO:35. Accordingly, in some embodiments, the polypeptide is at least 51% identical to SEQ ID NO:34. Accordingly, in other embodiments, the present invention provides a polypeptide at least 51%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:34. Accordingly, in some embodiments, the polypeptide at least 51% identical to SEQ ID NO:34 comprises a full-length polypeptide or a C-terminal fragment. The present invention is not limited to any particular C-terminal fragment. Indeed, a variety of C-terminal fragments are provided, including but not limited to polypeptides comprising 111-611 amino acids of the C-terminus. Accordingly, in other embodiments, the present invention provides a polypeptide comprising at least 100, 200, 300, 400, 500, 600 (or more) contiguous C-terminal amino acids. In some embodiments, the polypeptide at least 51% identical to SEQ ID NO:34 ranges in size from 4-712 contiguous C-terminal amino acids. In some embodiments, the interference is an inhibition of the polypeptide expression.
The inventions further provide a kit for identifying expression of an ATMIN nucleotide or protein. In some embodiments, the kit comprises an expression vector construct comprising an ATMIN polynucleotide. In some embodiments, the kit comprises an ATMIN polypeptide. In some embodiments, the kit comprises an expression vector of the present invention comprising an ATMIN nucleotide.



DESCRIPTION OF THE FIGURES
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 shows an exemplary analysis of HopM1 transgenic Arabidopsis plants. Bacterial multiplication in leaves of wild-type (WT) Arabidopsis plants (Col-0 gl1) and transgenic plants expressing full-length HopM1 (A), and in WT leaves of Col-0 gl1 and transgenic plants expressing deletion derivatives of HopM1 (C). Plants were sprayed with dexamethasone (DEX; see, Examples) 24 h before bacterial inoculation (1×106 cfu/ml). Bacterial populations were determined at day 3 after inoculation. (B) Immunoblot analysis of HopM1, H+ATPase, and Golgi-localized Arabidopsis thaliana xyloglucan xylosyltransferase (AtXTI, SEQ ID NO: 113) of HopM1 transgenic Arabidopsis leaf proteins separated into the indicated subcellular fractions. TM: Total membrane; S: Soluble fraction; PM: Plasma-membrane; and EM: Endomembranes.
FIG. 2 shows an exemplary analysis of physical interaction between HopM1 and AtMIN proteins and HopM1-dependent destabilization of AtMIN proteins. (A) Yeast two-hybrid (Y2H) assay of physical interaction between HopM1-300 expressed from pGILDA (Clontech) and AtMIN proteins expressed from pB42AD (Clontech; shown for AtMIN2, 7, 10, 12). A blue (dark) color indicates interaction, whereas a white (light) color indicates no interaction. The “+” symbol indicates positive control strain containing pLexA-p53 and pB42AD-T; (B) Immunoblot analysis of the physical interaction between AtMIN7-HA and 6×His-HopM11-300 (lane 1) or between AtMIN7-HA and 6×His-HopM1301-712 (lane 2) in N. benthamiana leaves using protein pull-down assay (see, Examples, Materials and Methods). AtMIN-HA and 6×His-HopM1 proteins were detected using the HA and 6×His epitope antibodies, respectively. AtMIN7-HA was pulled down with HopM1-300 but not with 6×His-HopM1301-712; and (C) Western blot and reverse transcription polymerase chain reaction analyses of HopM1-dependent destabilization of AtMIN7 in Arabidopsis plants. Leaves of Col-0 gl1 plants were infiltrated with water or 1×108 CFUs per milliliter of DCEL mutant bacteria with or without pORF43 and harvested 10 hours later. The endogenous AtMIN7 protein—detected with the use of a rabbit polyclonal AtMIN7 antibody—was absent in leaves infiltrated with DCEL mutant bacteria (pORF43) that produce HopM1; however, the AtMIN7 transcript level was not reduced. (D) Proteasome inhibitors (MG132 and epoxomicin) blocked the HopM1-mediated destabilization of AtMIN7 in N. benthamiana leaves, whereas a cocktail of inhibitors of serine-, cysteine-, aspartic-, and metallo-proteases did not. AtMIN7::HA and 6×His::HopM11-712 proteins were detected with HA and 6×His epitope antibodies, respectively.
FIG. 3 shows an exemplary analysis of AtMIN7 knockout (KO) plants. (A) Growth of Pst DC3000, the ΔCEL mutant, and the hrcC mutant in AtMIN7 KO plants or in Col-O plants. Bacteria were inoculated by dipping with 1×108 cfu/ml. Bacterial populations were determined at day 4. Two independent T-DNA insertion lines were analyzed with similar results; results from line #1 are shown here. (B) Disease symptoms (chlorosis and necrosis) in Col-0 plants and AtMIN7 KO plants at day four. (C) Effect of brefeldin A (BFA) treatment on bacterial multiplication.
FIG. 4 shows exemplary callose deposition in leaves of Col-0 and AtMIN7 KO plants. Arabidopsis Col-0 and AtMIN7 KO leaves were stained to show callose deposition 7 h after inoculation with 1×108 CFU/ml DC3000 and ΔCEL mutant bacteria. Average numbers of callose depositions per field of view (0.9 mm2) are presented with standard deviations displayed as errors.
FIG. 5 shows exemplary bacterial disease symptoms on Col-0 gl1 and HopM1 transgenic plants inoculated with Pst DC3000, the ΔCEL mutant, and the ΔCEL mutant (pORF43) which expresses HopM1 and the cognate chaperone ShcM (Ma et al., (1991) Mol. Plant-Microbe Interact. 4:69; Badel et al., (2003) Mol. Microbiol. 49:1239; herein incorporated by reference). Pink arrows (upper left lines) indicate those leaves illustrating the dominant-negative effect of HopM11-200 and HopM11-300 on the ΔCEL mutant (pORF43), whereas blue arrows (lower left lines) indicate those leaves that illustrate the ability of full-length HopM1 or HopM1101-712 to completely or partially complement the ΔCEL mutant (see, FIG. 1C for bacterial multiplication). Please note that leaves of HopM1101-712 plants infected with the ΔCEL mutant were only slightly yellow (discolored). The right panels show HopM1 protein levels, revealed by immunoblotting, 24 hours after spraying plants with 30 μM DEX (immediately before bacterial inoculation).
FIG. 6 (A) Yeast two-hybrid (Y2H) assay of physical interaction between HopM11-200 expressed from pGILDA (Clontech) and AtMIN proteins expressed from pB42AD (Clontech; shown for AtMIN2, 7, 10, 12). Yeast colonies were grown on complete minimal medium containing galactose and X-gal. A blue color indicates interaction, whereas a white color indicates no interaction. (B) AtMIN proteins were destabilized in yeast when co-expressed with full-length HopM1, but not with HopM11-300. AtMIN fusion proteins expressed from pB42AD were visualized by the HA epitope antibody. HopM1 fusion proteins expressed from pGilda were visualized by the LexA antibody. Coomassie Brilliant Blue-stained gels were used as loading controls. Arrows indicate lanes in which the amounts of AtMIN proteins are greatly reduced. AtMIN12 (a hypothetical protein predicted to be targeted to the chloroplast) is not destabilized. (C) Immunoblot analysis of 6×His-HopM1 and AtMIN::HA proteins in N. benthamiana leaves when AtMIN::HA proteins were transiently co-expressed with either full length 6×His::HopM1 or 6×His::HopM11-300. Total leaf proteins in these samples were visualized by Coomassie staining and used as loading controls (bottom panel). Arrows indicate lanes in which DEX-induced expression of full-length HopM1 destabilized AtMIN2, 7, and 10. (D) Immunoblot analysis of the stability of endogenous AtMIN7 in Arabidopsis leaves. Leaves of wild-type Col-0 gl1 plants were infiltrated with water or 1×108 CFU/ml ΔCEL mutant or ΔCEL mutant (pAVRE+pAVRF), which expresses AvrE but not HopM1 (FIG. 7). Treated leaves were harvested 10 hrs later. The endogenous AtMIN7 protein was detected using a rabbit polyclonal AtMIN7 antibody. AtMIN7 was not destabilized in leaves infiltrated with ΔCEL mutant (pAVRE+pAVRF). (E) Immunoblot analysis of HopM1-dependent destabilization of AtMIN10::HA in stable transgenic plants. Leaves of AtMIN10::HA transgenic plants were infiltrated with water or 1×108 CFU/ml ΔCEL mutant bacteria or ΔCEL mutant bacteria (pORF43) and harvested 10 hrs later. AtMIN10::HA was detected using the HA epitope antibody. Please note that membrane-associated AtMIN10::HA was preferably eliminated during bacterial infection.
FIG. 7 shows exemplary detection of polyubiquitinated AtMIN7 in planta. Left: AtMIN7::HA and 6×His::HopM1-300, 6×His::HopM301-712 or 6×His::HopM1-712 were transiently co-expressed in MG132-treated N. benthamiana leaves. Ubiquitinated proteins were detected by western blot (WB) with a polyclonal ubiquitin (Ub) antibody (Sigma Co.). Right: AtMIN7::HA and 6×His::HopM1-712 were transiently co-expressed in 1% DMSO(−)- or MG132-treated N. benthamiana leaves. AtMIN7 was immunoprecipitated (IP) using a polyclonal AtMIN7 antibody. Ubiquitinated AtMIN7 protein was detected by western blot (WB) with a polyclonal ubiquitin (Ub) antibody (Sigma Co.). See Materials and Methods for transient expression and immunoprecipitation of AtMIN7 and HopM1 in N. benthamiana leaves. Taken together, these results showed that full-length HopM1, but not nonfunctional HopM1 fragments, enhanced the polyubiquitination of AtMIN7 in vivo.
FIG. 8 shows exemplary characterization of Arabidopsis SALK lines carrying T-DNA insertions in the AtMIN7 gene. (A) The two T-DNA insertion lines used in this study carried T-DNA insertions in exon 1 (AtMIN7 KO #1) and exon 18 (AtMIN7 KO #3), respectively. (B) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, using primers indicated in blue showed no full-length AtMIN7 transcript in either of the two AtMIN7 knockout (KO) lines. Col-0 plants were used as a positive control. Ethidium bromide-stained total RNA profiles were used as loading controls. (C) Western blot analysis of AtMIN7 in wild-type (Col-0) and two KO Arabidopsis plants. The endogenous AtMIN7 protein was detected using a rabbit polyclonal antibody.
FIG. 9 shows (A) an exemplary phylogenetic tree indicating the relationship among Arabidopsis ADP-ribosylation factor (ARF) guanine nucleotide exchange factor (GEF) proteins, The Arabidopsis Book Article: pp. 1-35. Protein sequences (SEQ ID NOs:01, and 27-33, were aligned using the ClustalW program (http://align.genome.jp) to construct the tree. (B) Yeast two-hybrid interaction assay of the physical interaction between HopM11-300 expressed from pGILDA (Clontech) and selected Arf GEF proteins expressed from pB42AD (Clontech). Yeast colonies were grown on complete minimal medium containing galactose and X-gal. A blue color indicates interaction, whereas a white color indicates no interaction. (C) Immunoblot analysis of Arf GEF proteins in yeast when co-expressed with full-length HopM1 or HopM11-300. Arf GEF fusion proteins expressed from pB42AD were visualized by the HA epitope antibody. HopM1 fusion proteins expressed from pGilda were visualized by the LexA antibody. Coomassie Brilliant Blue-stained gels were used as loading controls. AtMIN7 was destabilized by full-length HopM1.
FIG. 10 shows a schematic diagram depicting a polarized vesicle trafficking pathway, in which AtMIN7 is a key component. The AtMIN7-dependent pathway is associated with plant immune responses, including the formation of callose deposits and probably release of antimicrobial phytoalexins (red dots in the papilla and plant cell wall). Pst DC3000 and presumably other P. syringae strains inject HopM1 into the host cell. Once inside the host cell, HopM1 is associated with an endomembrane compartment(s), binds to AtMIN7 through the N-terminus (in red), and destabilizes AtMIN7 and other AtMIN proteins. Brefeldin A (BFA) could mimic the effect of HopM1 by inhibiting the guanine nucleotide exchange factor (GEF) activity of the Sec7 protein family, of which AtMIN7 is a member.
FIG. 11 shows exemplary sequences for AtMIN7, Arabidopsis thaliana guanyl-nucleotide exchange factor (AT3G43300) and homologs in oilseed rape, tomato, and rice (SEQ ID NOs:01-12).
FIG. 12 shows exemplary nucleic acid and amino acid sequences for AtMIN2, AtMIN3, AtMIN4, AtMIN6, AtMIN9, AtMIN10, and AtMIN11, (SEQ ID NOs: 13-26) and exemplary amino acid sequences for ADP-ribosylation factor (ARF) guanine nucleotide exchange factor (GEF) proteins i.e., At1g01960, At3g60860, At4g38200, At4g35380, At1g13980 (GNOM), At5g39500, and At5g19610 (SEQ ID NOs: 27-33, respectively), see, exemplary phylogenetic tree constructions shown in FIG. 9.
FIG. 13 shows exemplary sequences for type III effector HopM1 virulence factor from Pseudomonas syringae pv. tomato str. DC3000 and Pseudomonas syringae pv. syringae B728a (SEQ ID NOs: 34-39).
FIG. 14 shows exemplary primer sequences for amplifying HOPM1 gene segments and AtMIN genes (SEQ ID NOs:40-79); exemplary amino acid sequences and nucleic acid sequences, respectively, encoding exemplary HopM1 fragments of the present inventions (SEQ ID NOs:80-103); an exemplary sequence used as a negative control for plasma membrane localization Xyloglucan 6-xylosyltransferase (AtXT1; At3g62720) (SEQ ID NOs:104 and 113); and exemplary related/homologous sequences for HopM1: Pseudomonas viridiflava HopPtoM-like protein1-300 (SEQ ID NOs:105 and 110); and Pseudomonas syringae pv. syringae B728a type III effector HopM11-300 (SEQ ID NOs:106 and 107), Pseudomonas syringae pv. phaseolicola 1448A HopM11-300 (SEQ ID NOs:108 and 109), and Pseudomonas syringae pv. phaseolicola 1448A HopM1 (SEQ ID NOs:111 and 112).
FIG. 15 shows pGILDA and pB42AD vector constructs for the yeast 2-Hybrid system, part of the MATCHMAKER LexA Two-Hybrid System (#K1609-1) (CLONTECHniques, OCTOBER 1999 p. 26-27, Clontech Laboratories Inc.; herein incorporated by reference in its entirety) used for providing expressed AtMIN proteins in yeast.
FIG. 16 shows plasmid pBI121.



DEFINITIONS
To facilitate an understanding of the present invention, a number of terms and phrases as used herein are defined below:
The use of the article “a” or “an” is intended to include one or more. As used herein, terms defined in the singular are intended to include those terms defined in the plural and vice versa.
The “effective amount” or “biologically effective amount” refers to the amount of a compound such as a protein that causes a desired biological effect, such as inhibiting pathogen growth on or in a plant. For instance, the effective amount of a peptide can be an amount necessary to inhibit bacterial proliferation, measurably decrease the progression of a bacterial infection, reduce the number of bacteria present or reduce the symptoms of bacterial infection in a plant.
The term “sample” is used in its broadest sense. In one sense it can refer to a plant cell or tissue. In another sense, it is meant to include a specimen, such as a bacterium or spore, or a culture obtained from any source, such as tissue culture or a bacterial culture, as well as biological samples, such as a protein sample, a nucleotide sample, a microbial sample, and the like, and environmental samples, such as microbial sampling. Biological samples may be obtained from plants, a leaf from infected plants, or microorganisms (including bacteria) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental material such as surface matter, soil, water, industrial, and agricultural samples. These examples are not to be construed as limiting the sample types applicable to the present invention.
The term “host cell” refers to any cell capable of replicating and/or transcribing and/or translating a heterologous gene. Thus, a “host cell” refers to any eukaryotic or prokaryotic cell (e.g., plant cells, such as Arabidopsis, tomato etc., bacterial cells such as E. coli, yeast cells, insect cells, etc.), whether located in vitro or in vivo. For example, host cells may be located in a transgenic plant.
The terms “eukaryotic” and “eukaryote” are used in it broadest sense. It includes, but is not limited to, any organisms containing membrane bound nuclei and membrane bound organelles. Examples of eukaryotes include but are not limited to plants, fungi, alga, diatoms, protists, and animals.
The terms “prokaryote” and “prokaryotic” are used in it broadest sense. It includes, but is not limited to, any organisms without a distinct nucleus. Examples of prokaryotes include but are not limited to bacteria, blue-green algae, archaebacteria, actinomycetes and mycoplasma. In some embodiments, a host cell is any microorganism.
As used herein the term “microorganism” refers to microscopic organisms and taxonomically related macroscopic organisms within the categories of algae, bacteria, fungi, protozoa, viruses, and subviral agents.
The terms “bacteria” and “bacterium” refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Actinomyces, Streptomyces, and Sporumosa and further including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within bacteria are prokaryotic organisms that are gram negative or gram positive.
As used herein, the terms “Gram negative” and “Gram positive” refer to staining patterns with the Gram-staining process that is well known in the art. (See, e.g., Finegold and Martin, Diagnostic Microbiology, 6th Ed., C V Mosby St. Louis, pp. 13-15 [1982]). “Gram positive bacteria” are bacteria that retain the primary dye used in the Gram stain, causing the stained cells to appear dark blue to purple under the microscope. “Gram negative bacteria” do not retain the primary dye used in the Gram stain, but are stained by the counterstain and appear red. In some embodiments, the bacteria are those capable of causing disease (i.e. pathogens) and those that cause product degradation or spoilage. Examples of gram-negative bacteria aerobic rods and cocci relevant to the present inventions include but are not limited to Pseudomonadaceae, such as Frateuria, Pseudomonas, Xanthomonas, Zooglea; Azotobacteriaceae, such as Azomonas, Azotobacter; Rhizobiaceae, such as Agrobacterium, Bradyrhizobium, Phyllobacterium, Rhizobium; Neisseriaceae, such as Acinetobacter Kingella, Moraxella and Neisseria. 
As used herein the terms “Pseudomonas species” or “P. spp.” refer to a Gram negative, aerobic, and motile bacterium. For example, virulent forms of Pseudomonas syringae are economically important plant pathogens causing diseases in tomato plants, bean plants and other susceptible plant species. For example, virulent forms of P. syringae pv. syringae cause disease such as brown spot on bean and snap bean plants and speck on tomatoes.
The term “pathovar” or “pv” refers to a strain or set of strains with the same or similar characteristics, differentiated on the basis of distinctive but not exclusive pathogenicity to one or more plant hosts. For example, Pseudomonas syringae pv. tomato, (Okabe, (1933) Bacterial diseases of plants occurring in Formosa. II. Bacterial leaf spot of tomato. Journal of the Society of Tropical Agriculture, Taiwan 5:26-36; Young, et al. Genus Pseudomonas Migula 1894. In: Young, et al. (1978) A proposed nomenclature and classification for plant pathogenic bacteria. New Zealand Journal of Agricultural Research 21:153-177; all of which are herein incorporated by reference), is a pathogen of tomato plants, causing disease such as bacterial speck, that also infects Arabidopsis plants and Nicotiana benthamiana plants, while P. syringae pv. glycinea infects soybean and may also infect Arabidopsis plants, see, Young et al. 1991, Rev. Pl. Pathol. 70:211-221 for a review of bacteria nomenclature; herein incorporated by reference.
The term “biovar” refers to a variety of a species; may be a name or number designation, for example, “biovar 2.”
The term “strain” or “Bacterium Strain” or “Bacterium Strain designation” refers to a designation, such as DC3000, listed after a species or pathovar designation, for example, Pseudomonas syringae pv. tomato DC3000, where Pseudomonas syringae pv. tomato may also refer to type strains such as CFBP 2212; ICMP 2844; LMG 5093; or NCPPB 1106; P. syringae pv. syringae B728a refers to strain B728a; P. syringae pv. phaseolicola 1448A is an isolate designated 1448 that may cause halo blight on bean.
The term “Race” in reference to a bacterium refers to naming a subdivision of a species, for example, P. syringae pv. phaseolicola 1448A, Race 6.
The terms “fungi” and plural “fungus” refer to organisms that form a large group of plant-like living organisms that do not contain chlorophyll, including yeasts, molds, and mushrooms
The term “mildew” refers to fungi that form a superficial, usually whitish growth on plants and various organic materials and also refers to a plant disease caused by such fungi.
The term “protist” refers to a heterogeneous group of organisms having relatively simple organization (unicellular, or multicellular), without highly specialized tissues, including unicellular algae, protozoa, slime molds, and water molds, animal-like protozoa, plant-like algae, and fungi-like mold, such as water mold, slime molds, diatoms, golden algae, brown algae, et cetera.
The term “water mold” or “Oomycota” refers to a fungus-like protist, for e.g., Phytopthana infestans that destroyed potato crops causing the Irish potato blight or Great Potato Famine, also referred to as “downy mildews” and “white rusts.”
The term “downy mildew” refers to a disease characterized by yellowish to brownish areas of irregular size and shape (oval to cylindrical) on infected leaves or seed stalks of susceptible plants, such as vine plants and vegetables that grow on vine-like plants, e.g. cucumbers, etc., caused by certain fungi and protists, such as several types of water mold, such as Plasmopara viticol that infects grape plants and Peronospora parasitica that infects Brassicae plants such as broccoli, Brussels sprouts, cabbage, and cauliflower plants.
The term “powdery mildew” refers to a disease characterized by spots or patches of white to grayish of superficial powdery growth on leaves and shoots caused by fungi that grow on the surface of a plant.
The term “avirulent” refers to mutants of a bacterium or virus that lost the capacity to infect a host productively, that is, to make more bacterium or virus.
As used herein, “Avr” or “Avirulence protein” refers to a protein found through the avirulence phenotype.
The term “virulence” refers to a degree of pathogenicity of a given pathogen.
The term “virulent” refers to a capability for causing a severe disease; e.g. strongly pathogenic.
The term “virulence factor” or “virulence protein” refers to molecules that are produced by pathogens and further allow pathogens to invade host organisms, cause disease, or evade immune responses, such factors include but are not limited to adhesion molecules that are involved in the adhesion of bacteria to host cells, e.g. host cell receptors for bacteria at the surface of host cells; colonization factors; invasion factors; immune response blockers, and toxins.
As used herein the term “pathogen” and grammatical equivalents refers to an organism, including microorganisms, that cause disease in another organism (e.g., plants) by directly infecting the other organism, or by producing agents that cause or enhance disease in another organism (e.g., bacteria that produce virulence proteins and/or pathogenic toxins and the like).
The term “pathogenicity” refers to a capability of a pathogen to cause disease.
The term “susceptible” refers to lacking an inherent ability to resist disease or attack by a given pathogen; e.g. nonimmune. When used in reference to a plant, such as a “susceptible plant” refers to a plant that is not able to resist infection of a pathogen and exhibits disease symptoms. A plant may be susceptible to one pathogen, but resistant to another.
The term “susceptibility” refers to an inability of a plant to resist the effect of a pathogen or other damaging factor, such as a virulence factor.
The term “resistance” refers to an ability of an organism to exclude or overcome, completely or in some degree, the effect of a pathogen or other damaging factor, e.g. immune. When used in reference to a plant gene, as in “resistance genes” or “r genes” resistance refers to a plant gene associated with recognition of pathogen avirulence factors, for example, putative receptors of avirulence factors such as leucine-rich repeat proteins and/or kinases.
The term “resistant” refers to possessing qualities that hinder the development of a given pathogen, e.g., a plant that is exposed to a pathogenic organism that does not become infected or shows few disease symptoms. When used in reference to a plant, as in a “resistant plant” refers to a plant that is able to resist pathogen infection and exhibits no or few disease symptoms. A plant may be resistant to one pathogen, but susceptible to another.
The term “symptom” in reference to an infection or disease refers to an external and internal reaction or alteration of a plant as a result of a disease, for example, formation of papilla, water-soaking, chlorosis, necrosis, et cetera.
The term “papilla” in reference to a plant papilla refers to a structure that may be induced and observed at the pathogen infection site between the primary cell wall and the plasma membrane of a host plant cell, where a papilla contains cell wall materials, such as callose and lignin.
The term “water-soaking” in reference to a plant refers to a disease symptom during a bacterial infection that may be caused by infected plant release of water into the apoplast.
As used herein the term “disease” refers to any malfunctioning of host cells and tissues that results from continuous irritation by a pathogenic agent or environmental factor and leads to development of symptoms (e.g., blight, leaf spot, seed spot, fruit spot and fruit scab, papilla, gall, crown gall, witches'-broom, canker, rot, leaf curl, mosaic, and yellows, wilt, stunting, mold, mildew, abnormal leaf color, abnormal vein patterns of leaves, mottling in leaves, spotting patterns in leaves, abnormal leaf shape, such as pronounced upward rolling and twisting of leaflets, stunted plant growth, abnormalities of flower color, abnormalities of fruit size, abnormalities of fruit shape, abnormalities of fruit color, etc). A disease may be caused or result from contact by microorganisms and/or pathogens, for example, fungi cause diseases such as Dutch elm disease, chestnut blight, rust, smut, certain mildews, and ergot.
The term “necrosis” refers to living tissues in plants that are undergoing nonapoptotic cell death. Necrosis may cause discoloring of stems or leaves or kill a plant entirely. A necrotic area refers to dead plant tissue or dead plant parts.
The term “chlorosis” refers to a yellowing of normally green tissues.
The term “Genomic Islands” or “GI” refer to mobile genetic elements that are transferred through horizontal gene transfer, such as by plasmid, phage, or a conjugative transposon, and have been integrated into an organism's genome, such as elements relating to the pathogenicity of an organism. A Genomic Island may confer upon an organism fitness to occupy a particular ecological niche (Hentschel et al. (2001) Microbes Infect. 3(7):545-8; herein incorporated by reference).
The term “genomic pathogenicity island” or “pathogenicity islands” or “PAIs” refers to a distinct class of genomic islands which are acquired by horizontal transfer. A pathogenicity island is incorporated in the genome of the majority of pathogenic microorganisms but are typically absent from or nonfunctional in those of non-pathogenic organisms of the same or closely related species. Pathogenicity islands usually occupy relatively large genomic regions ranging from 10-200 kb and encode genes which contribute to the virulence of the respective pathogen, for example, genes encoding adherence factors, toxins, iron uptake systems, invasion factors and secretion systems such as type III secretion-associated hrp/hrc genes, an exchangeable effector locus” or “EEL” encoding diverse putative effector proteins and a conserved effector locus” or “CEL” are located in pathogenicity islands.
The terms “horizontal gene transfer” or “HGT,” also “lateral gene transfer” or “LGT” refer to a process in which an organism transfers genetic material (i.e. DNA) to another cell that is not its offspring.
The term “gall” refers to a spherical-like overgrowth or swelling of plant cells that may be the result of an attack by certain insects, bacteria, fungi, or nematodes.
The term “host” or “subject,” as used herein, refers to a target of a pathogen or heterologous gene, or a susceptible organisms to be treated by the compositions of the present invention, such as organisms that are exposed to, or suspected of being exposed to, one or more pathogens or the subject of prophylactic treatment. Host organisms include, but are not limited to plants (e.g., crop plants), algae, yeast, and animals.
The term “host cell” refers to any cell capable of replicating and/or transcribing and/or translating a heterologous gene of a plant pathogen or a plant.
As used herein, the terms “contacted” and “exposed” refer to bringing one or more of the compositions of the present invention into contact with a pathogen or a sample to be protected against pathogens such that the compositions of the present invention may inactivate the microorganism or pathogenic agents, if present. The present invention contemplates that the disclosed compositions are contacted to the pathogens or microbial agents in sufficient volumes and/or concentrations to inactivate the pathogens or microbial agents.
The term “pathovar” or “pv” refers to a strain or set of strains with the same or similar characteristics, differentiated at an infrasubspecific level from other strains of the same species or subspecies on the basis of distinctive but not exclusive pathogenicity to one or more plant hosts. For example, Pseudomonas syringae pv. tomato DC3000 that is an economically destructive pathogen of tomato plant that also infects Arabidopsis plants. Pseudomonas syringae pv. tomato strains DC300, either containing or lacking the avirulence gene avrRPM.
As used herein, the terms “agronomic trait” and “economical trait” refers to any selected trait that is desirable in a plant, such that a desirable trait increases the commercial value of a plant or plant part, for example, a preferred oil content, protein content, seed protein content, seed fatty acid content, seed size, seed color, hilium color, seed coat thickness, seed sugar content, seed free amino acid content, seed germination rate, seed texture, seed fiber content, seed Vitamin E content, seed isoflavone content, seed phytate content, seed phytosterol content, seed isoflavone content, lecithin content, food-grade quality, hilium color, seed yield, plant type, plant height, lodging, shatter, herbicide resistance, disease resistance, insect resistance, nematode resistance, drought tolerance, drought resistance, water tolerance, water resistance, temperature tolerance, such as cold weather resistance, hot weather resistance, and the like, growth habit, maturity group, field tolerance, and growth in a hardiness zone.
As used herein, the terms “formulation” in reference to an “agronomic formulation” refers to a composition comprising a peptide or nucleic acid of the present invention for use on or around plants.
As used herein, “AtMIN” or “Arabidopsis thaliana HopM interactors” refers to at least 21 Arabidopsis thaliana AtMIN proteins, such as AtMIN2, AtMIN7, etc., and further may refer to a homolog, ortholog or paralog in Arabidopsis or other plants.
As used herein, “AtMIN7” refers to an Arabidopsis thaliana GEF.
As used herein, “GEF” or “guanyl-nucleotide exchange factor activity” or “guanyl-nucleotide release factor activity” or “guanyl-nucleotide releasing factor” or “GNRP” refers to a gene encoding for or a protein that stimulates (catalyse) an exchange of guanyl nucleotides by a GTPase, i.e. the exchange of GTP for GDP bound to the protein.
As used herein, “ADP-ribosylation factor” or “Arf” or “ARF” refers to a family of nucleotides and protein that encode a low molecular mass Ras-related GTPase.
As used herein, “exchange factors for ARF GTPases” or “ARF-GEFs” refers to a gene or protein of an ADP-ribosylation factor G.
As used herein, “GNOM” refers to a guanine nucleotide exchange factor (GEF) that acts on ADP ribosylation factor (ARF) type G proteins (ARF-GEF) (see, Busch et al. 1996; Shevell et al. 1994 and Steinmann et al. 1999; herein incorporated by reference).
As used herein, “Hop” or “hopPtoM” or “Hrp outer protein” refers generically to proteins translocated and/or secreted by a Hrp system of P. syringae and other plant pathogens with similar Hrp systems (e.g., Erwinia and Pantoea spp), see, “P. syringae Hop Identification and Nomenclature Home Page” at pseudomonas-syringae.org/pst_func_gen2.htm.
As used herein, “Hrp” or “hypersensitive response and pathogenicity” refers to mutations in the TTSS machinery that abolish the ability of P. syringae to elicit the “HR” in nonhosts or to be pathogenic in hosts.
As used herein, “hrc or “hrp conserved” refer to conserved genes associated with the pathogenicity of a pathogen, such as those found in P. syringae pv. syringae (causing brown spot of bean and other plants), Erwinia amylovora (causing fire blight of apple and pear), Ralstonia (Pseudomonas) solanacearum (causing bacterial wilt of tomato), and Xanthomonas campestris pv. vesicatoria (causing bacterial spot of pepper and tomato and also found in nonpathogenic bacteria such as Escherichia coli and Pseudomonas fluorescens (see, for reference, Bogdanove, et al., (1996) Mol. Microbiol. 20(3):681-3; herein incorporated by reference).
As used herein, “effector” refers to a virulence protein injected into host cells by a TTSS, which is broadly applicable to various plant and animal pathogens. As used herein, “TTSS” or “T3SS” type III secretion system” or “Type III secretion pathway” such as in “Hrp pathway in P. syringae pathovars” refers to secretion or translocation through this pathway is considered the defining characteristic for P. syringae effector proteins. Type III effector proteins are essential for the virulence of Pseudomonas syringae, Xanthomonas spp., Ralstonia solanacearum and Erwinia species. For the purposes of the present inventions, Gram-negative bacteria may deliver effector proteins into the cells of their eukaryotic hosts using the type III secretion system.
The terms “exchangeable effector locus” or “EEL” refer to a group of genes that flank a TTSS region, for example, three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL include HopPsyC, HopPsyE, and HopPsyV.
As used herein, “helper protein” or “translocator” refers to a term of convenience referring to extracellular accessory proteins (such as HrpA) plus other TTSS substrates (such as harpins) whose primary function is likely to be the translocation of true effectors through host barriers.
As used herein, “harpin” refers to a presumed helper proteins that are secreted by the TTSS in more abundance than true effectors, appear to interact with plant cell walls and membranes, are glycine-rich and devoid of cysteine, and possess a heat-stable ability to elicit the hypersensitive response when infiltrated into the intercellular (apoplastic) spaces of plant leaves.
The term “plant” is used in it broadest sense. A type of plant includes, but is not limited to, any species of woody plant, ornamental plant or decorative plant, crop or cereal plant, fruit plant or vegetable plant, and algae.
The term “crop” or “crop plant” is used in its broadest sense. The term includes, but is not limited to, any species of plant or algae edible by humans or used as a feed for animals or used or consumed by humans, or any plant or algae used in industry or commerce. A crop plant includes a family of plants, for example Brassicaceae, that includes but is not limited to cabbage, kale, radish, mustard plants and thale plants, or a genus of plants, such as Arabidopsis (rockcress) plants that includes but is not limited to Thale and Mouse Cress (Arabidopsis thaliana), Solanaceae including but is not limited to these examples, Nicotiana plants, such as Tobacco (Nicotiana spp., L.) plants, that refer to a genus of broad-leafed plants of the nightshade family, including but not limited to a Nicotiana benthamiana plant, and Lycopersicon spp., including but is not limited to Lycopersicon esculentum (tomato) plants and Poaceae (grass family), Oryza spp., including Oryza sativa and Oryza glaberrima rice plants.
The term “variety” refers to a biological classification for an intraspecific group or population, that can be distinguished from the rest of the species by any characteristic (for example morphological, physiological, cytological, etc.). A variety may originate in the wild but can also be produced through selected breeding (for example, see, cultivar).
The terms “cultivar,” “cultivated variety,” and “cv” refer to a group of cultivated plants distinguished by any characteristic (for example morphological, physiological, cytological, etc.) that when reproduced sexually or asexually, retain their distinguishing features to produce a cultivated variety.
A plant also refers to an intact living structure or a partial living structure, such as a plurality of plant cells that form a structure that is present at any stage of a plant's development, such as a plant part. Such structures include, but are not limited to, a leaf, shoot, stem, a fruit, flower, petal, et cetera.
The term “plant part” as used herein refers to a plant structure or a plant tissue. A plant part may comprise one or more of a leaf, stem, tiller, plug, rhizome, sprig, stolen, meristem, crown, and the like.
The term “seed” refers to a ripened ovule, consisting of the embryo and a casing.
The term “stem” refers to a main ascending axis of a plant.
The term “tiller” refers to a portion of a plant growing from the base of the stem of a plant, also referred to as a “shoot.” A tiller may also be described as a lateral stem (or shoot), usually erect, that develops from the central crown, and may also refer to the branch or shoot that originates at a basal node. A tiller is often used for propagation, such as vegetative propagation, of a plant.
The term “propagation” refers to the process of producing new plants, either by vegetative means involving the rooting or grafting of pieces of a plant, or by sowing seeds. The terms “vegetative propagation” and “asexual reproduction” refer to the ability of plants to reproduce without sexual reproduction, by producing new plants from existing vegetative structures that are clones, i.e., plants that are identical in all attributes to the mother plant and to one another. For example, the division of a clump, rooting of proliferations, or cutting of mature crowns can produce a new plant.
As used herein, the term “hybrid” in reference to a seed or plant is produced as the result of controlled cross-pollination as opposed to a “non-hybrid” seed produced as the result of natural pollination, as in a “hybrid seed” produced by breeding methods of the present invention.
As used herein, the terms “introgress” and “introgressing” refer to incorporating a genetic substance, such as germplasm, loci, allele, gene, DNA, and the like for introducing a trait into an organism, such as a plant, a plant cell, a yeast cell, and the like, for example, incorporating pathogen resistant transgenic material and/or transgenes into a previously pathogen susceptible plant variety. Introgression may refer to one of several types of breeding methods for a incorporating a genetic trait, such as pathogen resistance provided by expression of a transgene, including compositions and methods for identifying the expression of a heterolous transgene, such as by a Northern blot or immunoblotting or PCR analysis.
The terms “leaf” and “leaves” refer to a structure attached to a stem or branch of a plant where photosynthesis and transpiration take place.
The term “epidermis” refers to an outer most layer of cells of the leaf and of young stems and roots.
The term “cell wall” refers to a rigid layer of extracellular matrix material that completely surrounds a plant cell or fungal cell or a bacterium.
The terms “tissue culture” and “micropropagation” refer to a form of asexual propagation undertaken in specialized laboratories, in which clones of plants are produced from small cell clusters from very small plant parts (e.g. buds, nodes, leaf segments, root segments, etc.), grown aseptically (free from any microorganism) in a container where the environment and nutrition can be controlled.
The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.).
The term plant cell “compartments or organelles” is used in its broadest sense. The term includes but is not limited to vacuoles, the endoplasmic reticulum, Golgi apparatus, trans Golgi network, plastids, sarcoplasmic reticulum, glyoxysomes, mitochondrial, chloroplast, nuclear membranes, and the like.
For the purposes of the present invention, a “protoplast” refers to a cell, such as a plant, fungal or bacterium, that does not have a cell wall where a cell wall would have been present in its natural state.
The terms “allele” and “alleles” refer to each version of a gene for a same locus that has more than one sequence. For example, there are multiple alleles for eye color at the same locus.
The terms “recessive,” “recessive gene,” and “recessive phenotype” refer to an allele that has a phenotype when two alleles for a certain locus are the same as in “homozygous” or as in “homozygote” and then partially or fully loses that phenotype when paired with a more dominant allele as when two alleles for a certain locus are different as in “heterozygous” or in “heterozygote.”
The terms “dominant,” “dominant allele,” and “dominant phenotype” refer to an allele that has an effect to suppress the expression of the other allele in a heterozygous (having one dominant allele and one recessive allele) condition.
The terms “transgenic” when used in reference to a plant or leaf or fruit or seed or plant part for example a “transgenic plant,” “transgenic leaf,” “transgenic fruit,” “transgenic seed,” and a “transgenic host cell” refer to a plant or leaf or fruit or seed or part or cell that contains at least one heterologous or foreign gene in one or more of its cells.
The term “transgenic plant material” refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous gene in one or more of its cells.
The term “heterologous” when used in reference to a gene or nucleic acid refers to a gene that has been manipulated in some way. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.).
Heterologous genes may comprise plant gene sequences that comprise cDNA forms of a plant gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous genes are distinguished from endogenous plant genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
The term “accession” when used herein associated with sequences of genes and proteins refers to a gene or group of similar genes or proteins from these genes or proteins received from a single source at a single time. The term “accession number” when used herein refers to a unique identifier for protein and gene sequences and is assigned when an accession is entered into a database (for example GenBank at NCBI, European Molecular Biology Laboratory (EMBL), SWISS-PROT, and the like.
The term “gene” encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5′ of the coding region and which are present on the mRNA are referred to as 5′ non-translated sequences. The sequences which are located 3′ or downstream of the coding region and which are present on the mRNA are referred to as 3′ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region termed “exon” or “expressed regions” or “expressed sequences” interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide
The terms “variant” and “mutant” when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties. One type of conservative amino acid substitution refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. More rarely, a variant may have “non-conservative” changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on). Thus, nucleotide sequences of the present invention can be engineered in order to introduce or alter an AtMIN or HOPM1 coding sequence for a variety of reasons, including but not limited to initiating the production of tolerance to pathogens; alterations that modify the cloning, processing and/or expression of the gene product (such alterations include inserting new restriction sites and changing codon preference), as well as varying the protein function activity (such changes include but are not limited to differing binding kinetics to nucleic acid and/or protein or protein complexes or nucleic acid/protein complexes, differing binding inhibitor affinities or effectiveness, differing reaction kinetics, varying subcellular localization, and varying protein processing and/or stability) for enhancing pathogen resistance in a plant.
The term “fusion” when used in reference to a polypeptide refers to a chimeric protein containing a protein of interest joined to an exogenous protein fragment (the fusion partner). The term “chimera” when used in reference to a polypeptide refers to the expression product of two or more coding sequences obtained from different genes, that have been cloned together and that, after translation, act as a single polypeptide sequence. Chimeric polypeptides are also referred to as “hybrid” polypeptides. The coding sequences include those obtained from the same or from different species of organisms. The fusion partner may serve various functions, including enhancement of solubility of the polypeptide of interest, as well as providing an “affinity tag” to allow purification of the recombinant fusion polypeptide from a host cell or from a supernatant or from both. If desired, the fusion partner may be removed from the protein of interest after or during purification.
The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence of interest,” and “nucleic acid sequence of interest” refer to any nucleotide sequence (e.g., RNA or DNA), the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, etc.), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).
The term “structural” when used in reference to a gene or to a nucleotide or nucleic acid sequence refers to a gene or a nucleotide or nucleic acid sequence whose ultimate expression product is a protein (such as an enzyme or a structural protein), an rRNA, an sRNA, a tRNA, and the like.
The term “oligonucleotide” refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
The term “polynucleotide” refers to a molecule comprised of several deoxyribonucleotides or ribonucleotides, and is used interchangeably with oligonucleotide. Typically, oligonucleotide refers to shorter lengths, and polynucleotide refers to longer lengths, of nucleic acid sequences.
The term “an oligonucleotide (or polypeptide) having a nucleotide sequence encoding a gene” or “a nucleic acid sequence encoding” a specified polypeptide refers to a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence which encodes a gene product. The coding region may be present in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc., may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers, exogenous promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.
The “fragment” or “portion” in reference to a nucleotide sequence refers to a sequence that may range in size from an exemplary 100, 200, 300, or 399 contiguous nucleotide residues to the entire nucleic acid sequence coding region minus one nucleic acid residue. Thus, a nucleic acid sequence comprising “at least a portion of” a nucleotide sequence comprises from ten (10) contiguous nucleotide residues of the nucleotide sequence to the entire nucleotide sequence length of coding region minus one.
The terms “protein,” “polypeptide,” “peptide,” “encoded product,” “amino acid sequence” are used interchangeably to refer to compounds comprising amino acids joined via peptide bonds and a “protein” encoded by a gene is not limited to the amino acid sequence encoded by the gene, but includes post-translational modifications of the protein. Where the term “amino acid sequence” is recited herein to refer to an amino acid sequence of a protein molecule, the term “amino acid sequence” and like terms, such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. Furthermore, an “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein. The deduced amino acid sequence from a coding nucleic acid sequence includes sequences which are derived from the deduced amino acid sequence and modified by post-translational processing, where modifications include but not limited to glycosylation, hydroxylations, phosphorylations, and amino acid deletions, substitutions, and additions. Thus, an amino acid sequence comprising a deduced amino acid sequence is understood to include post-translational modifications of the encoded and deduced amino acid sequence. The term “X” may represent any amino acid.
The term “portion” or “fragment” when used in reference to a protein (as in “a fragment of a given protein”) refers to a sequence that may range in size from four amino acid residues to the entire amino sequence minus one amino acid.
The term “fragment” when in reference to a specific protein (such as “HopM1 proteins” etc.) refers to an exemplary 100, 200, 300, or 399 amino acid sequence. Accordingly, a fragment of a that protein may range in size from four (4) contiguous amino acid residues to the entire amino acid sequence minus one amino acid residue. Thus, a polypeptide sequence comprising “at least a portion of an amino acid sequence” comprises from four (4) contiguous amino acid residues of the amino acid sequence to the entire amino acid sequence.
The terms “homolog,” “homologue,” “homologous,” and “homology” when used in reference to amino acid sequence or nucleic acid sequence or a protein or a polypeptide refers to a degree of sequence identity to a given sequence, or to a degree of similarity between conserved regions, or to a degree of similarity between three-dimensional structures or to a degree of similarity between the active site, or to a degree of similarity between the mechanism of action, or to a degree of similarity between functions. In some embodiments, a homolog has a greater than 20% sequence identity to a given sequence. In some embodiments, a homolog has a greater than 30% sequence identity to a given sequence. In some embodiments, a homolog has a greater than 50% sequence identity to a given sequence. In some embodiments, a homolog has a greater than 70% sequence identity to a given sequence. In some embodiments, a homolog has a greater than 90% sequence identity to a given sequence. In some embodiments, a homolog has a greater than 95% sequence identity to a given sequence. In some embodiments, homology is determined by comparing internal conserved sequences to a given sequence. In some embodiments, homology is determined by comparing designated conserved functional regions. In some embodiments, homology is determined by comparing designated conserved “motif” regions.
The term “homology” when used in relation to nucleic acids or proteins refers to a degree of identity. There may be partial homology or complete homology. The following terms are used to describe the sequence relationships between two or more polynucleotides and between two or more polypeptides: “identity,” “percentage identity,” “identical,” “reference sequence,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” “Sequence identity” refers to a measure of relatedness between two or more nucleic acids or proteins, and is described as a given as a percentage “of homology” with reference to the total comparison length. A “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, the sequence that forms an active site of a protein or a segment of a full-length cDNA sequence or may comprise a complete gene sequence. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window,” as used herein, refers to a conceptual segment of in internal region of a polypeptide. In one embodiment, a comparison window is at least 77 amino acids long. In another embodiment, a comparison window is at least 84 amino acids long. In another embodiment, conserved regions of proteins are comparison windows. In a further embodiment, an amino acid sequence for a conserved transmembrane domain is 24 amino acids. Calculations of identity—may be performed by algorithms contained within computer programs such as the ClustalX algorithm (Thompson, et al. Nucleic Acids Res. 24, 4876-4882 (1997)); herein incorporated by reference); MEGA2 (version 2.1) (Kumar, et al. Bioinformatics 17, 1244-1245 (2001)); “GAP” (Genetics Computer Group, Madison, Wis.), “ALIGN” (DNAStar, Madison, Wis.), BLAST (National Center for Biotechnology Information; NCBI as described at http://www.ncbi.nlm.nih.g-ov/BLAST/blast_help.shtml) and MultAlin (Multiple sequence alignment) program (Corpet, Nucl. Acids Res., 16 (22), 10881-10890 (1988) at //prodes.toulouse.inra.fr/multalin/multalin.html), all of which are herein incorporated by reference).
For comparisons of nucleic acids, 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math. 2:482 (1981)) by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); herein incorporated by reference), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988); herein incorporated by reference), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.; herein incorporated by reference), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected. The term “sequence identity” means that two polynucleotide or two polypeptide sequences are identical (i.e., on a nucleotide-by-nucleotide basis or amino acid basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid, in which often conserved amino acids are taken into account, occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
The terms “substantial identity” as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence, for example, as a segment of the full-length sequences of the compositions claimed in the present invention.
The term “ortholog” refers to a gene in different species that evolved from a common ancestral gene by speciation. In some embodiments, orthologs retain the same function.
The term “paralog” refers to genes related by duplication within a genome. In some embodiments, paralogs evolve new functions. In further embodiments, a new function of a paralog is related to the original function.
The term “partially homologous nucleic acid sequence” refers to a sequence that at least partially inhibits (or competes with) a completely complementary sequence from hybridizing to a target nucleic acid and is referred to using the functional term “substantially homologous.” The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a sequence that is completely complementary to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial-degree of identity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-identical target.
The term “substantially homologous” when used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low to high stringency as described above.
The term “substantially homologous” when used in reference to a single-stranded nucleic acid sequence refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low to high stringency as described above.
The term “hybridization” in reference to a nucleic acid refers to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be “self-hybridized.”
The term “Tm” refers to the “melting temperature” of a nucleic acid. Melting temperature Tm is the midpoint of the temperature range over which nucleic acids are denatured (e.g. DNA:DNA, DNA:RNA and RNA:RNA, etc.). Methods for calculating the Tm of nucleic acids are well known in the art (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp. 9.50-51, 11.48-49 and 11.2-11.3; herein incorporated by reference).
The term “stringency” refers to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.
“Low stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5×Denhardt's reagent (50×Denhardt's contains per 500 ml:0.05 g Ficoll (Type 400, Pharmacia):0.05 g BSA (Fraction V; Sigma)) and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500 nucleotides in length is employed.
“Medium stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when a probe of about 500 nucleotides in length is employed.
“High stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when a probe of about 500 nucleotides in length is employed.
It is well known that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.). “Amplification” is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.
The term “amplifiable nucleic acid” refers to nucleic acids that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “sample template.”
The term “sample template” refers to nucleic acid originating from a sample that is analyzed for the presence of “target” (defined below). In contrast, “background template” is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
The term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
The term “probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
The term “expression” when used in reference to a nucleic acid sequence, such as a gene, refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein where applicable (as when a gene encodes a protein), through “translation” of mRNA. Gene expression can be regulated at many stages in the process.
“Up-regulation” or “activation” or “enhanced” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called “activators” and “repressors,” respectively.
The terms “in operable combination,” “in operable order” and “operably linked” refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
The terms “RNA interference” or “RNAi” and “interference” in reference to RNA refers to the silencing or decreasing of gene expression by siRNAs. It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in its duplex region to the sequence of the silenced gene. The gene may be endogenous or exogenous to the organism, present integrated into a chromosome or present in a transfection vector and/or an expression vector that is not integrated into the genome. The expression of the gene is either completely or partially inhibited. RNAi may also be considered to inhibit the function of a target RNA; the function of the target RNA may be complete or partial. In both plants and animals, RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, although the protein components of this activity are unknown. However, the 22-nucleotide RNA sequences are homologous to the target gene that is being suppressed. Thus, the 22-nucleotide sequences appear to serve as guide sequences to instruct a multicomponent nuclease, RISC, to destroy the specific mRNAs. Carthew has reported (Curr. Opin. (2001) Cell Biol. 13(2):244-248) that eukaryotes silence gene expression in the presence of dsRNA homologous to the silenced gene. Biochemical reactions that recapitulate this phenomenon generate RNA fragments of 21 to 23 nucleotides from the double-stranded RNA. These stably associate with an RNA endonuclease, and probably serve as a discriminator to select mRNAs. Once selected, mRNAs are cleaved at sites 21 to 23 nucleotides apart.
The term “regulatory element” refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, and the like.
Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237 (1987); herein incorporated by reference). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect, mammalian and plant cells. Promoter and enhancer elements have also been isolated from viruses and analogous control elements, such as promoters, are also found in prokaryotes. The selection of a particular promoter and enhancer depends on the cell type used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review, see Maniatis, et al., supra (1987); herein incorporated by reference).
The terms “promoter element,” “promoter,” or “promoter sequence” refer to a DNA sequence that is located at the 5′ end (i.e., precedes) of the coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.
The term “regulatory region” refers to a gene's 5′ transcribed but untranslated regions, located immediately downstream from the promoter and ending just prior to the translational start of the gene.
The term “promoter region” refers to the region immediately upstream of the coding region of a DNA polymer, and is typically between about 500 bp and 4 kb in length, and is preferably about 1 to 1.5 kb in length. Promoters may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., leaves). Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected.
The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue.
The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining. Briefly, tissue sections are embedded in paraffin, and paraffin sections are reacted with a primary antibody that is specific for the polypeptide product encoded by the nucleotide sequence of interest whose expression is controlled by the promoter. A labeled (e.g., peroxidase conjugated) secondary antibody that is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy. Promoters may be “constitutive” or “inducible.”
The term “constitutive” when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a transgene in substantially any cell and any tissue. Exemplary constitutive plant promoters include, but are not limited to SD Cauliflower Mosaic Virus (CaMV SD; see e.g., U.S. Pat. No. 5,352,605, incorporated herein by reference), mannopine synthase, octopine synthase (ocs), superpromoter (see e.g., WO 95/14098; herein incorporated by reference), and ubi3 promoters (see e.g., Garbarino and Belknap, Plant Mol. Biol. 24:119-127 (1994); herein incorporated by reference). Such promoters have been used successfully to direct the expression of heterologous nucleic acid sequences in transformed plant tissue.
In contrast, an “inducible” promoter is one that is capable of directing a level of transcription of an operably linked nucleic acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.) that is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus.
The term “regulatory element” refers to a genetic element that controls some aspect of the expression of nucleic acid sequence(s). For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, and the like.
The enhancer and/or promoter may be “endogenous” or “exogenous” or “heterologous.” An “endogenous” enhancer or promoter is one that is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer or promoter is one that is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of the gene is directed by the linked enhancer or promoter. For example, an endogenous promoter in operable combination with a first gene can be isolated, removed, and placed in operable combination with a second gene, thereby making it a “heterologous promoter” in operable combination with the second gene. A variety of such combinations are contemplated (e.g., the first and second genes can be from the same species, or from different species).
The term “naturally linked” or “naturally located” when used in reference to the relative positions of nucleic acid sequences means that the nucleic acid sequences exist in nature in the relative positions.
The presence of “splicing signals” on an expression vector often results in higher levels of expression of the recombinant transcript in eukaryotic host cells. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp. 16.7-16.8; herein incorporated by reference). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40. Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term “poly(A) site” or “poly(A) sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable, as transcripts lacking a poly(A) tail are unstable and are rapidly degraded. The poly(A) signal utilized in an expression vector may be “heterologous” or “endogenous.” An endogenous poly(A) signal is one that is found naturally at the 3′ end of the coding region of a given gene in the genome. A heterologous poly(A) signal is one which has been isolated from one gene and positioned 3′ to another gene. A commonly used heterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A) signal is contained on a 237 bp BamHI/BclI restriction fragment and directs both termination and polyadenylation (Sambrook, supra, at 16.6-16.7; herein incorporated by reference).
The term “vector” refers to nucleic acid molecules that transfer DNA segment(s). Transfer can be into a cell, cell to cell, et cetera. The term “vehicle” is sometimes used interchangeably with “vector.”
The terms “expression vector” and “expression cassette” refer to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome-binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
The term “expression vector” in reference to a construct, such as “expression vector construct” refers to an artificial vector engineered for expressing a nucleic acid in a particular organism, such as a plant, and can be more specifically engineered for expression within a particular type or species of plant or plant tissue.
The term “transfection” refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, glass beads, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, biolistics (i.e., particle bombardment) and the like.
The terms “stable transfection” and “stably transfected” refer to the introduction and integration of foreign DNA into the genome of the transfected cell. The term “stable transfectant” refers to a cell that has stably integrated foreign DNA into the genomic DNA.
The terms “transient transfection” and “transiently transfected” refer to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell. The foreign DNA persists in the nucleus of the transfected cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes. The term “transient transfectant” refers to cells that have taken up foreign DNA but have failed to integrate this DNA.
The terms “infecting” and “infection” when used with a bacterium refer to co-incubation of a target biological sample, (e.g., cell, tissue, etc.) with the bacterium under conditions such that nucleic acid sequences contained within the bacterium are introduced into one or more cells of the target biological sample.
The term “Agrobacterium” refers to a soil-borne, Gram-negative, rod-shaped phytopathogenic bacterium that causes crown gall. Agrobacterium is a representative genus of a soil-borne, Gram-negative, rod-shaped phytopathogenic bacterium family Rhizobiaceae. Its species are responsible for plant tumors such as crown gall and hairy root disease. In the dedifferentiated tissue characteristic of the tumors, amino acid derivatives known as opines are produced and catabolized. The bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. Agrobacterium tumefaciens causes crown gall disease by transferring some of its DNA to the plant host. The transferred DNA (T-DNA) is stably integrated into the plant genome, where its expression leads to the synthesis of plant hormones and thus to the tumorous growth of the cells. A putative macromolecular complex forms in the process of T-DNA transfer out of the bacterial cell into the plant cell.
The term “Agrobacterium” includes, but is not limited to, the strains Agrobacterium tumefaciens (which typically causes crown gall in infected plants), and Agrobacterium rhizogens (which causes hairy root disease in infected host plants). Infection of a plant cell with Agrobacterium generally results in the production of opines (e.g., nopaline, agropine, octopine, etc.) by the infected cell. Thus, Agrobacterium strains which cause production of nopaline (e.g., strain GV3101, LBA4301, C58, A208, etc.) are referred to as “nopaline-type” Agrobacteria; Agrobacterium strains which cause production of octopine (e.g., strain LBA4404, Ach5, B6, etc.) are referred to as “octopine-type” Agrobacteria; and Agrobacterium strains which cause production of agropine (e.g., strain EHA105, EHA101, A281, etc.) are referred to as “agropine-type” Agrobacteria. 
The terms “bombarding, “bombardment, and “biolistic bombardment” refer to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample. Methods for biolistic bombardment are known in the art (e.g., U.S. Pat. No. 5,584,807; herein incorporated by reference), and are commercially available (e.g. the helium gas-driven microprojectile accelerator, such as a PDS-1000/He, BioRad).
The term “microwounding” when made in reference to plant tissue refers to the introduction of microscopic wounds in that tissue. Microwounding may be achieved by, for example, particle bombardment as described herein.
The term “calcium phosphate co-precipitation” refers to a technique for the introduction of nucleic acids into a cell. The uptake of nucleic acids by cells is enhanced when the nucleic acid is presented as a calcium phosphate-nucleic acid co-precipitate. The original technique of Graham and van der Eb (in Virol., 52:456 (1973); herein incorporated by reference), is well-known to have been modified by several groups to optimize conditions for particular types of cells.
The term “transgene” refers to a foreign gene that is placed into an organism by the process of transfection.
The term “foreign gene” refers to any nucleic acid (e.g., gene sequence) that is introduced into the genome of an organism by experimental manipulations and may include gene sequences found in that organism so long as the introduced gene does not reside in the same location, as does the naturally occurring gene.
The terms “transformants” and “transformed cells” include the primary transformed cell and cultures derived from that cell without regard to the number of transfers. Resulting progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of transformants.
The term “selectable marker” refers to a gene which encodes an enzyme having an activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed, or which confers expression of a trait which can be detected (e.g., luminescence or fluorescence). Selectable markers may be “positive” or “negative.” Examples of positive selectable markers include the neomycin phosphotransferase (NPTII) gene that confers resistance to G418 and to kanamycin, and the bacterial hygromycin phosphotransferase gene (hyg), which confers resistance to the antibiotic hygromycin. Negative selectable markers encode an enzymatic activity whose expression is cytotoxic to the cell when grown in an appropriate selective medium. For example, the HSV-tk gene is commonly used as a negative selectable marker. Expression of the HSV-tk gene in cells grown in the presence of gancyclovir or acyclovir is cytotoxic; thus, growth of cells in selective medium containing gancyclovir or acyclovir selects against cells capable of expressing a functional HSV TK enzyme.
The term “reporter gene” refers to a gene encoding a protein that may be assayed. Examples of reporter genes include, but are not limited to, luciferase (See, e.g., deWet et al., Mol. Cell. Biol. 7:725 (1987) and U.S. Pat. Nos. 6,074,859; 5,976,796; 5,674,713; and 5,618,682; all of which are herein incorporated by reference), green fluorescent protein (e.g., GenBank Accession Number U43284; GFP variants commercially available from CLONTECH Laboratories, Palo Alto, Calif.; herein incorporated by reference), chloramphenicol acetyltransferase, β-galactosidase (lacZ gene), alkaline phosphatase, and horse radish peroxidase.
The term “antisense” refers to a deoxyribonucleotide sequence whose sequence of deoxyribonucleotide residues is in reverse 5′ to 3′ orientation in relation to the sequence of deoxyribonucleotide residues in a sense strand of a DNA duplex. A “sense strand” of a DNA duplex refers to a strand in a DNA duplex that is transcribed by a cell in its natural state into a “sense mRNA.” Thus, an “antisense” sequence is a sequence having the same sequence as the non-coding strand in a DNA duplex. The term “antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript or mRNA. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. In addition, as used herein, antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression. “Ribozyme” refers to a catalytic RNA and includes sequence-specific endoribonucleases. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein.
The term “siRNAs” refers to short interfering RNAs. In some embodiments, siRNAs comprise a duplex, or double-stranded region, of about 18-25 nucleotides long; often siRNAs contain from about two to four unpaired nucleotides at the 3′ end of each strand. At least one strand of the duplex or double-stranded region of a siRNA is substantially homologous to or substantially complementary to a target RNA molecule. The strand complementary to a target RNA molecule is the “antisense strand”. The strand homologous to the target RNA molecule is the “sense strand,” and is also complementary to the siRNA antisense strand. siRNAs may also contain additional sequences; non-limiting examples of such sequences include linking sequences, or loops, as well as stem and other folded structures. siRNAs appear to function as key intermediaries in triggering RNA interference in invertebrates and in vertebrates, and in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants.
The terms “hpRNA” and “hairpin RNA” refer to self-complementary RNA that forms hairpin loops and functions to silence genes (e.g. Wesley et al. (2001) The Plant Journal 27(6): 581-590; herein incorporated by reference). The term “ihpRNA” refers to intron-spliced hpRNA that functions to silence genes.
The term “target RNA molecule” refers to an RNA molecule to which at least one strand of the short double-stranded region of a siRNA is homologous or complementary. Typically, when such homology or complementary is about 100%, the siRNA is able to silence or inhibit expression of the target RNA molecule. Although it is believed that processed mRNA is a target of siRNA, the present invention is not limited to any particular hypothesis, and such hypotheses are not necessary to practice the present invention. Thus, it is contemplated that other RNA molecules may also be targets of siRNA. Such targets include unprocessed mRNA, ribosomal RNA, and viral RNA genomes.
The terms “posttranscriptional gene silencing” and “PTGS” refer to silencing of gene expression in plants after transcription, and appears to involve the specific degradation of mRNAs synthesized from gene repeats.
The term “cosuppression” refers to silencing of endogenous genes by heterologous genes that share sequence identity with endogenous genes.
The term “overexpression” generally refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
The term “cosuppression” refers to the expression of a foreign gene that has substantial homology to an endogenous gene resulting in the suppression of expression of both the foreign and the endogenous gene.
As used herein, the term “altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
The terms “overexpression” and “overexpressing” and grammatical equivalents, are specifically used in reference to levels of mRNA to indicate a level of expression approximately 2-fold higher than that typically observed in a given tissue in a control or non-transgenic animal. Levels of mRNA are measured using any of a number of techniques known to those skilled in the art including, but not limited to Northern blot analysis. Appropriate controls are included on the Northern blot to control for differences in the amount of RNA loaded from each tissue analyzed (e.g., the amount of 28S rRNA, an abundant RNA transcript present at essentially the same amount in all tissues, present in each sample can be used as a means of normalizing or standardizing the RAD50 mRNA-specific signal observed on Northern blots).
The terms “Southern blot analysis” and “Southern blot” and “Southern” refer to the analysis of DNA on agarose or acrylamide gels in which DNA is separated or fragmented according to size followed by transfer of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then exposed to a labeled probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support. Southern blots are a standard tool of molecular biologists (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp. 9.31-9.58; herein incorporated by reference).
The term “Northern blot analysis,” “Northern blot,” and “Northern” refer to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled probe to detect RNA species complementary to the probe used. Northern blots are a standard tool of molecular biologists (Sambrook, et al. supra, pp 7.39-7.52, (1989); herein incorporated by reference).
The terms “RACE” and “Rapid Amplification of cDNA Ends” refer to a PCR technique used to obtain the 3′ end of a cDNA as in 3′ RACE and to obtain the 5′ end of a cDNA as in 5′ RACE.
The terms “blot analysis,” “Western blot,” and “Western” refer to the analysis of protein(s) (or polypeptides) immobilized onto a support such as nitrocellulose or a membrane. A mixture comprising at least one protein is first separated on an acrylamide gel, and the separated proteins are then transferred from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized proteins are exposed to at least one antibody with reactivity against at least one antigen of interest. The bound antibodies may be detected by various methods, including the use of radiolabeled antibodies.
The term “isolated” when used in relation to a nucleic acid or polypeptide, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a particular protein includes, by way of example, such nucleic acid in cells ordinarily expressing the protein, where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded).
The term “purified” refers to molecules, either nucleic or amino acid sequences that are removed from their natural environment isolated or separated. An “isolated nucleic acid sequence” is therefore a purified nucleic acid sequence. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.
As used herein, the terms “purified” and “to purify” also refer to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percent of polypeptide of interest in the sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.
The term “reagent” in reference to a method and a kit, refers to a substance or molecule, such as a polynucleotide, an antisense nucleotide, peptide, an antibody, a chemical a buffer, an expression vector, and the like, necessary for various test methods and kits of the present invention, including those compounds used for measuring the expression level of the indicator gene, or protein, such as HOPM1, ATMIN7, and the like, are useful as reagents. These test reagents can be made into a kit for testing for altered trafficking, for pathogen infection, for a plant's response to a pathogen, the capability of a plant's response to a pathogen, for example, labeling a protein or nucleotide, with a substrate compound used for detection of the label, a buffer for diluting the sample, or a positive or negative standard sample. Furthermore, an instruction sheet and such indicating the method of using the kit can be packaged in the kit for the testing of this invention.
The peptide, polynucleotide, antibody, cell line, or model animal and plant, including animal and plant cells, which are necessary for the various methods of screening of this invention, can be combined in advance to produce a kit. More specifically, such a kit may comprise, for example, a cell that expresses the indicator gene, and a reagent for measuring the expression level of the gene or location of a protein. As a reagent for measuring the expression level of the indicator gene, for example, an oligonucleotide that has at least 15 nucleotides complementary to the polynucleotide comprising the nucleotide sequence of at least one indicator gene or to the complementary strand thereof may be used. Alternatively, an antibody that recognizes a peptide comprising amino acid sequence of at least one indicator protein may be used as a reagent.
In these kits may be packaged a substrate compound used for the detection of the indicator, medium and a vessel for cell culturing, positive and negative standard samples, and furthermore, a manual describing how to use the kit. A kit of this invention, for detecting the effect of a candidate compound on the expression level of the indicator gene or peptide of this invention, can be used for screening for a compound that modifies the expression level of the indicator gene of this invention. Test candidate compounds used in these methods include, in addition to compound preparations synthesized by known chemical methods, steroid derivatives and compound preparations synthesized by combinatorial chemistry, and mixtures of multiple compounds such as extracts from animal or plant tissues, or microbial cultures and their purified preparations.
GENERAL DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for enhancing plant defenses against pathogens. More particularly, the invention relates to enhancing plant immunity against bacterial pathogens, wherein AtMIN7 mediated protection is enhanced and/or there is a decrease in activity of an AtMIN7 associated virulence protein such as a Pseudomonas syringae pv. tomato DC3000 HopM1. Reagents of the present invention provide a means of studying cellular trafficking while formulations of the present inventions provide increased pathogen resistance in plants.
Pseudomonas syringae infects a wide range of economically important crop plant species, including but not limited to tomatoes, beans, cabbage and Brassica species. In the past two decades, P. syringae strains were used as an important model for the discovery of many fundamental mechanisms in host-pathogen interactions. Pseudomonas syringae is divided into pathovars differing in host specificity, for example, P. syringae pv. syringae (Psy) and P. syringae pv. tomato (Pto) represent particularly divergent pathovars that primarily infect beans and tomato plants, respectively, however both can cause pathogenic symptoms in Arabidopsis plants.
To render plant tissue suitable for microbial growth pathogens alter the physiology of the host. Such modifications include inhibiting anti-microbial defenses, releasing of water and/or nutrients into the apoplast, and inducing certain disease symptoms. Previous studies by others have revealed that P. syringae utilizes at least two different mechanisms to deliver virulence factors that promote these events: i) secretion of toxins into the apoplast and/or ii) direct injection of bacterial proteins into the host cell through a specialized delivery apparatus known as the Type III secretion system (TTSS).
In Arabidopsis, Pto DC3000 multiplies aggressively in leaves, in particular within spaces in between plant cells, a region referred to as “apoplast,” for about 2 days before the onset of disease symptoms. Symptoms include water soaking in the apoplast, followed by tissue necrosis and chlorosis (Whalen (1991) Plant Cell 3:49-59; Katagiri, et al. (2002) in The Arabidopsis Book, eds. Somerville and Meyerowitz, (Am. Soc. Plant Biologists, Rockville, Md.); all of which are incorporated by reference). The ability of DC3000 to infect Arabidopsis depends on TTSS as demonstrated by hrp mutants [e.g., hrpS and hrcC (formerly hrph) mutants] of DC3000 that do not multiply or cause disease in Arabidopsis plants (Yuan and He (1996) J. Bacteriol. 178:6399-6402, Roine, et al. (1997) Proc. Natl. Acad. Sci. USA 94:3459-3464; all of which are incorporated by reference). A TTSS of DC3000 is believed to secrete and/or translocate at least 30 effector proteins into the host cell (Boch, et al. (2002) Mol. Microbiol. 44:73-88; Fouts, et al. (2002) Proc. Natl. Acad. Sci. USA 99:2275-2280; Guttman, et al. (2002) Science 295:1722-1726; Petnicki-Ocwieja, et al. (2002) Proc. Natl. Acad. Sci. USA 99:7652-7657; Salanoubat, et al. (2002) Nature 415:497-502; Zwiesler-Vollick, et al. (2002) Mol. Microbiol. 45:1207-1218; all of which are herein incorporated by reference). Cumulatively, these effectors alter host cellular processes and promote disease development through unknown mechanisms.
Although the primary function of type III effectors is to promote plant susceptibility, some effectors may be recognized by the corresponding plant disease resistance proteins in resistant plants and trigger defense responses, including the hypersensitive response (HR) (Goodman and Novacky (1994) Am. Phytopathol. Soc., St. Paul); Greenberg (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:525-545; all of which are herein incorporated by reference). Further, many type III effector genes in P. syringae were discovered based on their ability to trigger a HR in resistant plants and have been named avr (for avirulence) genes (Ronald et al. (1992) J. Bacteriol. 174:1604-1611; herein incorporated by reference). For example, the type III effector, AvrPto, was identified based on its avirulence activity in plants (Ronald, et al. (1992) J. Bacteriol. 174:1604-1611; Scofield, et al. (1996) Science, 274:2063-2065; Tang, et al. (1999) Plant Cell 11:15-30; all of which are herein incorporated by reference). Although the ability of type III effectors to trigger defense responses in resistant plants is well understood, the mechanism by which type III effectors, as a group, enable plant pathogenic bacteria to proliferate in the intercellular space of a susceptible plant remains enigmatic. In addition to type III effectors, DC3000 also produces the phytotoxin coronatine (COR), which is required for full virulence in Arabidopsis (Ma, et al. (1991) Mol. Plant-Microbe Interact. 4:69-74; Mittal and Davis, (1995) Mol. Plant-Microbe Interact. 8:165-171; Bender, et al. (1999) Microbiol. Mol. Biol. Rev. 63:266-292; all of which are herein incorporated by reference).
The P. syringae strains examined during the course of studies for developing the present invention contain a common genomic pathogenicity island, which is composed of type III secretion-associated hrp/hrc genes, an exchangeable effector locus (EEL), and a conserved effector locus (CEL) (Alfano et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:4856; herein incorporated by reference). Deletion of the CEL in Pst DC3000 resulted in dramatic reduction of the bacterial population and complete elimination of disease symptoms (necrosis and chlorosis) in infected tomato and Arabidopsis plants, suggesting a particularly important role of CEL-encoded effectors in P. syringae interactions with different host plants (Alfano et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:4856; DebRoy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:9927; all of which are herein incorporated by reference). Hop (Hrp-dependent outer protein), such as HopPsyA, is one example of a protein encoded by pathogenicity island of Pseudomonas syringae that contributes to pathgenicity (For further examples, see, U.S. Pat. No. 6,852,835; herein incorporated by reference).
A virulence defect in ΔCEL mutant bacteria is caused by the deletion of the functionally redundant effector genes hopM1 (formerly hopPtoM) and avrE (DebRoy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:9927; herein incorporated by reference). pORF43 is a plasmid expressing HopM1 that with its cognate chaperone ShcM, is sufficient to fully complement the virulence defect of the ΔCEL mutant in Arabidopsis (DebRoy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:9927; herein incorporated by reference).
Pst DC3000 HopM1 is a novel 712-aa protein that lacks cysteine residues. Previous studies by the inventors showed that HopM1 is translocated into the host cell (Badel et al. Mol. Microbial. 49:1239 (2003); herein incorporated by reference). During the course of developing the present inventions, HopM1 expression was found to restore the virulence of the Pst DC3000 ΔCEL mutant in a host plant cell.
AtMIN7 encodes one of the eight members of the Arabidopsis Arf GEF protein family (Sanderfoot and Raikhel, in The Arabidopsis Book, Somerville, Meyerowitz, Eds., American Society of Plant Biologists, Rockville, Md., 2002; herein incorporated by reference), FIG. 9 and SEQ ID NOs:1 and 2. Adenosine dinucleotide (ADP) ribosylating factor (ARF) GEFs are key components of the vesicle trafficking system in eukaryotic cells and are the primary molecular targets of BrefeldinA (BFA), an inhibitor of vesicle trafficking well known in the art (Mossessova et al. (2003) Mol. Cell. 12:1403; Steinmann et al. (1999) Science 286:316; all of which are herein incorporated by reference).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for enhancing plant defenses against pathogens. More particularly, the invention relates to enhancing plant immunity against bacterial pathogens, wherein AtMIN7 mediated protection is enhanced and/or there is a decrease in activity of an AtMIN7 associated virulence protein such as a Pseudomonas syringae pv. tomato DC3000 HopM1. Reagents of the present invention provide a means of studying cellular trafficking while formulations of the present inventions provide increased pathogen resistance in plants.







TABLE 1







AtMIN proteins that are destabilized by HopMl. Further, these


proteins and not predicted to be targeted to organelles.
















Homology (number




SEQ ID


of putative gene
Putative


Name
NOs: XX
At locus
SALK lines used
family members)
function





AtMIN2
13 & 14
At1g161901
SALK_064980.56.00.x
RAD23/hHR23A
Binding to





SALK_066603.56.00.x
(3 members)
ubiquitin and







proteasome, p53







degradation,







DNA repair


AtMIN3
15 & 16
At1g18490
SALK_103109.23.60.x
Expressed protein
Not available





SALK_103215.33.55.x
(1 member)


AtMIN4
17 & 18
At2g14910
SALK_000496.38.95.x
Expressed protein
Not available





SALK_009273.19.95.x
(1 member)


AtMIN6
19 & 20
At2g47710
SALK_OI5279.54.75.X
Universal stress protein
Response to





SALK_099811.44.65.x
(USP) family protein,
stress






similar to ER6 protein






(1 member)


AtMIN7
1 & 2
At3g43300
SALK_OI2013.54.75.x
Guanine nucleotide
Guanyl-





SALK_013761.46.95.x
exchange factor (GEF)
nucleotide exchange






protein (8 members)
factor activity


AtMIN9
21 & 22
At5g64180
SALK_OI6899.19.70.x
Expressed protein
ATPbinding





SALK_092105.52.05.x
(1 member)


AtMIN10
23 & 24
At5g65430
SALK_036856.29.30.x
14-3-3 protein
Signal





SALK_092382.15.65.x
(14 members)
transduction







protein, binding







to phosphoproteins


AtMIN11
25 & 26
At5g66420
SALK_077054.31.05.x
Expressed protein
Hydrolase activity,





SALK_082859.26.60.x
(1 member)
hydrolyzing O-







glycosyl compounds,







carbohydrate metabolism,







defense response





1At1g16190: ‘At’ indicates Arabidopsis thaliana; ‘1g’ indicates that this gene is on chromosome 1.






The present invention relates to compositions and methods for increasing plant defenses against pathogens. More particularly, the invention relates to increasing plant immunity against bacterial pathogens, wherein ATMIN mediated protection is enhanced and/or there is a decrease in activity of an ATMIN associated virulence protein such as a Pseudomonas syringae pv. tomato DC3000 HopM1 virulence protein. Formulations of the present invention comprising a protective HopM1 fragment, such as HopM11-200 and HopM11-300, find use for providing plants with protection against pathogens and/or increasing pathogen resistance in plants. The present invention further relates to compositions and methods for enhancing plant defenses against pathogens, wherein ATMIN7 mediated protection is enhanced and/or there is a decrease in activity of an ATMIN7 associated virulence protein such as a Pseudomonas syringae pv. tomato DC3000 HopM1 (see, Nomura, et al., Science. 2006 Jul. 14; 313(5784):220-3, herein incorporated by reference in it's entirety).
The present invention relates to compositions and methods for increasing plant defenses against pathogens and protecting plants against pathogens, wherein HopM1 fragments mediate protection by decreasing activity of full-length HopM1, such as by providing HopM11-200 or a HopM11-300 protective fragments to a plant.
Reagents of the present invention comprising ATMIN and/or HopM1 and/or HopM1 fragments further provide methods for studying cellular trafficking.
I. ATMIN, HopM1, and Like Genes, Coding Sequences and Polypeptides.
The present invention is not limited to the use of any particular homolog or variant or mutant of an ATMIN or ATMIN-like gene or an ATMIN or ATMIN-like protein. Indeed, in some embodiments a variety of ATMIN or ATMIN-like genes or ATMIN or ATMIN-like proteins, homologs, variants and mutants may be used so long as they retain at least a portion of the activity of the corresponding wild-type protein. In particular, retaining activity that would increase resistance to a pathogen in a plant. In some embodiments, ATMIN or ATMIN-like genes and proteins encoded by the nucleic acids and amino acids of SEQ ID NOs:01-02, 03-12, and 13-36, find use in the present inventions.
In some embodiments, ATMIN7 or ATMIN7-like genes and proteins encoded by the nucleic acids and amino acids of SEQ ID NOs:01 and 02 find use in the present inventions. Accordingly, in other embodiments, nucleic acids that comprise sequences at least 57%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:02 find use in the present inventions. In other embodiments, nucleic acids encoding proteins that comprise polypeptides at least 38%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:01 find use in the present inventions. In other embodiments, the present invention provides polypeptides at least 38%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:01 find use in the present inventions. (See, Table 2).
The present invention is not limited to the use of HopM1 genes, proteins, and specific HopM1 fragments. Indeed, in some embodiments a variety of HopM1 proteins or HopM1 genes, homologs, variants and mutants may be used so long as they retain at least a portion of the activity of the corresponding wild-type HopM1 protein. Specifically, HopM1 is contemplated for use in identifying additional ATMIN or ATMIN-like genes and proteins that provide a plant with protection against pathogens. Further, HopM1 genes, homologs, variants and mutants may be used for identify control points in cellular trafficking, in particular the trafficking associated with increasing or decreasing virulence of pathogens. Accordingly, in some embodiments, HopM1 genes and proteins encoded by the nucleic acids and amino acids of SEQ ID NO:35 and 34 find use in the present inventions. In some embodiments, the present invention provides a nucleic acid at least 75%, 78%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:35. In other embodiments, nucleic acids encoding proteins that comprise polypeptides at least 510%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:34 find use in the present inventions. In other embodiments, the present invention provides polypeptides at least 51%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:34 find use in the present inventions. (See, Table 3).
The present invention also provides HopM1 protective fragments for protecting plants against pathogens. Furthermore, the present invention is not limited to a homolog or variant or mutant of a HopM1 protective fragment, such as a HopM11-300 and HopM11-200 protective fragments provided by SEQ ID NO:94 and polypeptide sequences comprising SEQ ID NO:82. In other embodiments, nucleic acids comprising sequences at least 74%, 79%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:94 find use in the present inventions. In other embodiments, polypeptides at least 46%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:82 find use in the present inventions. In other embodiments, the present invention provides a nucleic acid at least 46%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to any of SEQ ID NO:82 find use in the present inventions. Further, the present inventions provide homologs of HopM11-300 (See, Table 4). Functional variants can be screened by expressing the variant in an appropriate vector (described in more detail below) in a host cell, such as a yeast cell, plant cell, bacterium, and then analyzing the host cell's response to a pathogen or a virulence protein of a pathogen (e.g. Pseudomonas spp., full-length HopM1, etc.).
Further, the nucleic acid sequences and polypeptides of the present inventions provide compositions and methods for altering vesicular trafficking in a cell. In particular, the methods are used for identifying proteins and are contemplated for use in identifying protein binding domains that alter trafficking in a cell, such as proteins that alter trafficking of proteins produced by pathogens, for example, virulence proteins.
A. Nucleic Acid Sequences and Polypeptides:
1. ATMIN and HopM1 Genes:
The present invention provides plant ATMIN or ATMIN-like genes and proteins, including their homologs, orthologs, paralogs, variants and mutants. In some embodiments of the present invention, isolated nucleic acid sequences comprising ATMIN or ATMIN-like genes are provided. Mutations in these genes are contemplated that would alter the encoded ATMIN or ATMIN like proteins to provide increased resistance to pathogen infections. In some embodiments, isolated nucleic acid sequences comprising ATMIN7 or ATMIN7-like are provided. These sequences include sequences comprising ATMIN7 or ATMIN7-like and cDNA/genomic sequences, for example, SEQ ID NOs:2, 4, 6, 8, 10, and 12. In some embodiments of the present invention provide nucleic acid sequences that encode polypeptides that are homologous to at least one of SEQ ID NOs:1, 3, 5, 7, 9, and 11.
The present invention provides HopM1 or HopM1-like genes and polypeptides and fragments thereof, including their homologs, orthologs, paralogs, variants and mutants. In some embodiments of the present invention, isolated nucleic acid sequences comprising HopM1 or HopM1-like genes are provided. Mutations in these genes are contemplated that would alter the encoded HopM1 or HopM1-like proteins to provide increased resistance to pathogen infections. These sequences include sequences comprising HopM1 or HopM1-like and cDNA sequences, for example, SEQ ID NOs:35, 37, and 39. In some embodiments of the present invention provide nucleic acid sequences that encode polypeptides that are homologous to at least one of SEQ ID NOs:34, 36, and 38.
2. Additional ATMIN and ATMIN-Like Genes:
The present invention provides nucleic acid sequences comprising additional ATMIN, HopM1 and -like genes. For example, some embodiments of the present invention provide nucleic acid sequences that encode polypeptides that are homologous to at least one of SEQ ID NOs:01. In some embodiments, the polypeptides are at least 38%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% (or more) identical to SEQ ID NO:01. For example, some embodiments of the present invention provide nucleic acid sequences that encode polypeptides set forth in any one of SEQ ID NOs: 3, 5, 7, 9, 11 and 13, 15, 17, 19, 21, 23, 25, 27, and 28-33. For example, some embodiments of the present invention provide nucleic acid sequences that encode polypeptides that are homologous to at least one of SEQ ID NOs: 3, 5, 7, 9, 11 and 13, 15, 17, 19, 21, 23, 25, 27, and 28-33.
In other embodiments, the present invention provides nucleic acid sequences that hybridize under conditions ranging from low to high stringency to at least one of SEQ ID NO: 02, as long as the polynucleotide sequence capable of hybridizing to at least one of SEQ ID NOs: 02, 4, 6, 8, 10, 12, and 14, 16, 18, 20, 22, 24, and 26 encodes a protein that retains a desired biological activity of a protective pathogen response protein. In some preferred embodiments, the hybridization conditions are high stringency. In preferred embodiments, hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex and confer a defined “stringency” as explained above (See e.g., Wahl et al., (1987) Meth. Enzymol., 152:399-407; incorporated herein by reference). In other embodiments of the present invention, alleles of pathogen resistance genes, and in particular of an exogenous ATMIN or ATMIN-like gene, such as ATMIN7 or ATMIN7-like, or a protective fragment of a virulence protein, such as a protective HopM1 fragment (for example, HopM11-300), homologs or mutants or variants thereof, are provided. In preferred embodiments, alleles result from a mutation, (i.e., a change in the nucleic acid sequence) and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes that give rise to alleles are generally ascribed to deletions, additions, or insertions, or substitutions of nucleic acids. Each of these types of changes may occur alone, or in combination with the others, and at the rate of one or more times in a given sequence. Mutational changes in alleles also include rearrangements, insertions, deletions, additions, or substitutions in upstream regulatory regions.
In other embodiments of the present invention, the polynucleotide sequence encoding an exogenous ATMIN or ATMIN-like gene, such as ATMIN7 or ATMIN7-like, homologs or mutants or variants thereof, is extended utilizing the nucleotide sequences (e.g., SEQ ID NO:02) in various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, it is contemplated that for an exogenous ATMIN or ATMIN-like gene, such as ATMIN7 or ATMIN7-like, homologs or mutants or variants thereof, including related ATMIN OR ATMIN-like genes, the sequences upstream of the start site or downstream from the poly A tail can be identified using information in databases containing plant genomic information such as The Institute for Genomic Research (TIGR), Plant Functional Genomics Projects, Plant Gene Indices for rice, tomato, rape, wheat, barley, rye, maize, sorghum, soybean, potato, cotton, etc. (http://www.tigr.org/tdb/tgi/plant.shtml); GrainGenes for wheat, barley, rye, triticale, and oats (http://wheat.pw.usda.gov/QueryDB.shtml); Gramene: A Comparative Mapping Resource for Grains (http://www.gramene.org); rice (http://rgp.dna.affrc.gojp/), maize (MaizeGDB http://www.maizegdb.org/); barley (http://hordeum.oscs.montana.-edu/), soybean (http://stadler.agron.iastate.edulblast/blast.html); Arabidopsis (http://www.arabidopsis.org/) databases; and United Kingdom Crop Plant Bioinformatics Network (UK CropNet) at ukcrop.net/db.html; all of which are herein incorporated by reference.
An example of such a method for extending coding region information using a RACE PCR method is described herein for the identification of ATMIN or ATMIN-like segments upstream and downstream of the originally cloned segment. For ATMIN or ATMIN-like specific information, such as ATMIN7 or ATMIN7-like, (SEQ ID NO:4, 6, 8, 10, 12, and the like, or mutants or variants thereof, for which public genomic or expressed information is not available, or not complete, it is contemplated that polymerase chain reaction (PCR) methods in addition to RACE finds use in the present invention. In another embodiment, inverse PCR is used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., (1988) Nucleic Acids Res., 16:8186, herein incorporated by reference). In yet another embodiment of the present invention, capture PCR (Lagerstrom et al., PCR Methods Applic., 1:111-19 (1991), herein incorporated by reference) is contemplated for use in obtaining additional sequences. In still other embodiments, walking PCR is contemplated for use in obtaining additional sequences. Walking PCR is a method for targeted gene walking that permits retrieval of unknown sequence (Parker et al., Nucleic Acids Res., 19:3055-60 (1991), herein incorporated by reference). The PROMOTERFINDER kit (Clontech) uses PCR, nested primers and special libraries to “walk in” genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions. In yet other embodiments of the present invention, add TAIL PCR is used as a preferred method for obtaining flanking genomic regions, including regulatory regions (Liu and Whittier, Genomics, 25(3):674-81 (1995); Liu et al., Plant J., 8(3):457-63 (1995); all of which are herein incorporated by reference). Preferred libraries for screening for full-length cDNAs include libraries that have been size-selected to include larger cDNAs. Also, random primed libraries are preferred, in that they contain more sequences that contain the 5′ and upstream gene regions. A randomly primed library may be particularly useful in cases where an oligo d(T) library does not yield full-length cDNA. Genomic Libraries are useful for obtaining introns and extending 5′ sequence.
3. Variant ATMIN or ATMIN-Like Genes:
In some embodiments, the present invention provides isolated variants of the disclosed nucleic acid sequences encoding an ATMIN or ATMIN-like gene, such as ATMIN or ATMIN-like, or a protective fragment of a virulence protein, such as a protective HopM1 fragment (for example, HopM11-300), homologs or mutants or variants thereof. These variants include mutants, fragments, fusion proteins or functional equivalents of genes and gene protein products.
a. Mutants:
Some embodiments of the present invention contemplate compositions comprising and/or using nucleic acid sequences encoding mutant forms of ATMIN or ATMIN-like gene, such as ATMIN or ATMIN-like, or a protective fragment of a virulence protein, such as a protective HopM1 fragment (for example, HopM11-300), homologs or mutants or variants thereof, (i.e., mutants), and the polypeptides encoded thereby. In preferred embodiments, mutants result from mutation of the coding sequence (i.e., a change in the nucleic acid sequence) and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many variant forms. Common mutational changes that give rise to variants are generally ascribed to deletions, additions or substitutions of nucleic acids. Each of these types of changes may occur alone, or in combination with the others, and at the rate of one or more times in a given sequence.
Mutants of an ATMIN or ATMIN-like gene, such as ATMIN7 or ATMIN7-like, or a protective fragment of a virulence protein, such as a protective HopM1 fragment (for example, HopM11-300), homologs or mutants or variants thereof, can be generated by any suitable method well known in the art, including but not limited to EMS induced mutagenesis, site-directed mutagenesis, randomized “point” mutagenesis, and domain-swap mutagenesis. An example of domain-swap mutagenesis is contemplated in which portions of the ATMIN or ATMIN-like cDNA are “swapped” with the analogous portion of other ATMIN or ATMIN-like-encoding cDNAs such as used for identifying functional regions for pathogen resistance. Another example of domain-swap mutagenesis is contemplated in which portions of the mutants of a HopM1 or HopM1 fragment cDNA are “swapped” with the analogous portion of other HopM1 or HopM1 fragment-encoding cDNAs such as used for identifying functional regions for pathogen virulence or resistance. It is contemplated that is possible to modify the structure of a peptide having a protective activity (e.g., such as a HopM11-300 activity), for such purposes as increasing synthetic activity or altering the affinity of the ATMIN or ATMIN-like protein or protective peptide, HopM11-300, for a binding partner or a kinetic activity. Such modified peptides are considered functional equivalents of peptides having an activity of an ATMIN or ATMIN-like activity or HopM11-300 activity as defined herein. A modified peptide can be produced in which the nucleotide sequence encoding the polypeptide has been altered, such as by substitution, deletion, or addition. In some preferred embodiments of the present invention, the alteration increases or decreases the effectiveness of the ATMIN or ATMIN-like or HopM1 fragment gene product to exhibit a phenotype caused by altered responses of pathogen resistance genes and/or pathogen virulence genes and encoded proteins. In other words, construct “X” can be evaluated in order to determine whether it is a member of the genus of modified or variant ATMIN or ATMIN-like gene or HopM1 protective fragments of the present invention as defined functionally, rather than structurally. Accordingly, in some embodiments the present invention provides nucleic acids encoding a polypeptide comprising ATMIN or ATMIN-like binding domain sequence or a HopM11-300 binding domain sequence that can complement the polypeptides encoded by any one of SEQ ID NOs:01, and 82, as well as the polypeptides encoded by such nucleic acids.
Moreover, as described above, mutant forms of ATMIN or ATMIN-like proteins are also contemplated as being equivalent to those peptides that are set forth in more detail herein. For example, it is contemplated that isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. On the other hand, mutant forms of ATMIN or ATMIN-like proteins are contemplated as providing superior resistance to pathogens by affecting the biological activity of the resulting molecule, such that the altered biological activity increases pathogen resistance of a plant. It is contemplated that inhibiting the degradation rate of an ATMIN or ATMIN-like protein will increase pathogen resistance to the pathogen expressing a virulence protein that targets the wild-type ATMIN or ATMIN-like protein.
Accordingly, some embodiments of the present invention provide nucleic acids comprising sequences encoding variants of ATMIN or ATMIN-like gene products containing conservative replacements, as well as the amino acids of the proteins encoded by such nucleic acids. Such replacements are described herein. Whether a change in the amino acid sequence of a peptide results in a functional homolog can be readily determined by assessing the ability of the variant peptide to function in a fashion similar to the wild-type protein. Peptides having more than one replacement can readily be tested in the same manner.
b. Homologs:
In some embodiments, the present invention provides isolated variants of the disclosed nucleic acid sequence encoding a ATMIN or ATMIN-like gene, such as ATMIN7 or ATMIN7-like, or a protective fragment of a virulence protein, such as a protective HopM1 fragment (for example, HopM11-300), homologs or mutants or variants thereof, and the polypeptides encoded thereby; these variants include mutants, fragments, fusion proteins or functional equivalents genes and protein products.
Some homologs of encoded ATMIN or ATMIN-like gene, such as ATMIN or ATMIN-like, or a protective fragment of a virulence protein, such as a protective HopM1 fragment (for example, HopM11-300), homologs or mutants or variants thereof, have intracellular half-lives dramatically different than the corresponding wild-type protein. For example, the altered protein is rendered either more stable or less stable to proteolytic degradation or other cellular process that result in destruction of, or otherwise inactivate the encoded ATMIN or ATMIN-like or HopM1 product. Such homologs, and the genes that encode them, can be utilized to alter the activity of the encoded ATMIN or ATMIN-like or HopM1 products by modulating the half-life of the protein. For instance, a longer half-life may give rise to enhanced ATMIN or ATMIN-like biological effects. Other homologs have characteristics which are either similar to wild-type ATMIN or ATMIN-like or HopM1, or which differ in one or more respects from wild-type ATMIN or ATMIN-like or HopM1.
In some embodiments the combinatorial mutagenesis approach are contemplated for the present invention, the amino acid sequences for a population of ATMIN or ATMIN-like or HopM1 gene product homologs are aligned, preferably to promote the highest homology possible. Such a population of variants can include, for example, ATMIN or ATMIN-like or HopM1 gene homologs from one or more species or ATMIN or ATMIN-like or HopM1 gene homologs from the same species but which differ due to mutation. Amino acids that appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.
In a preferred embodiment of the present invention, the combinatorial ATMIN or ATMIN-like or HopM1 gene library is produced by way of a degenerate library of genes encoding a library of polypeptides that each include at least a portion of candidate encoded ATMIN or ATMIN-like or HopM1 protein sequence. For example, a mixture of synthetic oligonucleotides is enzymatically ligated into gene sequences such that the degenerate set of candidate ATMIN or ATMIN-like or HopM1 sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of ATMIN or ATMIN-like or HopM1 sequences therein.
There are many ways by which the library of potential ATMIN or ATMIN-like or HopM1 homologs can be generated from a degenerate oligonucleotide sequence. In some embodiments, chemical synthesis of a degenerate gene sequence is carried out in an automatic DNA synthesizer, and the synthetic genes are ligated into an appropriate gene for expression. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential ATMIN or ATMIN-like or HopM1 sequences or any combination of ATMIN or ATMIN-like sequences and ATMIN or ATMIN-like or HopM1 sequences. The synthesis of degenerate oligonucleotides is well known in the art (see e.g., Narang, Tetrahedron Lett., 39:3 9 (1983); Itakura et al., Recombinant DNA, in Walton (ed.), Proceedings of the 3rd Cleveland Symposium on Macromolecules, Elsevier, Amsterdam, pp 273-289 (1981); Itakura et al., Annu. Rev. Biochem., 53:323 (1984); Itakura et al., Science 198:1056 (1984); Ike et al., Nucl. Acid Res., 11:477 (1983); all of which are herein incorporated by reference). Such techniques have been employed in the directed evolution of other proteins (see e.g., Scott et al., Science, 249:386-390 (1980); Roberts et al., Proc. Natl. Acad. Sci. USA, 89:2429-2433 (1992); Devlin et al., Science, 249: 404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA, 87: 6378-6382 (1990); as well as U.S. Pat. Nos. 5,223,409; 5,198,346; and 5,096,815; all of which are herein incorporated by reference).
c. Directed Evolution:
Variants of ATMIN or ATMIN-like or HopM1 genes or coding sequences may be produced by methods such as directed evolution or other techniques for producing combinatorial libraries of variants. Thus, the present invention further contemplates a method of generating sets of nucleic acids that encode combinatorial mutants of the ATMIN or ATMIN-like or HopM1 proteins, as well as truncation mutants, and is especially useful for identifying potential variant sequences (i.e., homologs) that possess the biological activity of the encoded ATMIN or ATMIN-like or HopM1 proteins. In addition, screening such combinatorial libraries is used to generate, for example, novel encoded ATMIN or ATMIN-like gene product homologs that possess novel binding or other kinetic specificities or other biological activities. The invention further provides sets of nucleic acids generated as described above, where a set of nucleic acids encodes combinatorial mutants of the ATMIN or ATMIN-like or HopM1 proteins, or truncation mutants, as well as sets of the encoded proteins. The invention further provides any subset of such nucleic acids or proteins, where the subsets comprise at least two nucleic acids or at least two proteins.
It is contemplated that ATMIN or ATMIN-like gene, such as ATMIN7 or ATMIN7-like, or a protective fragment of a virulence protein, such as a protective HopM1 fragment (for example, HopM11-300), homologs or mutants or variants thereof, can be utilized as starting nucleic acids for directed evolution. These techniques can be utilized to develop encoded ATMIN or ATMIN-like or HopM1 product variants having desirable properties such as increased kinetic activity or altered binding affinity.
In some embodiments, artificial evolution is performed by random mutagenesis (e.g., by utilizing error-prone PCR to introduce random mutations into a given coding sequence). This method requires that the frequency of mutation be finely tuned. As a general rule, beneficial mutations are rare, while deleterious mutations are common. This is because the combination of a deleterious mutation and a beneficial mutation often results in an inactive enzyme. The ideal number of base substitutions for targeted gene is usually between 1.5 and 5 (Moore and Arnold, Nat. Biotech., 14, 458-67 (1996); Leung et al., Technique, 1:11-15 (1989); Eckert and Kunkel, PCR Methods Appln., 1:17-24 (1991); Caldwell and Joyce, PCR Methods Appln., 2:28-33 (1992); and Zhao and Arnold, Nuc. Acids. Res., 25:1307-08 (1997), all of which are herein incorporated by reference).
After mutagenesis, the resulting clones are selected for desirable activity (e.g., screened for abolishing or restoring hydroxylase activity in a constitutive mutant, in a wild type background where hydroxylase activity is required, as described above and below). Successive rounds of mutagenesis and selection are often necessary to develop enzymes with desirable properties. It should be noted that only the useful mutations are carried over to the next round of mutagenesis.
In other embodiments of the present invention, the polynucleotides of the present invention are used in gene shuffling or special PCR procedures (e.g., Smith, Nature, 370:324-25 (1994); U.S. Pat. Nos. 5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are herein incorporated by reference). Gene shuffling involves random fragmentation of several mutant DNAs followed by their reassembly by PCR into full-length molecules. Examples of various gene shuffling procedures include, but are not limited to, assembly following DNase treatment, the staggered extension process (STEP), and random priming in vitro recombination.
d. Screening Gene Products:
A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations, and for screening cDNA libraries for gene products having a certain property. Such techniques are generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ATMIN or ATMIN-like or HopM1 and/or ATMIN or ATMIN-like homologs, paralogs, and orthologs, and further for pathogen virulence proteins, such as HopM1 and/or HopM1 homologs, paralogs, and orthologs. The most widely used techniques for screening large gene libraries typically comprise cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
Each of the illustrative assays described below are amenable to high throughput analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques. Accordingly, in some embodiments of the present invention, the gene library is cloned into the gene for a surface membrane protein of a bacterial cell, (wherein the bacterial cell does not produce an endogenous virulence protein) and the resulting fusion protein detected by panning (WO 88/06630; Fuchs et al., (1991) BioTechnol., 9:1370-1371; and Goward et al., (1992) TIBS 18:136-140; all of which are herein incorporated by reference). In other embodiments of the present invention, fluorescently labeled molecules that bind encoded ATMIN or ATMIN-like or HopM1 products can be used to score for potentially functional ATMIN or ATMIN-like or HopM1 or HopM11-300 homologs, paralogs, and orthologs. Cells are visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, separated by a fluorescence-activated cell sorter.
In an alternate embodiment of the present invention, the gene library is expressed as a fusion protein on the surface of a viral particle. For example, foreign peptide sequences are expressed on the surface of infectious phage in the filamentous phage system, thereby conferring two significant benefits. First, since these phages can be applied to affinity matrices at very high concentrations, a large number of phage can be screened at one time. Second, since each infectious phage displays the combinatorial gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd, and fl are most often used in phage display libraries, as either of the phage gIII or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (See e.g., WO 90/02909; WO 92/09690; Marks et al., (1992) J. Biol. Chem., 267:16007-16010; Griffths et al., (1993) EMBO J., 12:725-734; Clackson et al., (1991) Nature 352:624-628; and Barbas et al., (1992) Proc. Natl. Acad. Sci., 89:4457-4461; all of which are herein incorporated by reference).
In another embodiment of the present invention, the recombinant phage antibody system (e.g., RPAS, Pharmacia Catalog number 27-9400-01) is modified for use in expressing and screening of encoded ATMIN or ATMIN-like and/or HopM1, and/or HopM1 protective fragment, homolog, paralog, and ortholog product combinatorial libraries. The pCANTAB 5 phagemid of the RPAS kit contains the gene that encodes the phage gIII coat protein. In some embodiments of the present invention, the ATMIN or ATMIN-like and/or HopM1, and/or HopM1 protective fragment, combinatorial gene library is cloned into the phagemid adjacent to the gIII signal sequence such that it is expressed as a gIII fusion protein. In other embodiments of the present invention, the phagemid is used to transform competent E. coli TG1 cells after ligation. In still other embodiments of the present invention, transformed cells are subsequently infected with M13KO7 helper phage to rescue the phagemid and its candidate ATMIN or ATMIN-like gene insert. The resulting recombinant phage containing phagemid DNA encoding a specific candidate ATMIN or ATMIN-like protein and display one or more copies of the corresponding fusion coat protein. In some embodiments of the present invention, the phage-displayed candidate proteins that display any property characteristic of an ATMIN or ATMIN-like or HopM1 or HopM11-300 protein are selected or enriched by panning. The bound phage is then isolated, and if the recombinant phages express at least one copy of the wild type gIII coat protein, they will retain their ability to infect E. coli. Thus, successive rounds of reinfection of E. coli and panning will greatly enrich ATMIN or ATMIN-like or HopM1 or HopM11-300 homologs, paralogs, and orthologs.
In light of the present disclosure, other forms of mutagenesis generally applicable will be apparent to those skilled in the art in addition to the aforementioned rational mutagenesis based on conserved versus non-conserved residues. For example, ATMIN or ATMIN-like or HopM1 or HopM11-300 homologs can be generated and screened using, for example, using alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochem, 33:1565-1572; Wang et al., (1994) J. Biol Chem, 269:3095-3099; Balint (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem., 218:597-601; Nagashima et al., (1993) J. Biol. Chem., 268:2888-2892; Lowman et al., (1991) Biochem, 30:10832-10838; and Cunningham et al., (1989) Science, 244:1081-1085; all of which are herein incorporated by reference), by linker scanning mutagenesis (Gustin et al., (1993) Virol., 193:653-660; Brown et al., (1992) Mol. Cell. Biol., 12:2644-2652; McKnight and Kingsbury (1982) Science, 217(4557):316-24; all of which are herein incorporated by reference), or by saturation mutagenesis (Myers et al., (1986) Science, 232(4750):613-618; herein incorporated by reference).
In some preferred embodiments, the ability of the ATMIN or ATMIN-like or HopM1 or HopM11-300 sequence to bind to its response element is tested in vitro. In some preferred embodiments, the ability of the ATMIN or ATMIN-like or HopM1 or HopM11-300 sequence to bind to its response element is tested in vivo. A response element of an ATMIN or ATMIN-like sequence may be a pathogen protein binding domain and/or an endogenous cellular protein domain. A response element of a HopM1 and/or HopM1 fragment sequence may be an endogenous cellular protein domain.
e. Truncation Mutants of HopM1 Proteins and/or ATMIN or ATMIN-Like Proteins:
In addition, the present invention provides isolated nucleic acid sequences encoding truncated fragments of encoded HopM1 polypeptides and contemplated truncated fragments of ATMIN or ATMIN-like genes (i.e., truncation mutants) and the polypeptides encoded by such truncated nucleic acid sequences. In preferred embodiments, the HopM1 and/or ATMIN or ATMIN-like fragment is biologically active.
In some embodiments of the present invention, when expression of a portion of a HopM1 and/or ATMIN or -like protein is desired, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al., J. Bacteriol., 169:751-757 (1987), herein incorporated by reference) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al., (1990) Proc. Natl. Acad. Sci. USA, 84:2718-1722, herein incorporated by reference). Therefore, removal of an N-terminal methionine, if desired, can be achieved either in vivo by expressing such recombinant polypeptides in a host that produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP.
f. Fusion Proteins Containing HopM1 Proteins and/or ATMIN or ATMIN-Like Proteins:
The present invention also provides nucleic acid sequences encoding fusion proteins incorporating all or part of ATMIN or ATMIN-like and/or HopM1 or HopM11-300, and the polypeptides encoded by such nucleic acid sequences. In some embodiments of the present invention, chimeric constructs code for fusion proteins containing a portion of an ATMIN or ATMIN-like and/or HopM1 or HopM11-300 protein and a portion of another gene. In some embodiments, the fusion proteins have biological activity similar to the wild type ATMIN or ATMIN-like and/or HopM1 or HopM11-300 (e.g., have at least one desired biological activity of the protein). In other embodiments, the fusion protein has altered biological activity. In addition to utilizing fusion proteins to alter biological activity, it is widely appreciated that fusion proteins can also facilitate the expression and/or purification of proteins, such as the ATMIN or ATMIN-like and/or HopM1 or HopM11-300 protein of the present invention. Accordingly, in some embodiments of the present invention, it is contemplated that an ATMIN or ATMIN-like and/or HopM1 or HopM11-300 protein is generated as a glutathione-S-transferase (i.e., GST fusion protein). It is also contemplated that such a GST fusion proteins would enable easy purification of the ATMIN or ATMIN-like and/or HopM1 or HopM11-300 protein, such as by the use of glutathione-derivatized matrices (See e.g., Ausabel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1991), herein incorporated by reference).
In some embodiments, the fusion proteins have an ATMIN or ATMIN-like and/or HopM1 or HopM11-300 functional domain with a fusion partner. Accordingly, in some embodiments of the present invention, the coding sequences for the polypeptide (e.g., an ATMIN or ATMIN-like functional domain) are incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. It is contemplated that such a single fusion product polypeptide is able to provide a transgenic plant with an increased resistance to pathogen infections.
In another embodiment of the present invention, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of an ATMIN or ATMIN-like and/or HopM1 or HopM11-300 protein allows purification of the expressed ATMIN or ATMIN-like and/or HopM1 or HopM11-300 fusion protein by affinity chromatography using a Ni2+ metal resin. In still another embodiment of the present invention, the purification leader sequence is then subsequently removed by treatment with enterokinase (See e.g., Hochuli et al., (1987) J. Chromatogr., 411:177; and Janknecht et al., (1991) Proc. Natl. Acad. Sci. USA, 88:8972; all of which are herein incorporated by reference). In yet other embodiments of the present invention, a fusion gene coding for a purification sequence appended to either the N or the C terminus allows for affinity purification; one example is addition of a hexahistidine tag to the carboxy terminus of an ATMIN or ATMIN-like and/or HopM1 or HopM11-300 protein that is optimal for affinity purification, see EXAMPLES for a description and use of a 6× Histidine tagged protein.
Techniques for making fusion genes are well known. Essentially, the joining of various nucleic acid fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment of the present invention, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, in other embodiments of the present invention, PCR amplification of gene fragments is carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed to generate a chimeric gene sequence (See, e.g., Current Protocols in Molecular Biology, supra, herein incorporated by reference).
B. Encoded ATMIN or ATMIN-Like and/or HopM1 or HopM11-300 Gene Polypeptides:
The present invention provides isolated ATMIN or ATMIN-like and/or HopM1 or HopM11-300 polypeptides, as well as variants, homologs, mutants or fusion proteins thereof, as described above. In some embodiments of the present invention, the polypeptide is a naturally purified product, while in other embodiments it is a product of chemical synthetic procedures, and in still other embodiments it is produced by recombinant techniques using a prokaryotic or eukaryotic host (e.g., by bacterial, yeast, higher plant, insect and mammalian cells in culture). In some embodiments, depending upon the host employed in a recombinant production procedure, the polypeptide of the present invention is glycosylated or non-glycosylated. In other embodiments, the polypeptides of the invention also include an initial methionine amino acid residue.
1. Purification of ATMIN, Hop M1 and HopM11-300 Polypeptides:
The present invention provides or contemplates purified ATMIN, Hop M1 and HopM11-300, and/or homologs thereof, polypeptides as well as variants, homologs, mutants or fusion proteins thereof, as described above. In some embodiments of the present invention, HopM11-300 and/or HopM11-300-like polypeptides purified from recombinant organisms are provided. In other embodiments, HopM11-300 and/or HopM11-300-like polypeptides purified from recombinant bacterial extracts transformed with Pseudomonas HopM1 and/or HopM11-300-like cDNA, and in particular any one or more of HopM11-300, and/or HopM11-300-like and or related HopM11-300, are provided.
The present invention also contemplates methods for recovering and purifying ATMIN, Hop M1 and HopM11-300, and/or homologs thereof, from recombinant cell cultures including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
The present invention further provides nucleic acid sequences having the coding sequence (or a portion of the coding sequence) for a ATMIN or ATMIN-like and/or HopM1 protein (including a fragment) and/or HopM11-300-like protein fused in frame to a marker sequence that allows for expression alone or for both expression and purification of the polypeptide of the present invention. A non-limiting example of a marker sequence is a hexahistidine (6×HIS) tag that is supplied by a vector, for example, a pQE-30 vector which adds a hexahistidine nucleotide tag to the N terminal of an ATMIN or ATMIN-like gene and/or HopM1 or HopM1 fragment gene which results in expression of the polypeptide with a 6×HIS tag, or, for another example, the marker sequence is a hemagglutinin (HA) tag. A HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37:767 (1984), herein incorporated by reference). See, Examples, for 6×HIS and HA tags.
2. Chemical Synthesis of HopM11-300 and/or HopM11-300-Like Nucleotide Sequences and Polypeptides:
In an alternate embodiment of the invention, a coding sequence of protective fragments, such as HopM11-300 and HopM11-200, genes and/or HopM11-300-like genes (see, examples in Table 4), are synthesized, in whole or in part, using chemical methods well known in the art (See, e.g., Caruthers et al., (1980) Nucleic Acids Symp Ser., 7:215-223; Crea and Horn, (1980) Nucl. Acids Res., 8(10):2331-2348; Matteucci and Caruthers, (1980) Tetrahedron Lett., 21:719; and Chow et al., (1981) Nucl. Acids Res., 10(21):6695-714, all of which are herein incorporated by reference). In other embodiments of the present invention, the protein itself is produced using chemical methods to synthesize an entire HopM11-300 or HopM11-200 and/or HopM11-300-like amino acid sequence (for examples, SEQ ID NOs:82, 105, 106, and 108) or a variant thereof. For example, peptides are synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (See e.g., Creighton, Proteins Structures And Molecular Principles, W.H. Freeman and Co, New York N.Y. (1983), herein incorporated by reference). In other embodiments of the present invention, the composition of the synthetic peptides is confirmed by amino acid analysis or sequencing (See e.g., Creighton, supra, herein incorporated by reference).
Direct peptide synthesis can be performed using various solid-phase techniques (Roberge et al., (1995) Science, 269:202-204, herein incorporated by reference) and automated synthesis may be achieved, for example, using ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequence of HopM11-300 and/or HopM11-300-like, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with other sequences to produce a variant polypeptide.
3. Generation of ATMIN or ATMIN-Like and/or HopM1 and/or HopM1 Fragment Antibodies:
In some embodiments of the present invention, antibodies are generated to allow for the detection and characterization of an ATMIN or ATMIN-like and/or HopM1 and/or HopM1 fragment proteins. The antibodies may be prepared using various immunogens. In one embodiment, the immunogen is an Arabidopsis ATMIN or ATMIN-like peptide (e.g., an amino acid sequence as depicted in SEQ ID NOs:01, or HopM1 or a fragment thereof, such as HopM11-300 SEQ ID NOs:82, to generate antibodies that recognize an ATMIN or ATMIN-like and/or a HopM1 protein. Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fab expression libraries.
Various procedures known in the art may be used for the production of polyclonal antibodies directed against an ATMIN or ATMIN-like or HopM1 protein. For the production of antibody, various host animals can be immunized by injection with the peptide corresponding to the ATMIN or ATMIN-like or HopM1 protein epitope including but not limited to rabbits, mice, rats, sheep, goats, et cetera. In a preferred embodiment, the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface-active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum).
For preparation of monoclonal antibodies directed toward an ATMIN or ATMIN-like protein and/or HopM1 or HopM1-like protein, it is contemplated that any technique that provides for the production of antibody molecules by continuous cell lines in culture finds use with the present invention (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., herein incorporated by reference). These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Kohler and Milstein, (1975) Nature, 256:495-497, herein incorporated by reference), as well as the trioma technique, the human B-cell hybridoma technique (See e.g., Kozbor et al., (1983) Immunol Today, 4:72, herein incorporated by reference), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp: 77-96, herein incorporated by reference).
In an additional embodiment of the invention, monoclonal antibodies are produced in germ-free animals utilizing technology such as that described in PCT/US90/02545). Furthermore, it is contemplated that plant tissue antibodies may be generated (e.g. Canas and Malmberg, (1992) Plant Sci 83:195-203, herein incorporated by reference) or by producing plant protein specific monoclonal antibodies by using mouse hybridomas (Lund et al., (1998) Plant Physiol 116:1097-1110, herein incorporated by reference). In addition, it is contemplated that techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778, herein incorporated by reference) find use in producing an ATMIN or ATMIN-like and/or HopM1 or HopM1-like protein-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., (1989) Science, 246:1275-1281, herein incorporated by reference) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for an ATMIN or ATMIN-like and/or HopM1 or HopM1-like protein.
It is contemplated that any technique suitable for producing antibody fragments finds use in generating antibody fragments that contain the idiotype (antigen binding region) of the antibody molecule. For example, such fragments include but are not limited to: F(ab′)2 fragment that can be produced by pepsin digestion of the antibody molecule; Fab′ fragments that can be generated by reducing the disulfide bridges of the F(ab′)2 fragment, and Fab fragments that can be generated by treating the antibody molecule with papain and a reducing agent.
In the production of antibodies, it is contemplated that screening for the desired antibody is accomplished by techniques known in the art (e.g., radioimmunoassay), ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, et cetera.
In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. As is well known in the art, the immunogenic peptide should be provided free of the carrier molecule used in any immunization protocol. For example, if the peptide was conjugated to KLH, it may be conjugated to BSA, or used directly, in a screening assay. In some embodiments of the present invention, the foregoing antibodies are used in methods known in the art relating to the expression of an ATMIN or ATMIN-like protein (e.g., for Western blotting), measuring levels thereof in appropriate biological samples, etc. The antibodies can be used to detect an ATMIN or ATMIN-like and/or HopM1 or HopM1-like protein in a biological sample from a plant. The biological sample can be an extract of a tissue, or a sample fixed for microscopic examination.
The biological samples are then tested directly for the presence of an ATMIN or ATMIN-like or HopM1 protein or HopM1 fragment using an appropriate strategy (e.g., ELISA or radioimmunoassay) and format (e.g., microwells, dipstick (e.g., as described in WO 93/03367 herein incorporated by reference)), etc. Alternatively, proteins in the sample can be size separated (e.g., by polyacrylamide gel electrophoresis (PAGE), in the presence or not of sodium dodecyl sulfate (SDS), and the presence of an ATMIN or ATMIN-like or HopM1 polypeptide detected by immunoblotting (Western blotting). Immunoblotting techniques are generally more effective with antibodies generated against a peptide corresponding to an epitope of a protein, and hence, are particularly suited to the present invention.
C. Expression of Cloned ATMIN or ATMIN-Like or HopM1:
In some embodiments, genes described above may be used to generate recombinant DNA molecules that direct the expression of the encoded protein product in appropriate host cells. As will be understood by those of skill in the art, it may be advantageous to produce ATMIN or ATMIN-like or HopM1-encoding nucleotide sequences possessing non-naturally occurring codons. Therefore, in some preferred embodiments, codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., (1989) Nucl. Acids Res., 17(2):477-498, herein incorporated by reference) can be selected, for example, to increase the rate of ATMIN or ATMIN-like or HopM1 expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
1. Vectors for Production of ATMIN or ATMIN-Like or HopM1:
The nucleic acid sequences of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the nucleic acid sequence may be included in any one of a variety of expression vectors for expressing a polypeptide.
In some embodiments of the present invention, vectors include, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences (e.g., derivatives of plant tumor sequences, T-DNA sequences, derivatives of SV40, bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies). It is contemplated that any vector may be used as long as it is replicable and viable in the host.
In particular, some embodiments of the present invention provide recombinant constructs comprising one or more of the nucleic sequences as broadly described above. In some embodiments of the present invention, the constructs comprise a vector, such as a plasmid or eukaryotic vector, or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In preferred embodiments of the present invention, the appropriate nucleic acid sequence is inserted into the vector using any of a variety of procedures. In general, the nucleic acid sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art.
Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Such vectors for incorporation into host cells include, but are not limited to, the following vectors and their derivatives: 1) Prokaryotic and other host cells—pBI221, pBI121 (Clonetech), pYeDP60, pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH45A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pBI2113Not, pBI2113, pBI101, pBI121, pGA482, pGAH, PBIG, and 2) Eukaryotic and other host cells—pHISi-1, pMLBART, Agrobacterium tumefaciens strain GV3101, pSV2CAT, pOG44, PXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, and pSVL (Pharmacia); pLGV23Neo, pNCAT, and pMON200. Any other plasmid or vector may be used as long as they are replicable and viable in the host.
In some preferred embodiments of the present invention, plant expression vectors comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences for expression in plants. In other embodiments, DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
In certain embodiments of the present invention, the nucleic acid sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. Promoters useful in the present invention include, but are not limited to, the LTR of SV40 promoter, the E. coli lac or trp, the phage lambda PL and PR, T3 and T7 promoters, and the cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, and mouse metallothionein-I promoters and other promoters known to control expression of gene in prokaryotic or eukaryotic cells or their viruses. In other embodiments of the present invention, recombinant expression vectors include origins of replication and selectable markers permitting transformation of the host cell (e.g., dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli).
In some embodiments of the present invention, DNA encoding the polypeptides of the present invention is expressed with plant promoters. Plant promoters can by constitutive, leaky and transient. In some embodiments, a promoter is a transient promoter (e.g. transient rd29A promoter as in U.S. Pat. No. 6,495,742B1; U.S. Pat. No. 6,670,528; herein incorporated by reference). Examples of constitutive promoters contemplated for the present invention include a “cauliflower mosaic virus 35S promoter” and “CaMV35S promoter.” In some embodiments, promoters of the present invention are stress response promoters and comprise one or more of a rd29A gene promoter (Yamaguchi-Shinozaki, et al., (1994) The Plant Cell 6:251-264); rd29B gene promoter (Yamaguchi-Shinozaki, et al., (1994) The Plant Cell 6:251-264); rd17 gene promoter (Iwasaki, et al., (1997) Plant Physiol., 115:1287); rd22 gene promoter (Iwasaki, et al., (1995) Mol. Gen. Genet., 247:391-398); DREB1A gene promoter (Shinwari, et al., (1988) Biochem. Biophys. Res. Com. 250:161-170); cor6.6 gene promoter (Wang, et al., (1995) Plant Mol. Biol. 28:619-634); cor15a gene promoter (Baker, et al., (1994) Plant Mol. Biol. 24:701-713); erd1 gene promoter (Nakashima et al., (1997) Plant J. 12:851-861); kin1 gene promoter (Wang, et al., (1995) Plant Mol. Biol. 28:605-617); all of which are herein incorporated by reference.
In some embodiments of the present invention, transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Enhancers useful in the present invention include, but are not limited to, the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
In other embodiments, the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. In still other embodiments of the present invention, the vector may also include appropriate sequences for amplifying expression.
2. Host Cells for Production of ATMIN or ATMIN-Like or HopM1:
In a further embodiment, the present invention provides host cells containing the above-described constructs. In some embodiments of the present invention, the host cell is a higher eukaryotic cell (e.g., a plant cell). An example of a transgenic plant cell and methods thereof are provided in U.S. Patent Application Pub. No. 20030144192A1, herein incorporated by reference. In other embodiments of the present invention, the host cell is a lower eukaryotic cell (e.g., a yeast cell). In still other embodiments of the present invention, the host cell can be a prokaryotic cell (e.g., a bacterial cell). Specific examples of host cells include, but are not limited to, Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, as well as Saccharomycees cerevisiae, Schizosaccharomyces pombe, Drosophila S2 cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells, COS-7 lines of monkey kidney fibroblasts, (Gluzman, (1981) Cell 23:175, herein incorporated by reference), 293T, C127, 3T3, HeLa and BHK cell lines, NT-1 (tobacco cell culture line), root cell and cultured roots in rhizosecretion (Gleba et al., (1999) Proc Natl Acad Sci USA 96: 5973-5977, herein incorporated by reference).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. In some embodiments, introduction of the construct into the host cell can be accomplished by calcium phosphate transfection; DEAE-Dextran mediated transfection, or electroporation (See e.g., In Davis et al., (1986) Basic Methods in Molecular Biology, Elsevier, N.Y., herein incorporated by reference). Alternatively, in some embodiments of the present invention, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Proteins can be expressed in eukaryotic cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989), herein incorporated by reference.
In some embodiments of the present invention, following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. In other embodiments of the present invention, cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. In still other embodiments of the present invention, microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonification, mechanical disruption, or use of cell lysing agents.
II. Methods of Modifying a Pathogen Resistance Phenotype by Manipulating ATMIN or ATMIN-Like and/or HopM1 Protective Fragment Gene Expression.
The present invention also provides methods of using ATMIN or ATMIN-like and/or HopM1 and/or HopM1 protective fragments, homologs, orthologs, and variants thereof, of genes and proteins. In some embodiments, the sequences are used for research purposes. For example, nucleic acid sequences comprising coding sequences of an ATMIN or ATMIN-like and/or HopM1 or HopM1 protective fragments orthologs, for example any one or more of ATMIN or ATMIN-like and/or HopM1, and/or HopM1 protective fragments or related pathogenic virulence polypeptide may be used to discover other genes that affect pathogen resistance.
In other embodiments, ATMIN or ATMIN-like gene sequences are utilized to alter pathogen resistance. In some embodiments, ATMIN or ATMIN-like sequences increase resistance to a pathogen. Thus, it is contemplated that nucleic acids encoding an ATMIN or ATMIN-like polypeptide of the present invention may be utilized to either increase or decrease the level of ATMIN or ATMIN-like mRNA and/or protein in transfected cells as compared to the levels in wild-type cells.
In yet other embodiments, the present invention provides methods to alter pathogen resistance in plants in which ATMIN or ATMIN-like or HopM1 or HopM11-300 proteins are not usually found and/or add a novel pathogen resistance protein, such as a HopM1 protective fragment, in which pathogen resistance to a particular pathogen is not otherwise found, by expression of at least one heterologous ATMIN or ATMIN-like gene or protective fragment, such as HopM11-300. Thus, in some embodiments, nucleic acids comprising coding sequences of at least one ATMIN or ATMIN-like gene or HopM1 or HopM1 fragment, for example any one or more of ATMIN or ATMIN-like, are used to transform plants without a pathway for producing a pathogen resistance to a particular pathogen. It is contemplated that some particular plant species or cultivars do not express any ATMIN or ATMIN-like genes or protective pathogen derived fragments. For these plants, it is necessary to transform a plant with the necessary ATMIN or ATMIN-like genes or HopM1 protective gene fragments required to confer the preferred pathogen resistance phenotype. It is contemplated that other particular plant species or cultivars may possess at least one ATMIN or ATMIN-like gene; thus, for these plants, it is necessary to transform a plant with those ATMIN or ATMIN-like genes that can interact with endogenous ATMIN or ATMIN-like genes or HopM1 protective gene fragments in order to confer a preferred pathogen resistance phenotype.
The presence of ATMIN or ATMIN-like and/or HopM1 genes, including HopM1 gene fragments, in a species or cultivar can be tested by a number of ways, including but not limited to using probes from genomic and cDNA from ATMIN or ATMIN-like and/or HopM1 and downstream ATMIN or ATMIN-like and/or HopM1 activated genes, or by using PCR analysis or by using Northern blotting, or antibodies specific to ATMIN or ATMIN-like and/or HopM1 polypeptides. The additional ATMIN or ATMIN-like and/or HopM1 genes needed to confer the desired phenotype can then be transformed into a plant to confer the phenotype. In these embodiments, plants are transformed with ATMIN or ATMIN-like and/or HopM1 and/or HopM1 truncated and fragment genes as described herein.
As described above, in some embodiments, it is contemplated that the nucleic acids encoding an ATMIN or ATMIN-like and/or HopM1 polypeptide of the present invention may be utilized to increase the level of ATMIN or ATMIN-like mRNA and/or protein in transfected cells as compared to the levels in wild-type cells.
A. Transgenic Plants, Seeds, and Plant Parts:
The present invention also provides a transgenic plant, a transgenic plant part, a transgenic plant cell, or a transgenic plant seed, comprising any of the nucleic acid sequences of the present invention described above, wherein the nucleic acid sequence is heterologous to the transgenic plant, a transgenic plant part, a transgenic plant cell, or a transgenic plant seed. In some embodiments, the nucleic acid sequence is operably linked to any of the promoters described above. In other embodiments, the nucleic acid is present in any of the vectors described above.
The present invention also provides a method for producing ATMIN and HopM1 genes and gene fragments and their encoded polypeptides, comprising culturing a transgenic host cell comprising a heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence is any of the nucleic acid sequences of the present invention described herein which encode an ATMIN or ATMIN-like and/or a HopM1 polypeptide or variant thereof, including fragments, under conditions sufficient for expression of an encoded ATMIN or ATMIN-like and/or a HopM1 polypeptide, and producing ATMIN or ATMIN-like and/or a HopM1 polypeptide in the transgenic host cell.
The present invention also provides a method for altering the phenotype of a plant, comprising providing an expression vector comprising any of the nucleic acid sequences of the present invention described above, and plant tissue, and transfecting plant tissue with the vector under conditions such that a plant is obtained from the transfected tissue and the nucleic acid sequence is expressed in the plant and the phenotype of the plant is altered. In some embodiments, the nucleic acid sequence encodes ATMIN or ATMIN-like and/or a HopM1 polypeptide or variant thereof. The present invention also provides a method for altering the phenotype of a plant, comprising growing a transgenic plant comprising an expression vector comprising any of the nucleic acid sequences of the present invention described above under conditions such that the nucleic acid sequence is expressed and the phenotype of the plant is altered. In some embodiments, the nucleic acid sequence is an ATMIN or ATMIN-like and/or a HopM1 polypeptide or variant thereof. In other embodiments, the nucleic sequence encodes a nucleic acid product which interferes with the expression of a nucleic acid sequence encoding full-length HopM1 polypeptide or variant thereof, wherein the interference is based upon the coding sequence of full-length HopM1 polypeptide or variant thereof.
Accordingly, in some embodiments, the present invention provides plants transformed with at least one heterologous gene encoding an ATMIN or ATMIN-like and/or a HopM1 gene, or encoding a sequence designed to increase ATMIN or ATMIN-like ATMIN or ATMIN-like and/or a HopM1 protective fragment gene expression. It is contemplated that these heterologous genes are utilized to increase the level of the polypeptide encoded by heterologous genes, or to decrease the level of the protein encoded by endogenous genes.
1. Plants and Seeds:
The present invention is not limited to any particular plant comprising a heterologous nucleic acid (e.g., plants comprising a heterologous nucleic acid encoding a polypeptide comprising SEQ ID NOs:01 or 82, or nucleic acids corresponding to SEQ ID NOs:02 and 94). Indeed, a variety of plants are contemplated, including but not limited to Brassica sp., such as Arabidopsis, oil seed rape, and the like, rice and tomato. The present invention is not meant to limit the varieties of plants and include natural, cultivated, selectively bred, engineered (transgenic), natural mutants, cultivated mutants, engineered mutants and the like.
The present invention is not limited to any particular use of the transgenic plant. Indeed, a variety of purposes are contemplated. In some embodiments, the transgenic plant is for food production. For example, oilseed rape, rice and tomatoes. In further embodiments, the transgenic plant is for use in breeding programs to increase pathogen resistance for a particular pathogen and for use in any plant used by humans and animals.
2. Vectors:
The methods of the present invention contemplate the use of at least one heterologous gene encoding ATMIN or ATMIN-like gene and/or HopM1 gene and/or HopM1 gene fragments thereof, or encoding a sequence designed to increase, ATMIN or ATMIN-like gene expression. Heterologous genes include but are not limited to naturally occurring coding sequences, as well variants encoding mutants, variants, truncated proteins, and fusion proteins, as described above.
Heterologous genes intended for expression in plants are first assembled in expression cassettes comprising a promoter. Methods, which are well known to or developed by those skilled in the art, may be used to construct expression vectors containing a heterologous gene and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Exemplary techniques are widely described in the art (see e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) and Ausubel, et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., herein incorporated by reference).
In general, these vectors comprise a nucleic acid sequence encoding an ATMIN or ATMIN-like gene and/or HopM1 gene and/or gene fragments thereof, or encoding a sequence designed to increase ATMIN or ATMIN-like or HopM1 or protective HopM1 fragment gene expression, (such as HopM11-300) operably linked to a promoter and other regulatory sequences (e.g., enhancers, polyadenylation signals, etc.) required for expression in a plant.
Promoters include but are not limited to constitutive promoters, tissue-, organ-, and developmental-specific promoters, and inducible promoters. Examples of promoters include but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine amino peptidase (“LAP,” Chao et al., Plant Physiol 120: 979-992 (1999), herein incorporated by reference); a chemically-inducible promoter from tobacco, Pathogenesis-Related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); a heat shock promoter (e.g. U.S. Pat. No. 5,187,267, herein incorporated by reference); a tetracycline-inducible promoter (e.g. U.S. Pat. No. 5,057,422, herein incorporated by reference); and seed-specific promoters, such as those for seed storage proteins (e.g., phaseolin, napin, oleosin, and a promoter for soybean beta conglycin (Beachy et al., (1985) EMBO J. 4: 3047-3053, herein incorporated by reference).
The expression cassettes may further comprise any sequences required for expression of mRNA. Such sequences include, but are not limited to transcription terminators, enhancers such as introns, viral sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
A variety of transcriptional terminators are available for use in expression of sequences using the promoters of the present invention. Transcriptional terminators are responsible for the termination of transcription beyond the transcript and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants include, but are not limited to, the CaMV 35S terminator, the tm1 terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminator (see e.g., Odell et al., (1985) Nature 313:810; Rosenberg et al., (1987) Gene 56:125; Guerineau et al., (1991) Mol. Gen. Genet. 262:141; Proudfoot, (1991) Cell 64:671; Sanfacon et al., (1991) Genes Dev. 5:141; Mogen et al., (1990) Plant Cell 2:1261; Munroe et al., (1990) Gene, 91:151; Ballas et al., Nucleic Acids Res. (1989) 17:7891; Joshi et al., (1987) Nucleic Acid Res., 15:9627; all of which are incorporated herein by reference).
In addition, in some embodiments, constructs for expression of the gene of interest include one or more of sequences found to enhance gene expression from within the transcriptional unit. These sequences can be used in conjunction with the nucleic acid sequence of interest to increase expression in plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells (Callis et al., (1987) Genes Develop. 1:1183; herein incorporated by reference). Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
In some embodiments of the present invention, the construct for expression of the nucleic acid sequence of interest also includes a regulator such as a nuclear localization signal (Kalderon et al., (1984) Cell 39:499; Lassner et al., (1991) Plant Molecular Biology 17:229; all of which are herein incorporated by reference), a plant translational consensus sequence (Joshi, (1987) Nucleic Acids Research 15:6643; herein incorporated by reference), an intron (Luehrsen and Walbot, (1991) MolGen Genet. 225:81; herein incorporated by reference), and the like, operably linked to the nucleic acid sequence encoding an ATMIN or ATMIN-like gene.
In preparing the construct comprising the nucleic acid sequence encoding an ATMIN or ATMIN-like gene, or encoding a sequence designed to decrease ATMIN or ATMIN-like gene expression, various DNA fragments can be manipulated, so as to provide for the DNA sequences in the desired orientation (e.g., sense or antisense) orientation and, as appropriate, in the desired reading frame. For example, adapters or linkers can be employed to join the DNA fragments or other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, or the like is preferably employed, where insertions, deletions or substitutions (e.g., transitions and transversions) are involved.
Numerous transformation vectors are available for plant transformation. The selection of a vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selection markers used routinely in transformation include the nptII gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra, (1982) Gene 19: 259; Bevan et al., (1983) Nature 304:184, all of which are incorporated herein by reference), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., (1990) Nucl Acids Res. 18:1062; Spencer et al., (1990) Theor. Appl. Genet. 79: 625, all of which are incorporated herein by reference), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann, (1984) Mol. Cell. Biol. 4:2929; herein incorporated by reference), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., (1983) EMBO J., 2:1099, herein incorporated by reference).
In some preferred embodiments, the (Ti (T-DNA) plasmid) vector is adapted for use in an Agrobacterium mediated transfection process (see e.g., U.S. Pat. Nos. 5,981,839; 6,051,757; 5,981,840; 5,824,877; and 4,940,838; all of which are herein incorporated by reference). In some embodiments, strains of Agrobacterium tumefaciens are C58, LBA4404, EHA101, C58C1RifR, EHA105, and the like. Examples of Agrobacterium mediated transfection in grasses are provided in International Patents WO 00/04133; WO 00/11138; and U.S. Patent Application Nos. 20030106108A1; 20040010816A1; and U.S. Pat. No. 6,646,185; all of which are herein incorporated by reference.
Construction of recombinant Ti and Ri plasmids in general follows methods typically used with the more common vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with the native plasmids and sometimes constructed from foreign sequences. These may include but are not limited to structural genes for antibiotic resistance as selection genes.
There are two systems of recombinant Ti and Ri plasmid vector systems now in use. The first system is called the “cointegrate” system. In this system, the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the pMLJ1 shuttle vector and the non-oncogenic Ti plasmid pGV3850. The use of T-DNA as a flanking region in a construct for integration into a Ti- or Ri-plasmid has been described in EPO No. 116,718 and International Appln. Nos. WO 84/02913, 02919 and 02920 all of which are herein incorporated by reference). See also Herrera-Estrella, Nature 303:209-213 (1983); Fraley et al., Proc. Natl. Acad. Sci, USA 80:4803-4807 (1983); Horsch et al., Science 223:496-498 (1984); and DeBlock et al., EMBO J. 3:1681-1689 (1984), all of which are herein incorporated by reference).
A second system is called the “binary” system in which two plasmids are used; the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector and the non-oncogenic Ti plasmid PAL4404. Some of these vectors are commercially available. In other embodiments of the invention, the nucleic acid sequence of interest is targeted to a particular locus on the plant genome. Site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, for example, homologous recombination using Agrobacterium-derived sequences. Generally, plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described (e.g. U.S. Pat. No. 5,501,967, herein incorporated by reference). Homologous recombination may be achieved using targeting vectors that contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known.
In yet other embodiments, the nucleic acids of the present invention are utilized to construct vectors derived from plant (+) RNA viruses (e.g., brome mosaic virus, tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, tomato mosaic virus, and combinations and hybrids thereof). Generally, the inserted ATMIN or ATMIN-like polynucleotide can be expressed from these vectors as a fusion protein (e.g., coat protein fusion protein) or from its own subgenomic promoter or other promoter. Methods for the construction and use of such viruses are described in U.S. Pat. Nos. 5,846,795; 5,500,360; 5,173,410; 5,965,794; 5,977,438; and 5,866,785; all of which are incorporated herein by reference.
In some embodiments of the present invention the nucleic acid sequence of interest is introduced directly into a plant. One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is a modified version of the plasmid pCIB246, with a CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator (e.g. WO 93/07278; herein incorporated by reference).
3. Transformation Techniques:
Once a nucleic acid sequence encoding an ATMIN or ATMIN-like gene and/or HopM1 gene and/or gene fragments thereof, is operatively linked to an appropriate promoter and inserted into a suitable vector for the particular transformation technique utilized (e.g., one of the vectors described above), the recombinant DNA described above can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant targeted for transformation. In some embodiments, the vector is maintained episomally. In other embodiments, the vector is integrated into the genome.
In some embodiments, direct transformation in the plastid genome is used to introduce the vector into the plant cell (See e.g., U.S. Pat. Nos. 5,451,513; 5,545,817; 5,545,818; and International Patent WO 95/16783; all of which are incorporated herein by reference). The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleic acid encoding the RNA sequences of interest into a suitable target tissue (e.g., using biolistic or protoplast transformation with calcium chloride or PEG). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., PNAS, 87: 8526-8530 (1990); Staub and Maliga, Plant Cell, 4: 39-45 (1992), all of which are incorporated herein by reference). The presence of cloning sites between these markers allowed creation of a plastid targeting vector introduction of foreign DNA molecules (Staub and Maliga, (1993) EMBO J., 12:601; herein incorporated by reference). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab and Maliga, (1993) PNAS, 90: 913-917; herein incorporated by reference). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the present invention. Plants homoplasmic for plastid genomes containing the two nucleic acid sequences separated by a promoter of the present invention are obtained, and are preferentially capable of high expression of the RNAs encoded by the DNA molecule.
In other embodiments, vectors useful in the practice of the present invention are microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (e.g. Crossway, (1985) Mol. Gen. Genet, 202:179). In still other embodiments, the vector is transferred into the plant cell by using polyethylene glycol (e.g. Krens et al., (1982) Nature, 296:72; Crossway et al., (1986) BioTechniques, 4:320; all of which are herein incorporated by reference)); fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies (e.g. Fraley et al., Biochemistry, (1980) 19(26):6021-6029; herein incorporated by reference); protoplast transformation (EP 0 292 435); direct gene transfer (e.g. Paszkowski et al., (1992) Biotechnology 24:387-392; Potrykus et al., Mol Gen Genet. (1985) 199(2):169-177; all of which are herein incorporated by reference) including direct gene transfer into protoplasts (e.g. in Arabidopsis thaliana, Damm et al., (1989) Mol Gen Genet. 217(1):6-12; in rice, Meijer et al., (1991) Plant Mol Biol 16(5):807-820); all of which are herein incorporated by reference).
In still further embodiments, the vector may also be introduced into the plant cells by electroporation (e.g. Fromm, et al., (1985) Proc. Natl. Acad. Sci. USA, 82(17):5824-5828 and (1986) Nature 319(6056):791-793); Riggs and Bates, (1986) Proc. Natl. Acad. Sci. USA 83(15):5602-5606; all of which are herein incorporated by reference). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
In yet other embodiments, the vector is introduced through ballistic particle acceleration using devices (e.g., available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,945,050; and McCabe et al., (1988) Biotechnology 6:923; Weissinger et al., (1988) Annual Rev. Genet. 22:421; Sanford et al., (1987) Particulate Science and Technology, 5:27 (onion); Svab et al., (1990) Proc. Natl. Acad. Sci. USA, 87:8526 (tobacco chloroplast); Christou et al., (1988) Plant Physiol., 87:671 (soybean); McCabe et al., (1988) Bio/Technology 6:923 (soybean); Klein et al., (1988) Proc. Natl. Acad. Sci. USA, 85:4305 (maize); Klein et al., (1988) Bio/Technology, 6:559 (maize); Klein et al., (1988) Plant Physiol., 91:4404 (maize); Fromm et al., (1990) Bio/Technology, 8:833; and Gordon-Kamm et al., (1990) Plant Cell, 2:603 (maize); Koziel et al., (1993) Biotechnology, 11:194 (maize); Hill et al., (1995) Euphytica, 85:119; Koziel et al., Annals of the New York Academy of Sciences 792:164 (1996); Shimamoto et al., (1989) Nature 338: 274 (rice); Christou et al., (1991) Biotechnology, 9:957 (rice); Datta et al., (1990) Bio/Technology 8:736 (rice); European Appln. EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al., (1993) Biotechnology, 11: 1553 (wheat); Weeks et al., (1993) Plant Physiol., 102:1077 (wheat); Wan et al., (1994) Plant Physiol., 104:37 (barley); Jahne et al., (1994) Theor. Appl. Genet. 89:525 (barley); Knudsen and Muller, (1991) Planta, 185:330 (barley); Umbeck et al., (1987) Bio/Technology 5:263 (cotton); Casas et al., (1993) Proc. Natl. Acad. Sci. USA, 90:11212 (sorghum); Somers et al., (1992) BioTechnology 10:1589 (oat); Torbert et al., (1995) Plant Cell Reports, 14:635 (oat); Weeks et al., (1993) Plant Physiol., 102:1077 (wheat); Chang et al., WO 94/13822 (wheat) and Nehra et al., (1994) The Plant Journal, 5:285 (wheat); all of which are herein incorporated by reference).
In addition to direct transformation, in some embodiments, vectors comprising a nucleic acid sequence encoding an ATMIN or ATMIN-like gene or are transferred using Agrobacterium-mediated transformation (Hinchee et al., Biotechnology, 6:915 (1988); Ishida et al., Nature Biotechnology 14(6):745-50 (1996); all of which are herein incorporated by reference). Heterologous genetic sequences (e.g., nucleic acid sequences operatively linked to a promoter of the present invention) can be introduced into appropriate plant cells, by means of the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Schell, Science, 237: 1176 (1987); herein incorporated by reference). Species, which are susceptible infection by Agrobacterium, may be transformed in vitro. The transformed cells are then cultured as suspension cells or regenerated as transgenic plants.
4. Regeneration:
After selecting for transformed plant material that can express a heterologous gene encoding an ATMIN or ATMIN-like gene, such as ATMIN7 or ATMIN7-like, or a protective fragment of a virulence protein, such as a protective HopM1 fragment (for example, HopM11-300) or variant thereof, including but not limited to methods described herein, whole plants are regenerated. Plant regeneration from cultured protoplasts was described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III, 1986, herein incorporated by reference. It is known that many plants can be regenerated from cultured cells or tissues or parts, including but not limited to major species of grasses, such as rice, fodder plants; vegetables, such as tomato; and crop plants, such as CanolaTM (Canadian Oil Low Acid) plants, a cultivar of a rapeseed variants from which rapeseed oil is obtained, also known as “LEAR” oil (for Low Erucic Acid Rapeseed). Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is provided first, then callus tissue is formed for inducing shoots and leaves for subsequent rooting and plant formation.
Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate and form mature plants, such as oilseed rape plant regeneration. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. The reproducibility of regeneration depends on the control of these variables.
5. Generation and Evaluation of Transgenic Plant Lines/Cultivars:
a. Generation of Pathogen Resistance and Agronomic Traits:
Transgenic plants for developing plant lines with agronomic value for commercial use are established from transgenic plants by tissue culture propagation. Further, the presence of nucleic acid sequences encoding an exogenous ATMIN or ATMIN-like gene, such as ATMIN7 or ATMIN7-like), or a protective fragment of a virulence protein, such as a protective HopM1 fragment (for example, HopM11-300), homologs or mutants or variants thereof, may be transferred to related varieties by traditional plant breeding techniques. Examples of transgenic plant lines are described herein. These transgenic lines are then utilized for evaluation of pathogen resistance and other agronomic traits.
b. Evaluation of Pathogen Resistance and Agronomic Traits:
The transgenic plants, lines, and hybrid plants thereof, will be tested for the effects of the transgene on pathogen resistance and phenotype. The parameters evaluated for pathogen resistance are compared to those in control untransformed plants and lines. Parameters evaluated include evaluating numbers of multiplying bacteria in plant parts following inoculation protocols such as those described in the EXAMPLEs, in addition to selected general agronomic traits such as effects of heat, cold, drought, salt, light; effects on growth rates, and specific traits such as yield, seed color, and the like, depending upon the plant. Ranges of pathogen resistance can be expressed as a CFU per area, or callose deposits, or plant phenotype, in a particular tissue or at a developmental state; for example, pathogen resistance can be measured in young plants and in mature plants. The tests described herein were conducted in the greenhouse and are contemplated for field-testing.
III. Biocontrol Formulations of the Present Inventions:
The protective polypeptides of the present inventions may be provided in a biocontrol formulation for agronomic use. It is contemplated that the biocontrol formulation is a composition comprising the protective polypeptide as the active ingredient. The composition may be formulated for agronomic use in a variety of ways to provide an effective amount of the polypeptide to a plant. Polypeptides may be used in formulations as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include but are not limited to spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersant, or polymers.
Alternatively, the polypeptides of the present inventions may be prepared by recombinant bacterial expression systems in vitro and isolated for subsequent field application. Such polypeptides may be either in crude cell lysates, suspensions, colloids, etc., or alternatively may be purified, refined, buffered, and/or further processed, before formulating in an active biocontrol formulation. Likewise, under certain circumstances, it may be desirable to isolate peptide clumps and/or spores from bacterial cultures expressing the protective polypeptides and apply solutions, suspensions, or colloidal preparations of such peptides and/or spores as the active ingredient(s) of a biocontrol formulation.
The compositions of the biocontrol formulations may be made by formulating either the recombinant bacterial cell, peptide, and/or spore suspension, or an isolated polypeptide component with the desired agriculturally acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term “agriculturally-acceptable carrier” covers all adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in biocontrol formulation technology; these are well known to those skilled in biocontrol formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the biocontrol composition with suitable adjuvants using conventional formulation techniques.
It is contemplated that the biocontrol formulations of the present inventions will be applied to the environment of the target pathogen, typically onto the foliage of the plant or crop to be protected, by conventional methods, preferably by spraying. The strength and duration of biocontrol application will be set with regard to conditions specific to the particular pest(s), crop(s) to be treated and particular environmental conditions. The proportional ratio of active ingredient to carrier will naturally depend on the chemical nature, solubility, and stability of the active ingredient, as well as the particular formulation contemplated for use.
Other application techniques, e.g., dusting, sprinkling, soaking, soil injection, seed coating, seedling coating, spraying, aerating, misting, atomizing, and the like, are also feasible and may be required under certain circumstances such as when targeting pathogens that cause root or stalk infestation, or for applications to delicate vegetation or for applying to ornamental plants. These application procedures are well-known to those of skill in the art.
Regardless of the method of application, the amount of the active ingredient(s) are applied in an effective amount, which will vary depending on such factors as, for example, the specific pathogen to be controlled, the specific plant or crop to be treated, the environmental conditions, and the method, rate, and quantity of application of the active composition. The biocontrol formulation may be administered to a particular plant or target area in one or more applications as needed. An effective amount may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount may depend on the composition applied or administered, the plant being treated, the severity and type of the infection, and the manner of administration.
The biocontrol formulations of the invention may be employed in the method of the invention singly or in combination with other compounds, including and not limited to other biocides. The method of the invention may also be used in conjunction with other treatments such as surfactants, detergents, polymers or time-release formulations. The biocontrol formulations of the present invention may be formulated for either systemic or topical use.
The concentration of biocontrol formulations which is used for environmental, systemic or foliar application will vary widely depending upon the nature of the particular formulation, means of application, environmental conditions, and degree of bio activity. Typically, the active ingredients will be present in the applied formulation at a concentration of at least about 0.5% by weight and may be up to and including about 99% by weight. Dry formulations of the compositions may be from about 0.5% to about 99% or more by weight of the composition, while liquid formulations may generally comprise from about 0.5% to about 99% or more of the active ingredient by weight.
EXPERIMENTAL
The following examples serve to illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosures which follow, the following abbreviations apply: N (normal); M (molar); mM (millimolar); μM (micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg (micrograms); ng (nanograms); pg (picograms); L and l (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); U (units); min (minute); s and sec (second); k (kilometer); deg (degree); ° C. (degrees Centigrade/Celsius), colony-forming units (cfu), optical density (OD), polymerase chain reaction (PCR), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), potassium hydroxide (KOH), phenylmethylsulfonyl fluoride (PMSF).
Example I
This example describes the exemplary types of Pseudomonas bacteria used with materials and methods used for growing bacteria, inoculating plants and determining the magnitude of bacterial growth in infected plants of the present invention (Katagiri et al., in The Arabidopsis Book, Somerville, Meyerowitz, Eds. (American Society of Plant Biologists, Rockville, Md., 2002), website at dx.doi.org/10.1199/tab.0039; all of which are herein incorporated by reference in their entirety).
Bacterial Strains:
Pseudomonas syringae strains used for these examples and for exemplary inventions described herein were wild-type (WT) Pst DC3000 (Ma et al., Mol. Plant-Microbe Interact. 4:69 (1991); herein incorporated by reference), a Pst DC3000 ΔCEL mutant strain described in Alfano et al. (2000) Proc. Natl. Acad. Sci. USA. 97:4856; herein incorporated by reference, a Pst DC3000 ΔCEL mutant carrying pORF43, thus further expressed hopPtoM-shcM in a pUCP19, as described in Badel, et al. (2003) Molecular Microbiology 49(5):1239-1251 and used in DebRoy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:9927; all of which are herein incorporated by reference, and a Pst DC3000 hrcC mutant (formerly a hrpH mutant; Yuan et al. (1996) J. Bacteriol. 178:6399; herein incorporated by reference).
Pseudomonas Inoculum Preparation:
Each inoculum was prepared by calculating the proper dilution necessary for a desired bacterial concentration and then diluting that volume of bacteria in sterile water. In brief: 1) Bacteria were streaked out from a −80° C. glycerol stock onto a plate of low salt Luria Berating (LB) medium (10 g/L Tryptone, 5 g/L Yeast Extract and 5 g/L NaCl pH=7.0), with antibiotics (used as indicated at the following concentrations: ampicillin, 200 μg/ml; chloramphenicol, 34 μg/ml; rifampicin, 100 μg/ml; spectinomycin, 50 μg/ml) or without antibiotics depending upon the experimental design, and grown for 1 or 2 days at 30° C.; 2) Bacteria from the fresh streak were transferred to a liquid culture with appropriate antibiotics and grown with shaking at 30° for 8 to 12 hours then harvested when bacterial culture reached mid to late log phase growth (OD600=0.6 to 1.0), (for growth on solid medium, bacteria were plated and grown on solid medium where confluent bacteria were then scraped off the plate for use in preparation of the inoculum); 3) A bacterial culture was centrifuged at 2500×g for 10 minutes in a swinging bucket rotor to pellet the bacteria; 4) The culture supernatant was poured off, the bacteria were resuspended in sterile water or 10 mM MgCl2; 5) Under certain conditions, cells were washed 1 or 2 times in water (in volumes equal to that used to grow the bacteria) by repeating steps 3 and 4; and 6) Optical density of the bacterial cell suspension was quantified using a spectrophotometer set at 600 nm. For Pst DC3000 an OD600=0.2 was approximately 1×108 CFU)/mLiter.
Methods of Spray or Dipping Inoculation (Infection):
Natural infection routes for Pseudomonas syringae and other foliar bacterial pathogens are through wounds or natural openings such as stomata. Dipping or spraying bacterial suspensions on Arabidopsis leaves mimics this natural method of entry into the apoplastic space.
Spray Inoculation:
Plants were grown with a bacterial suspension prepared as previously described. Plants in pots or entire flats were sprayed with a bacterial suspension containing 2 to 5×108 CFU/mL in water with 0.02 to 0.05% Silwet L-77 (Union Carbide) using a spray bottle with a fine mist setting to spray the bacterial suspension onto leaves until there was imminent runoff. Leaf surfaces were coated with the bacterial suspension and appeared evenly wet.
Dipping Inoculation:
Plants grown in pots with a mesh covering the pot were dipped upside down into a bacterial suspension similar to that used for spray inoculation. The inverted pot of plants were fully submerged in the bacterial suspension for 2 to 3 seconds and then removed. Leaf surfaces were evenly coated with the bacterial suspension. Following inoculation, plants were immediately placed under a plastic dome to maintain high humidity for 2 to 3 days. The high humidity (80 to 90% but not 100%) supported bacterial induced disease symptom development without saturating leaf intracellular spaces that mimicked abnormal disease symptom development.
Vacuum Infiltration:
The following is a brief outline of infiltration procedures: 1) inoculum was prepared as described above with the addition of a surfactant Silwet L-77 at a level of 0.004% (40 μl/L); 2) vacuum infiltration apparatus was assembled; the refrigerated condensation trap was turned on; 3) inoculum was poured into a container (such as a 1-L glass beaker), which also supported the inverted pot so that the whole pot was not submerged while the plants was entirely immersed in the inoculum; 4) the beaker with the immersed plants was placed in the vacuum chamber, sealed with the valve stopcock, and the vacuum pump was turned on; 5) when vacuum pressure reached a level of approximately 20 inches of mercury, it was maintained for 1 minute while the pump continued to pull a vacuum. After 1 minute, the vacuum pressure gauge read 22 to 25 inches mercury with bubbles that appeared on the surface of the leaves as well as on the top of the inoculum; 6) after 1 minute, the vacuum pressure was rapidly released by removing the valve stopcock. When the vacuum pressure returned to zero, the plants were removed from the chamber. During the rapid return to atmospheric pressure leaves became infiltrated with the bacterial suspension; 7) successful inoculation resulted in almost all the leaves being fully infiltrated with the inoculum. Effectiveness of the vacuum treatment was easily assessed by examining the plant leaves; infiltrated leaves look darker green (water-soaked) due to the presence of the bacterial suspension within the leaf intercellular spaces; 8) soil-contaminated bacterial suspension was discarded and replaced with fresh inoculum and steps 4 through 7 were repeated for inoculating additional plants; and 9) after inoculation, the plants were completely dried (for 1 to 3 hours), until the leaves did not appear to be water-soaked. The inoculated plants were then covered with a plastic dome for 2 to 3 days to maintain high humidity. As one example, Col-0 plants inoculated with Pst DC3000 at a dose of OD600=0.002 Pst DC3000 (106 cfu/mL) showed a water-soaked disease symptom within 2 to 3 days followed by chlorosis and necrosis of the inoculated tissue that occurred 3 to 4 days post-inoculation.
Syringe Injection:
Plants were grown by standard techniques and the inoculum was prepared as described above. Individual leaves were infiltrated with bacteria using a syringe. Briefly: 1) A leaf was selected and marked for identification using a blunt-ended permanent marker; 2) The leaf was carefully inverted, exposing the abaxial (under) side. A 1-mL needleless syringe that contained a bacterial suspension was used to pressure-infiltrate the leaf intracellular spaces at the same time avoiding the vascular system of the leaf where damage of the midrib would have obvious detrimental effects on the viability of the leaf tissue; 3) as a small amount of inoculum (approximately 10 μL) infiltrated the leaf a water-soaking-like discoloration of the leaf was apparent; and 4) intercellular spaces of the infiltrated leaves were dried and the plants were covered with a plastic dome to maintain humidity for 2 to 3 days.
Bacterial Pathogen Enumeration Procedure:
A standard enumeration procedure involves pathogen inoculation, using any one of described methods, supra, followed by assaying bacterial populations present within host tissues at regular intervals. The population present within the tissue was calculated based on the dilution factor divided by the amount of tissue present in each sample. Plotting log (culturable bacterial number/cm2 leaf tissue) against time (usually in days) after pathogen inoculation produced an unfitted curve, i.e. growth curve. For a review of methods, see, Katagiri et al., in The Arabidopsis Book, Somerville, Meyerowitz, Eds. (American Society of Plant Biologists, Rockville, Md., 2002), website://dx.doi.org/10.1199/tab.0039; herein incorporated by reference.
Following inoculation, infected plants were monitored daily over a 3- to 4-day period for symptom development and bacterial multiplication. For experiments described in FIGS. 1 and 5, plants were sprayed with 30 μM dexamethasone (DEX) 24 h before bacterial inoculation (1×106 cfu/ml). Spraying transgenic plants expressing full-length HopM1 with 30 μM DEX induced rapid leaf necrosis within 10 h, which prevented bacterial multiplication. Therefore, in further experiments 0.003 μM MDEX was used for spraying plants, an amount that did not induce leaf necrosis, but induced complementation of the Pst DC3000 ΔCEL mutant (FIG. 1A).
Detailed Bacterial Counting Procedure:
Leaves were harvested and surface sterilized as follows: 1) whole leaves were removed from a host plant and gently mixed in a 70% ethanol solution for 1 minute. Leaves were blotted briefly on paper towels then rinsed in sterile distilled water for 1 minute, then blotted dry on paper towels. Leaf disks were excised from leaves with a 0.5 cm2 or smaller cork borer, depending on the size of the sample leaves; 2) leaf disks from the leaves of 2 or more independent replicate plants were pooled for a single tissue sample and placed in a 1.5-mL microfuge tube with 100 μL sterile distilled water, the amount of leaf tissue per tube was recorded as leaf surface area. Three or more samples were harvested for each time point. Steps 1 and 2 were repeated for each sample; 3) Tissue samples were ground with a microfuge tube plastic pestle, by hand or by using a small hand-held electric drill. The samples were thoroughly macerated until pieces of intact leaf tissue were no longer visible; 4) the pestle was rinsed with 900 μL of water, with the rinse being collected in the original sample tube such that the sample was in a volume of approximately 1 mL; 5) steps 3 and 4 were repeated for harvesting additional samples; 6) following grinding of the tissue, samples were vortexed to evenly distribute the bacteria within the water/tissue sample. A 100-μl sample was removed and diluted in 900 μl sterile distilled water. A serial 1:10 dilution series was created for each sample by repeating this process. The number of serial dilutions necessary to get countable colonies were determined for each sample, however dilutions to 10−7 were usually sufficient for any bacterial strain; 7) The samples were plated on the appropriate medium (e.g., Low salt Luria Bertani) supplemented with the necessary antibiotics to select for the specific inoculated bacterial strain. Plating was done in the traditional way (100 μL of a single sample was spread on a single plate) or several 10 μL aliquots of the 1:10 serial dilutions were spotted on to a single plate and allowed to dry onto the surface; and 8) Plates were placed at 30° C. for approximately 2 days with a cfu value determined for each dilution of each sample. For the 10-μL spotting technique, a single spot was used for estimating the bacterial population when it had >10 and <70 colonies present in the spotted sample dilution.
Yeast Plasmids and Systems:
pGILDA and pB42AD vector constructs for the yeast 2-Hybrid system are shown in FIG. 15.
AtMIN DNA fragments were amplified by PCR using the primers listed below, standard PCR procedures were used, then fragments were isolated and cloned into a pB42AD vector.
Yeast colonies were grown on complete minimal medium containing galactose and Xgal according to manufacture's instructions. A blue (dark) color indicated a protein-protein interaction, whereas a white (light) color indicates no such interaction. Further, a “+” symbol indicates positive control strain containing pLexA-p53 and pB42AD-T (AD/SV40 large T-antigen fusion) based on the known interaction of murine p53 and SV40 large T-antigen (CLONTECHniques, JULY 1996; Clonetech Labs; herein incorporated by reference in its entirety) (see, FIG. 2A).
AtMIN fusion proteins expressed from pB42AD were visualized by the HA epitope antibody, as was AtMIN10-HA. HopM1 fusion proteins expressed from pGilda were visualized by the LexA antibody. Coomassie Brilliant Blue-stained gels were used as loading controls. Arrows indicate lanes in which the amounts of AtMIN proteins are greatly reduced (FIG. 6A). AtMIN12 (a putative protein predicted to be targeted to the chloroplast) was not destabilized.
Brefeldin A (BFA) Treatment:
Leaves were infiltrated with 1×106 cfu/ml of bacteria, detached, and placed in microtiter wells with petioles immersed in the 36 μM BFA (Sigma Co.) solution. At 24 h intervals over 3 days, leaves were transferred to fresh BFA solutions. Bacterial populations and disease symptoms were determined on day 0 and day three.
Callose Staining:
Callose staining was performed 7-9 hours after bacterial inoculation as described previously (Hauck et al., Proc. Natl. Acad. Sci. USA 100:8577 (2003); herein incorporated by reference), with the exception of no application of DEX. Leaves were examined with a Zeiss Axiophot D-7082 Photomicroscope with an A3 fluorescence cube. The number of callose depositions was determined with ImagePro Plus software. The values presented in FIG. 4 are averages and standard deviations from at least four independent leaves evaluated for each treatment.
Reverse Transcription (RT)-PCR of Arabidopsis SALK Lines:
Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions RNeasy® Mini Handbook, Fourth Edition April 2006, p. 52-55; herein incorporated by reference. First-strand cDNAs were synthesized from 200 ng of total RNA by using oligo dT primer and AMV reverse transcriptase from an RNA LA PCR Kit Ver. 1.1 (Takara) according to the manufacturer's instruction (see, RNA LA PCR Kit Ver. 1.1 Manual USA Version, v.02.08; herein incorporated by reference) in its entirety. PCR amplification was carried out using oligonucleotide primers specific to each AtMIN transcript. The following primers were used for obtaining an AtMIN7, described and used herein: sense primer, 5′-CGCCCAGCATATGCCAAGGATTGGTACTC-3′ (NdeI site underlined) SEQ ID NO:78; antisense primer, 5′-TGAATTCTTACTGTTGCAAAAGTGGCTTC-3′ (EcoRI site underlined) SEQ ID NO:79.
Example II
This example briefly describes plants with materials and methods used for growing plants and for providing and then analyzing transgenic plants (see, Katagiri et al., in The Arabidopsis Book, Somerville, Meyerowitz, Eds. (American Society of Plant Biologists, Rockville, Md., 2002), at website dx.doi.org/10.1199/tab.0039; herein incorporated by reference).
Arabidopsis and Nicotiana Plants:
Arabidopsis thaliana plant lines used for the present inventions were wild-type ecotype Columbia (Col-0) with a glabrous (gl1) morphological marker. Arabidopsis thaliana SALK lines were obtained that were previously transformed with Agrobacterium T-DNA with a kanamycin-resistance gene (NPTII) insertion in each of the AtMIN genes listed in Table 1 providing a knock-out (KO) line for each AtMIN gene (Alonso et al., Science 301:653 (2003); herein incorporated by reference) (see, Arabidopsis Biological Resource Center (ABRC) (website at: /signal.salk.edu/)).
Nicotiana benthamiana plants were obtained and grown under conditions similar to Arabidopsis plants.
Soil and Pot Preparation:
Soil mix was an equal mix of BACCTO Premium Potting Soil (Michigan Peat Company) high porosity professional plant mix, perlite and vermiculite. Moist soil mix was mounded into 3-inch square pots followed by a thin layer of fine vermiculite spread over the top of the soil that rose in the center about 0.5 to 1 inch above the edge of the pot. Pots that were destined for providing plants for bacterial inoculation were covered with mesh, such as plastic window screen, held firmly to the surface of the soil with a rubber band. The pots were placed in flats and soaked with a fertilizer solution. For syringe injection or spray inoculation no mesh was used in pots. For plants used for dipping or vacuum infiltration mesh was used in pots. This was important for helping contain the soil during inversion in the inoculum.
Growing Plants:
Seed was sown in the pots and covered with a screen and a plastic dome that maintained a high humidity for efficient germination. For synchronizing germination, the flats were placed in the cold (4° C.) for 2 days and then moved to a growth chamber. Growth chamber conditions were 30° C. and 70-80% relative humidity with 12 hours of fluorescent light (a light intensity of approximately 100 to 150 μEinstein/m2/sec). After about 1 week, when seedlings emerged through the screen the plastic domes were opened slightly for a few days and then removed completely. At this time excess plants were removed from the pot to leave 4 to 6 well-distributed plants in each pot. The plants were watered, from the bottom up (adding water to the flat without overwatering) once or twice a week without letting the soil completely dry out between watering. Fertilizer was added during watering every two weeks. Plants 4 to 6 weeks old were used for inoculation (at this point they had numerous large leaves but did not have flowers).
Agrobacterium: A. tumefaciens C58.C1 (C58C1) used for these EXAMPLES were a derivative of A. tumefaciens C58 lacking a full Ti plasmid pAtC58. C58.C1 is a nonpathogenic A. tumefaciens strain lacking the Ti portion of pAtC58 and instead harbored a cryptic pAtC58 (Vaudequin-Dransart, et al. 1998 Mol. Plant-Microbe Interact. 11:583-591; herein incorporated by reference). A. tumefaciens bacteria were cultivated at 30° C. using standard Agrobacterium growth medium, such as a Trypticase soy agar.
E. coli: Routine cloning and gene expression for HOPM1 and AtMIN genes, including those destined for expression and transformation used standard E. coli, such as DHSα (Invitrogen, Corp.).
Plasmid Preparation:
Plasmids were isolated from Pseudomonas sp. and other bacteria using well-known methods (Kado and Liu (1981) J. Bacteriol. (1981) 145(3):1365-73; and Casse, et al., (1979) J. Gen. Microbiol. 113:229-242; all of which are herein incorporated by reference in their entirety).
Binary Plasmids (Vectors) for Inserting Heterologous Genes into Agrobacterium: 
pBI121: Shown in FIG. 16, originally obtained from Clontech Laboratories, Inc., was used for providing pBI121-AtMIN vectors using standard cloning methods.
pTA7002: a DEX-inducible expression vector, via an ava promoter, that expressed nucleotide inserts upon DEX exposure (see, Aoyama and Chua (1997) The Plant Journal 11:605; herein incorporated by reference in its entirety) that was used for providing pTA7002 deletion derivatives (for example, pTA7002-HopM11-300). Six× Histidine (6×His)-tagged proteins were provided by first cloning sequences, such as full-length HopM1 or HopM11-300, into pET-3 (publication TB095 December 1998, Novagen; herein incorporated by reference), for attaching the 6 Histidine coding regions, such as for providing expressed 6×His-HopM1 or 6×His-HopM11-300, then subcloning these nucleotide sequence comprising the HIS-tag into pTA7002 using standard molecular biology techniques. Transformation procedures are described below.

Transgenic Plants:

Arabidopsis plants were stably transformed with HopM1, HopM1 deletion fragments, and AtMIN genes. In brief, a floral dip Agrobacterium-mediated transformation protocol was used for inserting genes and gene fragments of HopM1 or AtMIN into Arabidopsis plants using methods, such as described by Clough and Bent (Clough et al., (1998) Plant J. 16:735; herein incorporated by reference).
N. benthamiana plants were transiently transformed with HopM1 and AtMIN proteins. In brief, fully expanded N. benthamiana leaves were co-infiltrated with Agrobacterium tumefaciens C58 C1 (for an example of co-infiltration techniques, see, Hellens, et al. (2005) Plant Methods, 1:13; herein incorporated by reference) comprising plasmids and genes described herein (for example, see, Bechtold, et al. (1993) Comptes Rendus De L Academie Des Sciences Serie Iii-Sciences De La Vie-Life Sciences 316(10):1194-1199; herein incorporated by reference).
[Western] Immunoblot Analysis of Leaf Discs:
In brief: leaf disc fractions were homogenized in 1×SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, boiled for 5 min, and centrifuged for 2 minutes. Proteins in the supernatant were separated on SDS-PAGE gels and transferred to Immobilon-P membrane for immunoblotting procedures (Millipore Corp., Bedford, Mass.).
Primary antibodies used were a mouse 6×His epitope antibody (to detect the 6×His-HopM1 proteins; purchased from Clontech Laboratories, Inc.), a chicken HA epitope antibody for recognizing AtMIN fusion proteins expressed from pB42AD (AtMIN-HA proteins) was purchased from Aves Labs, Inc., a rabbit LexA binding domain (BD) antibody to detect BD-HopM1 fusion proteins expressed by pGilda were purchased from Clontech Laboratories, Inc.), a rabbit AtMIN7 antibody was raised against recombinant AtMIN7 protein expressed in E. coli at Cocalico Biologicals, Inc., antibodies for recognizing PM-localized H+-ATPase (Dr. Marc Boutry), and antibodies for recognizing Golgi-localized xyloglucan xylosyltransferase (AtXT1) (Dr. Ken Keegstra). The secondary antibody used for detection of primary mouse antibody binding (for example, mouse 6×His epitope antibody and the like) was a goat anti-mouse IgG antibody conjugated with alkaline phosphatase (Sigma Co.); primary chicken antibody binding (for example, chicken HA epitope antibody and the like) was an alkaline Phosphatase (AP)-labeled anti-chicken IgY (Aves Labs, Inc.); and primary rabbit antibody binding (for example, mouse LexA antibody and the like) was a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (Sigma Co.).
Example III
This example demonstrates pathogen susceptibility of transgenic plants that expressed full-length HopM1 showing compensation for the virulence defect of a Pst DC3000 ΔCEL mutant.
Transgenic Expression of HopM1 and AtMIN Proteins in Arabidopsis and Nicotiana: 
Transgenic Arabidopsis plants (Col-0 gl1) were produced that expressed a full-length 6×His tagged HopM1 using method described above. These transgenic HopM1 plants were highly susceptible to Pseudomonas infection as were certain transgenic plants expressing pTA7002 deletion derivatives, described below.
Specifically, 6×His tagged HopM1 transgenic plants were infected with one of Pst DC3000, Pst DC3000 ΔCEL, or Pst hrcC. Arabidopsis plants that expressed full-length HopM1 almost fully complemented the virulence defect of a Pst DC3000 ΔCEL mutant, see, FIG. 1A. Moreover, the complementation was specific to the Pst DC3000 ΔCEL mutant because multiplication of the TTSS-defective hrcC mutant (Yuan and He, (1996) J. Bacterial. 178:6399; herein incorporated by reference), which does not secrete any effectors, did not show this effect nor did Pst DC3000 (FIG. 1A).
In order to determine where HopM1 protein was located within the transgenic plant cell, immunoblot studies were used to located the His tags of the expressed transgene in leaves collected from transgenic Arabidopsis plants that expressed 6×His HopM1. Subcellular fractionation experiments followed by immunoblotting, see below for procedure, revealed that HopM1 expression was enriched in the endomembrane fraction in the transgenic plants (FIG. 1B). Taken together, these results suggest that bacterial HopM1 acts in a host endomembrane compartment(s) to promote bacterial pathogenesis.
Subcellular Localization of HopM1:
Five-week-old HopM1 transgenic plants were sprayed with 30 μM DEX. The leaves were collected 6 hours later and homogenized in ice-cold homogenization buffer (0.5M sucrose, 0.6%[w/v] polyvinylpyrrolidone, 1.0 mM dithiothreitol, 5.0 mM ascorbic acid, 50 mM HEPES/KOH, pH 7.5, 1 mM PMSF). The homogenate was centrifuged at 4° C. for 10 min at 1,500×g and the supernatant was filtered through Miracloth (Calbiochem, San Diego, Calif.) to remove plant debris. The filtrate was centrifuged again at 13,000×g for 30 min at 4° C. The supernatant was collected and centrifuged for 30 min at 100,000×g to yield soluble (supernatant) and microsomal (pellet) protein fractions. An aqueous two-phase partitioning procedure was used to separate the plasma membrane (PM) and endomembranes (EMs) according to Larsson et al. (Larsson et al., Methods Enzymol. 228:451 (1994); herein incorporated by reference) with a polymer concentration of 6.2% (w/vol). The microsomal protein pellets were resuspended in buffer R (250 mM sucrose, 5 mM potassium phosphate, pH=7.5, 6.0 mM KCl) and subjected to phase partitioning. Both the upper phase (enriched for the PM) and the lower phase (enriched for the EM) were further partitioned for two more times with lower phase buffer and upper phase buffer, respectively. The PM and EM fractions were harvested at the end of the third partitioning from their corresponding upper and lower phases by centrifugation at 4° C. for 60 min at 150,000×g.
Fractions were applied to 12% SDS-PAGE gels for protein separation using standard methods, then transferred onto Immobilon-P membranes (Millipore Corp.) using standard protein transfer methods. Membranes were processed using immunoblot procedures briefly described herein, see Example II. The secondary antibody used was a goat-anti-rabbit antibody conjugated with alkaline-phosphatase (Sigma).
Example IV
This example demonstrates the discovery of a N-terminal truncated derivative of HopM1 that when expressed in transgenic plants interfered with the virulence function of full-length HopM1 during an infection with pathogenic Pseudomonas bacteria.
The inventors investigated the virulence function of HopM1 as defined by the following experiments using truncation derivatives of HopM1. Numerous transgenic Arabidopsis plant lines (12+) were produced where each line expressed one of at least 12 different C- and N-terminally truncated derivatives of HopM1 in a pTA7002 expression vector (see, FIG. 14 for primers used to produce sequences for HopM1 truncation derivatives).
Following evaluation of the HopM1 truncated deletion sequences of transgenic Arabidopsis plants, the inventors discovered that Arabidopsis plants expressing HopM1101-712, SEQ ID NO:98 (produced using SEQ ID NOs:54 and 55 that lacked the coding region for the first 100 aa) partially restored the multiplication and disease chlorosis symptom of the Pst DC3000 ΔCEL mutant (FIG. 1C and FIG. 5). None of the other eleven truncated derivatives complemented the virulence defect of the Pst DC3000 ΔCEL mutant (FIG. 1C and FIG. 5).
Further analysis of the transgenic plants expressing truncation mutants revealed that when Arabidopsis plants expressed N-terminal regions of HopM1 (HopM11-200 and HopM11-300) there was a dominant-negative effect exerted on the function of full-length HopM1 delivered from the infecting Pst DC3000 ΔCEL mutant-pORF43 bacteria, expressing HopM1 and its cognate chaperone ShcM (i.e. no AvrE) (FIG. 1C and FIG. 5).
Thus, disease symptoms (necrosis and chlorosis) on plants and bacterial multiplication within plants were significantly reduced in HopM11-200 and HopM11-300 Arabidopsis transgenic plants, compared with those in Col-0 gl1 or Arabidopsis transgenic plants expressing other HopM1 truncated derivatives, such as truncated derivatives from the C-terminal regions (for example, HopM1101-712 plants shown in FIG. 1C and FIG. 5). The dominant-negative effect was specific to HopM1 because HopM11-200 and HopM11-300 plants were still susceptible to Pst DC3000, which produces AvrE, in addition to HopM1 (FIG. 1C and FIG. 5). These results demonstrated that the N-terminal100-300 aa (SEQ ID NO:82) of HopM1 functioned as an independent domain in vivo interfering with the virulence function of full-length HopM1 delivered from bacteria.
These results were replicated in N. benthamiana leaves that were co-infiltrated with Agrobacterium tumefaciens C58C1 carrying either pBI121-AtMIN or pBAR1-AtMIN and A. tumefaciens C58C1 carrying either pTA7002-HopM11-712, pTA7002-HopM11-300, or pTA7002-HopM11-712. Oligonucleotide primers used for amplifying hopM1 and AtMIN genes from Pst DC3000 genomic DNA or Arabidopsis total cDNA, as applicable, used for creating these expression vectors are shown in FIG. 14. Two days after leaf infiltration, 0.3 μM DEX was applied to induce the expression of HopM11-712, or HopM11-300 or HopM11-712. Three hours after DEX treatment, leaf discs were taken for subsequent analyses using immunoblotting or co-immunoprecipitation experiments (see, EXAMPLES herein for procedures).
Example V
This example demonstrates using a yeast two-hybrid (Y2H) screening for obtaining HopM1 interacting proteins of the present invention, such as AtMIN genes and proteins that associated with either pathogen resistance or pathogen susceptibility in plants.
Yeast Two-Hybrid (Y2H) Screening Analysis:
A LexA-based yeast two-hybrid system was used for screening an Arabidopsis Y2H cDNA library using full-length HopM1 and the dominant-negative domain of HopM1 (HopM11-300) as bait, in separate screenings. This system was based upon a pGilda Lex A expression vector (CLONTECHniques, OCTOBER 1999 p. 26-27, Clontech Laboratories Inc.; herein incorporated by reference in its entirety) for expressing HopM1 proteins in combination with a lacZ reporter gene on a separate plasmid that autonomously replicated in yeast (CLONTECHniques, OCTOBER 1999 p. 26-27, Clontech Laboratories Inc.; herein incorporated by reference in its entirety), see below for addition information (see, MATCHMAKER LexA Two-Hybrid System Catalog #K1609-1 and MATCHMAKER LexA Libraries User Manual (PT3040-1) Version #PR67300 and (Yeast Protocols Handbook, Protocol #PT3024-1 Version #PR13103, published 14 Mar. 2001; herein incorporated by reference in its entirety).
hopM1 DNA fragments were amplified by PCR using the primers listed below and standard PCR procedures then fragments were isolated and cloned into a bait vector pGilda Lex A (resistance to ampicillin (100 μg/ml) to E. coli hosts; Protocol #PT3147-5; Version #PR81829; Clontech Laboratories, Inc.; herein incorporated by reference in its entirety). The following primers were used: Full-length hopM1: Sense primer, 5′-GGAATTCATGATCAGTTCGCGGATCGGC-3′ (EcoRI site underlined) SEQ ID NO:74; Antisense primer, 5′-CCTGCTCGAGTGACGGATGTTATTCAAAG-3′ (XhoI site underlined) SEQ ID NO:75; hopM11-300: Sense primer, 5′-GGAATTCATGATCAGTTCGCGGATCGGC-3′ (EcoRI site underlined) SEQ ID NO:76; Antisense primer, 5′-GGCCCTCGAGCTTACCAGCCACCCACCG-3′ (XhoI site underlined) SEQ ID NO:77.
Plasmid constructs were transformed into EGY48[p8op-lacZ] competent yeast cells (EGY48; Clontech Laboratories, Inc.) using standard yeast transformation procedures. Library screening procedures followed the instructions described in the Y2H manual provided by Clontech, (Yeast Protocols Handbook, Protocol #PT3024-1 Version #PR13103, published 14 Mar. 2001; herein incorporated by reference in its entirety).
However, yeast-2-hybrid (Y2H) screens of an Arabidopsis cDNA library failed to recover target interactor host proteins using full-length HopM1 bait. This failure to isolate interacting host proteins using full-length HopM1 was unexpected. However, a dominant-negative effect in a cellular process can be caused by unproductive protein-protein interactions as shown in Shpak et al. (2003) Plant Cell 15:1095 and Wang et al. (2005) Dev Cell 8:855; all of which are herein incorporated by reference. Therefore, the dominant-negative domains of HopM11-1200 and HopM11-300 were suspected to compete with full-length HopM1 for interaction with full-length HopM1 targeted host proteins.
In contrast to yeast screens using full-length HopM1 bait, Y2H screens using HopM11-300 as bait caught 21 strong interactors of HopM11-300. For the purpose of the present inventions, these 21 interactors were named “AtMIN” for Arabidopsis thaliana HopM interactors with at least 8 of the AtMIN genes listed in Table 1.
Example VI
This example demonstrates HopM1-dependent destabilization of AtMIN proteins by demonstrating protein-protein interactions between HopM1 and/or HopM11-300 with AtMIN proteins. In particular, this example demonstrates HopM1-dependent destabilization of AtMIN proteins in yeast cells and in N. benthamiana leaves transiently expressing HopM1 and AtMIN proteins. This example further demonstrates HopM1-dependent destabilization of AtMIN proteins in yeast two-hybrid (Y2H) systems (A) and in N. benthamiana leaves transiently expressing HopM1 and AtMIN proteins (B) and between HopM1 and AtMIN proteins in Arabidopsis thaliana cells and plants (C) of the present invention. These experiments contributed to the identification of AtMIN genes and proteins associated with whether a plant responded to a pathogen by resistance or allowing an infection (susceptibility).
Each AtMIN protein was amplified by PCR using primers, such as those shown in FIG. 14, isolated and then individually cloned into a pB42AD vector using standard methods.
A. Interactions Between HopM11-300 and AtMIN Proteins were Observed in Yeast Two-Hybrid (Y2H) Assays.
AtMIN proteins were destabilized in yeast when co-expressed with full length HopM1, but not with HopM11-300. AtMIN12 (a hypothetical protein predicted to be targeted to the chloroplast) was not destabilized.
A yeast two-hybrid (Y2H) assay was performed for determining the physical interaction between HopM11-300 compared to full-length HopM1 expressed by pGILDA and each AtMIN protein expressed by a pB42AD vector in yeast cells. HopM11-300 or full-length HopM1 pGILDA and each individual test AtMIN pB42AD were co-transformed into yeast cells as described in EXAMPLE III. Exemplary results showed a loss of AtMIN2, AtMIN7, and AtMIN10 in cells that co-expressed HopM1 (FIG. 2A). AtMIN proteins that were predicted to be chloroplast or mitochondrial proteins did not appear to be different between yeast strains that did or did not co-express full-length HopM1 (see, AtMIN12 in FIG. 2A).
Immunoblot analysis was then performed on yeast cells lines that expressed one each of the 21 AtMIN proteins co-expressed with either HopM11-300 or full-length HopM1(1-712). AtMIN proteins demonstrated destabilization in yeast when co-expressed with full length HopM1, but not with HopM11-300. For comparison, AtMIN12 (a hypothetical protein predicted to be targeted to the chloroplast) was not destabilized (FIG. 6A).
In yeast cells that expressed HopM11-300 with any one of eight of the following AtMIN proteins; AtMIN2, AtMIN3, AtMIN4, AtMIN6, AtMIN7, AtMIN9, AtMIN10, and AtMIN11, the inventors further observed an unexpected result. Eight AtMIN proteins (AtMIN2 represented by SEQ ID NOs:13 and 14; AtMIN3 represented by SEQ ID NOs:15 and 16; AtMIN4 represented by SEQ ID NOs:17 and 18; AtMIN6 represented by SEQ ID NOs:19 and 20; AtMIN7 represented by SEQ ID NOs:13 and 14; AtMIN9 represented by SEQ ID NOs:21 and 22; AtMIN10 represented by SEQ ID NOs:23 and 24; and AtMIN11 represented by SEQ ID NOs:25 and 26) either disappeared or were present in much smaller amounts in yeast cells expressing full-length HopM1 as opposed to yeast cells co-expressing those proteins and HopM11-300.
B. Interactions Between HopM11-300 and AtMIN Proteins were Observed in Nicotiana benthamiana Plant Cells.
Transient transgene expression of HopM11-300 or full-length HopM1 and AtMIN proteins in Nicotiana benthamiana cells showed that AtMIN7 interacted with HopM11-300 but not HopM1301-712 These assays were based on transient expression experiments in Nicotiana benthamiana leaf cells followed by pull down assays and immunoblot analysis (see FIG. 2B for AtMIN7 and FIG. 6B for AtMINs 2, 7, and 10).
Plant cells expressing 6×His-HopM1 and AtMIN-HA proteins in N. benthamiana leaves were engineered to co-express a second transiently expressed protein, either full-length 6×His-HopM1 or 6×His-HopM11-300. Immunoblot analysis of leaves expressing protein pairs demonstrated a physical interaction between AtMIN7-HA and 6×His-HopM11-300 (lane 1) but no interaction between AtMIN7-HA and 6×His-HopM1301-712 (lane 2) (FIG. 2B). Specifically, AtMIN7-HA was pulled down with HopM1-300, but not with 6×His-HopM1301-712. Please note that membrane associated AtMIN 10-HA was preferably eliminated during bacterial infection. Arrows indicate lanes in which DEX-induced expression of full-length HopM1 destabilized AtMIN2, AtMIN7, and AtMIN 10 (FIG. 6B).
Expression Plasmids for Nicotiana cells: For transient expression studies in Nicotiana benthamiana; expression plasmids were engineered to express C-terminal HA epitope-tagged AtMIN proteins using a constitutive CaMV 35S promoter operably linked to AtMIN sequences. Plasmids used were a pBAR1 provided by Jeff Dangl, University of North Carolina, Chapel Hill and a pBI121 (described in Jefferson et al. (1987) EMBO 6:3901; herein incorporated by reference). Leaves of AtMIN10-HA transgenic plants were infiltrated with water or 1×108 CFU/ml ΔCEL mutant bacteria or ΔCEL mutant bacteria (pORF43).
Protein pull-down analysis methods: N. benthamiana leaf discs were homogenized in lysis buffer (50 mM Tris-HCl pH=8.0, 250 mM NaCl, 10 mM β-mercaptoethanol, 1% Triton X100, 1 mM PMSF, plant protease inhibitor cocktail [Sigma Co.]). Total protein extracts were collected after centrifugation of the homogenate at 20,000-x g for 15 min at 4° C. to remove insoluble materials. The supernatant was incubated with Ni-NTA agarose beads (Qiagen) with gentle shaking for 1 hour at 4° C., followed by centrifugation at 15,000-x g for 1 min to pull down 6×His-HopM1 and its interacting proteins. Beads were then washed three times with lysis buffer and resuspended in 1×SDS-PAGE sample buffer for SDS-PAGE gel and/or immunoblot analyses. Equal amounts of total extracts were used for immunoblot analysis of HopM1 and AtMIN7, whereas the amount of pull-down sample used in the AtMIN7 blot was 15-fold higher than that used in the HopM1 blot. Total leaf proteins in these samples was visualized by Coomassie staining and used as loading controls (bottom panel). AtMIN10-HA was detected using the HA epitope antibody.
C. HopM1 Destabilizes AtMIN Proteins in Arabidopsis Transgenic Cells and Plants.
Western blot analysis of HopM1 transgenic Arabidopsis plants showed HopM1-dependent destabilization of AtMIN7 and transgene AtMIN10-HA.
AtMIN7 is a low-abundance protein in Arabidopsis plants, however it is detected with a rabbit polyclonal antibody, described herein. In order to show that HopM1 destabilized AtMIN7, leaves of Col gl1 plants were infiltrated with water or 1×108 CFU/ml ΔCEL mutant bacteria or ΔCEL mutant bacteria (pORF43, expressing HopM1 and the cognate chaperone ShcM). AtMIN7 was absent on the immunoblots of leaves infiltrated with ΔCEL mutant bacteria (pORF43) but not the leaves infiltrated with water or ΔCEL mutant bacteria (FIG. 2C).
Furthermore AtMIN10-HA stably expressed in transgenic plants was also destabilized by HopM1. Leaves of AtMIN10-HA transgenic plants were infiltrated with water or 1×108 CFU/ml ΔCEL mutant bacteria or ΔCEL mutant bacteria (pORF43). AtMIN10-HA was detected using the HA epitope antibody. Moreover, subcellular fractionation analysis of AtMIN-HA transgenic plants, in which AtMIN-HA is localized in both soluble and membrane fractions, showed that membrane-associated AtMIN-HA was preferably eliminated during bacterial infection (FIG. 6). This result is consistent with the membrane localization of HopM1, as shown in FIG. 1B.
This example provides an explanation as to why these AtMIN proteins were not detected or isolated when full-length HopM1 was used in the previous Y2H screening (EXAMPLE V).
Example VII
This Example shows that destruction of specific AtMIN protein(s) is necessary for HopM1-mediated promotion of Pst DC3000 pathogenesis in Arabidopsis plants. This information was obtained by the inventors using Arabidopsis SALK lines carrying TDNA insertions in AtMIN genes and AtMIN KO plants, examples listed in Table 1.
The inventors analyzed Arabidopsis SALK lines (Alonso et al. (2003) Science 301:653; herein incorporated by reference) carrying T-DNA insertions in each of the AtMIN genes listed in Table 1. Col-0 plants were used as a positive control. For example, two T-DNA insertion lines used in this study carried T-DNA insertions in exon 1 (AtMIN7 KO #1) and exon 18 (AtMIN7 KO #3), respectively (FIG. 8A).
Plants from each AtMIN knockout (KO) line were infected with the Pst DC3000 ΔCEL mutant. When KO plants were infected with the ΔCEL mutant, the AtMIN knockout (KO) lines, except for the AtMIN7 KO line, restricted the growth of the ΔCEL mutant in a manner similar to the wild-type Col-0 plants. Unlike the other lines, the AtMIN7 KO plant line did not restrict growth of the Pst DC3000 ΔCEL mutant in a manner similar to the wild-type Arabidopsis Col-0 plants. Instead, AtMIN7 KO plants (FIG. 8) infected by the Pst DC3000 ΔCEL mutant showed markedly increased bacterial multiplication and chlorotic and necrotic disease symptoms when compared to wild-type Arabidopsis Col-0 plants (FIGS. 3A and 3B). Further, AtMIN7 KO plants responded to both Pst DC3000 bacteria and Pst hrcC mutant bacteria in a manner similar to the wild-type Arabidopsis Col-0 plants (FIG. 3, A and B). These demonstrations showed that increased susceptibility to bacterial infection in AtMIN7 KO plants is specific to Pst DC3000 ΔCEL mutant bacteria, mirroring the results shown in FIG. 1A, thus AtMIN7 is directly related to the virulence function of HopM1.
Specifically, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, that used primers indicated in blue showed no full-length AtMIN7 transcript in either of the two AtMIN7 knockout (KO) lines (FIG. 8B). Western blot analysis of AtMIN7 in wild-type (Col-0) and two KO Arabidopsis plants showed that AtMIN7 was absent in leaves of KO lines (FIG. 8C) where endogenous AtMIN7 protein in controls was detected using a rabbit polyclonal antibody.
This result demonstrates that the increased susceptibility to bacteria in AtMIN7 KO plants is specific to ΔCEL mutant bacteria, mirroring the results shown in FIG. 1A, and therefore is biologically relevant to the virulence function of HopM1. Further, the inventors believe this is the first demonstration of a host-target mutation specifically complementing the virulence loss of a plant-pathogen mutant lacking the cognate TTSS effector.
Example VIII
This example demonstrates that BFA treatment significantly enhanced the virulence (both multiplication and disease symptoms) of the Pst DC3000 ΔCEL mutant in wild-type Col-0 gl1 plants (FIG. 3C) that mimicked the results of similar experiments using HopM11-300.
In order to test whether the virulence defect of the Pst DC3000 ΔCEL mutant is caused by its inability to inhibit host vesicle traffic, BFA treatment was performed in order to observe whether the virulence of this bacterial mutant was restored when proteins were inhibited from translocation out of the Golgi apparatus.
The HopM1-mediated destruction of AtMIN7 and the ability of BFA to restore the virulence of the Pst DC3000 ΔCEL shows that HopM1 is involved in the inhibition of a host vesicle trafficking pathway. Accelerated vesicle traffic is associated with polarized cell wall-associated defense in plants (Bestwick et al. (1995) Plant Physiol. 108:503; Collins et al. (2003) Nature 425:973; all of which are herein incorporated by reference) previous studies by the inventors showed that a major function of HopM1 is suppression of this defense (DebRoy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:9927; herein incorporated by reference). This result is consistent with the demonstration of increased susceptibility of AtMIN7 KO plants to the Pst DC3000 ΔCEL mutant (FIG. 3) and establishes an active role of AtMIN7 in host immune response.
Unexpectedly, the restoration of bacterial virulence by BFA was also specific to the Pst DC3000 ΔCEL mutant, because there were no significant differences in the multiplication or disease symptoms caused by Pst DC3000 or the hrcC mutant in Col-0 gl1 plants treated with water or BFA (FIG. 3C).
Example IX
This example demonstrates that AtMIN7 is required for cell wall-associated defense in Arabidopsis plants.
Callose deposition (a cellular marker of this defense) in leaves of Col-0 and AtMIN7 KO plants infected by Pst DC3000 or the Pst DC3000 ΔCEL mutant. As observed previously (DebRoy et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:9927; herein incorporated by reference), Col-0 leaves accumulated a high number of callose deposits in response to the ΔCEL mutant, whereas Pst DC3000 suppressed callose deposition in Col-0 leaves (FIG. 4).
Leaves of AtMIN7 KO plants were reduced in the ability to mount an active callose response to the ΔCEL mutant, whereas their response to Pst DC3000 was similar to that of Col-0 plants (FIG. 4).
Example X
This example demonstrates that several Arabidopsis plant factors may contribute to enhancing resistance to bacterial infections.
In EXAMPLE VIII, inhibition of vesicle trafficking in Arabidopsis AtMIN7 KO plants restored a lower level of virulence. Specifically, inhibition of vesicle trafficking in Arabidopsis plants significantly enhanced the virulence (both multiplication and disease symptoms) of the Pst DC3000 ΔCEL mutant in wild-type Arabidopsis Col-0 gl1 plants (FIG. 3C). Further, there were no significant differences in the multiplication or disease symptoms caused by Pst DC3000 or the hrcC mutant in wild-type Arabidopsis Col-0 gl1 plants treated with water or BFA (FIG. 3C) showing that BFA restoration of bacterial virulence was unique to the Pst DC3000 ΔCEL mutant bacteria.
The restoration of the virulence of the Pst DC3000 ΔCEL mutant in BFA-treated leaves was greater than that in the AtMIN7 KO plants, therefore the inventors contemplated that additional Arf GEFs are targeted by BFA that would represent proteins targeted by HopM1. One or more of these Arf GEFs are contemplated to be partially redundant in function to AtMIN7. Therefore, the inventors constructed an exemplary schematic phylogenetic tree showing the relationship among Arabidopsis Arf guanine nucleotide exchange factor (GEF) proteins (FIG. 9). Protein sequences were aligned using the ClustalW program (website at align.genome.jp) to construct this phylogenetic tree.
Example XI
The majority of plant pathogenic bacteria, including Pst DC3000, are extracellular pathogens that reproduce in apoplast areas of the plant after the bacterium has initially transversed the outermost layer of cell wall encased epidermal cells. However, the results obtained during the development of the inventions described herein, showed that P. syringae has an effective strategy to overcome a cell wall-associated host defense by suppression and/or elimination of AtMIN proteins that in turn are a component of an immunity-associated vesicle traffic pathway. Thus the inventors developed an exemplary model for demonstrating AtMIN protein function and interaction within a cell (see, an exemplary schematic diagram in FIG. 10).
In brief, the inventors contemplate a polarized vesicle trafficking pathway, in which AtMIN7 is a key component. By using the information from the examples described herein, an AtMIN7-dependent pathway is now associated with plant immune responses, including the formation of callose deposits and release of antimicrobial phytoalexins (red (darker) dots in the papilla and plant cell wall, FIG. 10). Thus, Pst DC3000, and likely other P. syringae strains, inject HopM1 into the host cell. Once inside the host cell, HopM1 is associated with an endomembrane compartment(s), binds to AtMIN7 through the N-terminus (in red/dark area), and destabilizes AtMIN7 and other AtMIN proteins. Brefeldin A (BFA) revealed further information when its use mimicked the effect of HopM1 by inhibiting the GEF activity of the Sec7 protein family, of which AtMIN7 is a member.
The HopM1-dependent elimination of a host plant AtMIN7 protein that is a member of the Sec7/Arf GEF family protein provides a bridge to the recent demonstrations that vesicle trafficking and extracellular secretion play important roles in plant immune response (Collins et al. (2003) Nature 425:973; Wang et al. (2005) Science 308:1036; all of which are herein incorporated by reference).
The results provided herein are in contrast to previously published studies that showed an intracellular human pathogen, Salmonella enterica, using TTSS effectors to interfere with host vesicle trafficking for bacterium induced biogenesis and established maintenance of a specialized membrane-bound compartment in which bacteria survived and multiplied (Cossart and Sansonetti (2004) Science 304:242; Knodler and Steele-Mortimer (2005) Mol. Biol. Cell 16:4108; all of which are herein incorporated by reference). Despite the difference in proposed mechanisms, the results shown herein showed that plant bacterial protein modulation of host vesicle trafficking is a goal of infectious pathogens for creating a host environment favorable for bacterial survival and multiplication; a type of modulation that is contemplated to be shared by human pathogens.







TABLE 2







AtMIN7 and Homolog identity.













Protein aa

mRNA na


Genus sp. and gene/
SEQ ID
identity
SEQ ID
identity


protein name
NO: XX
(%)
NO: XX
(%)














Arabidopsis thaliana
1
100% 
2
100% 


(AT3G43300)


AtMIN7


Q9LXK4_ARATH


oilseed_rape
5
94%
6
92%


homologue to


UP|Q9LXK4


(Q9LXK4)


Arabidopsis thaliana
3
93%
4
Not


Guanine nucleotide-



pro-


exchange-like protein



vided


Oryza sativa
11
69%
12
76%


(japonica cultivar-


group)


Putative guanine


nucleotide-exchange


protein GEP2


Lycopersicon
7
61%
8
68%


esculentum


tomato mixed elicitor,


BTI


Lycopersicon
9
38%
10
57%


esculentum


cDNA clone


LePU0380 similar to


Acc#


ref|NP_195533.1|;


guanine nucleotide-


exchange protein -


like; protein id:


At4g38200.1
















TABLE 3







HopM1 and Homolog identity.













Protein aa

mRNA na


Genus sp. and gene/
SEQ ID
identity
SEQ ID
identity


protein name
NO: XX
(%)
NO: XX
(%)





HopM1
34
100% 
35
100% 


Pseudomonas syringae
36
64%
37
75%


pv. syringae B728a,


type III effector


HopM1


Pseudomonas viridiflava
38
51%
39
78%


HopPtoM-like protein
















TABLE 4







HopM11-300 and Homolog identity.













Protein aa

mRNA na


Genus sp. and gene/
SEQ ID
identity
SEQ ID
identity


protein name
NO: XX
(%)
NO: XX
(%)














HopM11-300
82
100% 
94
100% 


Pseudomonas syringae
108
83%
109
85%


pv. phaseolicola


1448A coding for


1-300


Pseudomonas syringae
106
58%
107
74%


pv. syringae B728a,


type III effector


HopM1 coding for


1-300


Pseudomonas viridiflava
105
46%
110
79%


HopPtoM-like protein


coding for 1-300









All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry, chemistry, molecular biology, plant biology, plant disease, and plant pathogens or related fields are intended to be within the scope of the following claims.

## Claims

1. An expression vector construct comprising a nucleic acid molecule at least 95% identical to SEQ ID NO:02, wherein said nucleic acid encodes a polypeptide that alters pathogen resistance in a plant.

2. The expression vector construct of claim 1, wherein said nucleic acid molecule encodes a polypeptide that is at least 95% identical to SEQ ID NO:01, wherein said polypeptide alters pathogen resistance in a plant.

3. The expression vector construct of claim 2, wherein said polypeptide increases pathogen resistance in a plant.

4. The expression vector construct of claim 1, wherein said nucleic acid is operably linked to an exogenous promoter.

5. The expression vector construct of claim 4, wherein said exogenous promoter is a eukaryotic promoter.

6. The expression vector construct of claim 5, wherein said eukaryotic promoter is active in a plant.

7. The expression vector construct of claim 1, wherein said vector is a eukaryotic vector.

8. The expression vector construct of claim 7, wherein said vector is a plant vector.

9. The expression vector construct of claim 8, wherein said plant vector is a prokaryotic vector.

10. The expression vector construct of claim 1, wherein said vector is a T-DNA vector.

11. A transgenic plant comprising a heterologous nucleic acid molecule encoding a polypeptide that is at least 95% identical to SEQ ID NO:01, wherein said polypeptide expression provides pathogen resistance.

12. The plant of claim 11, wherein said pathogen is a microbial pathogen.

13. The plant of claim 12, wherein said microbial pathogen is a bacterium.

14. The plant of claim 13, wherein said bacterium is a Pseudomonas species.

15. A transformed plant part of the plant of claim 11.

16. A transformed seed of the plant of claim 11.

17. A transformed tiller of the plant of claim 11.

18. The transgenic plant of claim 11, wherein said nucleic acid molecule is derived from the group consisting of Brassicaceae, Solanaceae, and Poaceae.

19. The transgenic plant of claim 11, wherein said plant is selected from the group consisting of a crop plant, a decorative plant, and a tree.

20. The expression vector construct of claim 1, wherein said nucleic acid molecule is 100% identical to SEQ ID NO:02.

21. The expression vector construct of claim 1, wherein said nucleic acid molecule encodes a polypeptide that is 100% identical to SEQ ID NO:01, wherein said polypeptide alters pathogen resistance in a plant.

22. The transgenic plant of claim 11, wherein said nucleic acid molecule is 100% identical to SEQ ID NO:02.