ANTIBODIES AGAINST AVIRULENCE/PATHOGENICITY PROTEINS OF PLANT PATHOGENS

Abstract: The subject invention pertains to materials and methods that provide plants with resistance to plant pathogens and pests. Antibodies and aptamers that immunoreact or bind and inhibit the action of protein expression products from avirulence and/or phatogenicity genes, including, but not limited to, the Xanthomonas avr/pth family of such genes, are described. The antibodies of the subject invention function by blocking the action of the primary protein products of avr/pth genes by intercepting and denaturing them prior to their translocation to the plant nucleus. The method of the subject invention concerns transforming a plant with polynucleotide molecules that encode the antibodies. Expression of the antibodies in the plant confers resistance from pathogens and pests. The subject invention also pertains to polynucleotide molecules encoding the subject antibodies, as well as plants and plant tissue transformed with the polynucleotide molecules encoding the subject antibodies.

Description: DESCRIPTION ANTIBODIES AGAINST AVIRULENCE/PATHOGENICITY PROTEINS OF PLANT PATHOGENS This invention was made with government support under grant numbers 86-CRCR-l- 2234, 58-43YK-5-3 and 58-7B30-3-465 awarded by the U.S. Department of Agπculture. The government has certain rights m this invention. Background ofthe Invention Avirulence (avr) genes have been demonstrated in many different microbial plant pathogens, and have been predicted to be found in viruses and insects avr genes were originally descnbed by Flor in 1942 (Flor, 1942) and have been demonstrated or suggested to exist m at least 27 different plant host/parasite systems, including diseases caused by rusts, smuts, bunts, mildews, scabs, nematodes, insects, bactena, viruses, and even by other plants (Day, 1974). avr genes are often pathogenicity genes. The idea that pests and pathogens should carry genes that encode avirulence, as opposed to virulence, has always been enigmatic, and several different working hypotheses to explain the enigma have been proposed (Gabπel and Rolfe, 1990). Recent work has shown that an entire family of avr genes are also pathogenicity (pth) genes (Swamp et al., 1991, 1992; Yang et ai, 1994; Gabnel et ai, 1993) This Xanthomonas avr/pth gene family compπses the largest number of avr genes cloned and sequenced to date All ofthe members of the Xanothomonas avr/pth gene family encode proteins that have nuclear localizing signals (NLSs) (Yang and Gabπel, 1995) These genes have been demonstrated to be essential for pathogenicity of several different xanthomonads. The NLSs encoded by the genes function to direct the proteins to the plant cell nucleus. A review of published predicted peptide sequences of other known avr/pth genes disclosed that these genes also encoded putative NLSs Most notable among these are avr4 of the fungal pathogen Cladosporiumfulvum (Joosten et al., 1994), hrpN from Erwima amylovora (Wei et al , 1992), and the hrpZ family from Pseudomonas syringae (pathovars syπngae, glycmea and tomato, Preston et al., 1995). The archetypal Avr/Pth proteins that carry NLSs are PthA and Avrbδ, which represent the Xanothomonas avr/pth gene family. Recently, it has been demonstrated using hothpthA and avrbβ that the NLSs encoded by these genes are required for symptoms of citrus canker (pthA), and cotton blight (avrbδ), respectively (Yang and Gabπel, 1995 and Gabπel lab, unpublished) Furthermore, nearly identical NLSs may be observed m the published DNA sequences of avrBs3 (Bonas et al, 1989), avrXal (Hopkins et al , 1992) and in all unpublished DNA sequences from work done m my lab at the University of Floπda on avrB4, pthB,pthC,pthP and pthN. In fact, the NLS coding regions are conserved in all members ofthe Xanothomonas avr/pth gene family examined Pubhshed and unpublished research in my lab has shown that pthA or a homologue is required for Asiatic citrus canker disease (Swarup et al , 1992), pthB or a homologue is required for False citrus canker disease, pthC or a homologue is required for Mexican Lime cancrosis, pthPC or a homologue is required for bacteπal poplar canker disease, mdpthP or a homologue is required for common bean blight (Yang et al., 1996). It is also known that avrXal is also a member ofthe Xanthomonas avr/pth gene family, and that it contributes to the pathogenicity of X. oryzae (bactenal blight of πce; Leach et al , 1996). By extension of the results of our work with pthA and avrbβ, it is likely that export from the pathogen, import into the plant cell, and translocation to the plant nucleus are required events in all cases where these avr/pth genes are known to be either required for pathogenicity or contribute to pathogenicity (all forms of citrus canker, cotton blight, bacteπal blight of πce, poplar canker, and common bacteπal blight). Furthermore, the pathogenicity of many, if not most, microbes, fungi, viruses, and even insect pests that attack their host(s) is likely due to proteins that are guided to the plant nucleus as an obligatory step. De Wit et al. (WO 91/15585, 1991) descπbe a method for protecting plants agamst pathogens whereby a polynucleotide sequence of an avirulence gene that encodes a specific ehcitor protem is incorporated into the genome of a plant containing a corresponding resistance gene. The avirulence genes are regulated in such a manner that the expression ofthe genes only occurs at the site of pathogen infection on the plant. The purpose of the De Wit method is to activate the plant defense response by providing avirulence genes and providing for their transcπption and translation mto active protem products. There is no teaching or suggestion in the De Wit method that the avirulence genes or their products should be blocked or otherwise interfered with. By contrast, the purpose of the present invention is to inhibit or block the normal function of avirulence gene products under circumstances where these products help condition pathogenicity. The transfer of genes which encode antibodies effective against specific target viruses has been descπbed in plants. Tavladoraki et al (1993) first demonstrated the effective use of antibodies agamst a viral disease plants. However, there is no teaching or suggestion in the art directed to the in planta expression of antibodies that are immunoreactive against avirulence or pathogenicity proteins of plant pathogens. Nucleic acids and proteins often carry the ability to bind other molecules with the same high affinity and degree of molecular specificity as is exhibited by antibodies. An entirely new genetic technology is developing around the ability to isolate extremely rare nucleic acid sequences with specific ligand binding properties (similar to antibodies) from very large pools of random sequences. The process used is an iterative selection and amplification scheme sometimes called SELEX (for Systematic Evolution of Ligands by Exponential Enrichment; refer to Tuerk & Gold[ 1990]; Gold [1995]). The selected molecules with specific ligand binding properties are called "aptamers" (from the Latin aptus, to fit; refer Szostak, J.W. 1992). Although originally the term aptamer was used to describe nucleic acid molecules, it is also applied to proteins as well (refer Tuerk & Gold, 1990; Colas et al. 1996) Genes which encode RNA or protein aptamers (capable of high affinity binding with target proteins) that are capable of inhibiting protein functions have been described (Conrad et al. [1996]; Colas et al. [1996]). The function of aptamers is similar to that of antibodies in terms of target protein binding specificity. Aptamers have not been expressed in plants and there is no teaching or suggestion in the art directed to the in planta expression of aptamers that bind avirulence or pathogenicity proteins of plant pathogens. Brief Summary ofthe Invention The subject invention concerns antibodies expressed in plants, wherein the antibodies are immunoreactive with avr/pth primary (protein) gene products of plant pathogens and pests. The Avr Pth proteins encoded by these genes typically comprise NLSs that play a critical role in directing the proteins to the plant nucleus. Avr Pth proteins are actively exported from the invading pathogen, imported into the plant cytoplasm, and transported to the plant nucleus. Avr/Pth proteins are susceptible to inactivation by binding with the expressed anti-Avr/Pth antibodies, thereby interrupting an essential step in the production of disease in the plant. The subject invention also concerns the polynucleotide molecules that encode antibodies or aptamers that bind to the Avr Pth proteins or proteins that contain NLSs. The subject invention also concerns a method for protecting plants from pathogenic organisms. Pathogens to which resistance can be conferred in plants include any bacteria, fungi, viruses, insects or other organisms which require for pathogenicity the expression of avr/pth genes or genes that encode NLSs. The subject invention further concerns plants and plant tissue transformed with polynucleotide molecules that encode the anti-Avr/Pth antibodies and aptamers ofthe invention. Another aspect of the present invention is plants and plant tissue expressing the anti-Avr/Pth antibodies or anti-Avr/Pth aptamers. Brief Description of the Drawings Figure 1 shows an Western blot of crude lysate from Xanthomonas cells using rabbit polyclonal anti-PthA antiserum. Lane 1, X. citri strain 3213 (Gabriel et ai, 1989); lane 2, marker-interruption derivative of 3213, Xcl.2 (pthA: :npt-sac) (Yang et ai, 1995); lane 3, X. campestris strain 3048 (Gabriel et ai, 1989); lane 4, transconjugant 3048/pZit45 (PthA + ) (Swarup et al., 1992). Asterisks indicate PthA or its truncated derivative. The thick, top band in lane 1 is a doublet. Brief Description ofthe Sequences SEQ ID NO. 1 is a PCR primer that can be used in accordance with the subject invention. SEQ ID NO. 2 is a PCR primer that can be used in accordance with the subject invention. SEQ ID NO. 3 is a PCR primer that can be used in accordance with the subject invention. SEQ ID NO. 4 is a PCR primer that can be used in accordance with the subject invention. SEQ ID NO. 5 is a PCR primer that can be used in accordance with the subject invention. SEQ ID NO. 6 is a PCR primer that can be used in accordance with the subject invention. SEQ ID NO. 7 is a PCR primer that can be used in accordance with the subject invention. SEQ ID NO. 8 is a PCR primer that can be used in accordance with the subject invention. Detailed Description ofthe Invention The subject invention concerns antibodies that are immunoreactive with the protein products of the avr/pth genes. Typically, the avr/pth gene products have nuclear localizing signals (NLSs) which function to direct the gene product to the plant nucleus. The antibodies and aptamers ofthe subject invention bind to and inactivate the Avr/Pth proteins encoded by the avr/pth genes, thereby preventing the proteins from being transported to the plant nucleus The antibodies and aptamers ofthe subject invention can also be used for purifying Avr/Pth proteins or for use in lmmunohistological studies to identify Avr/Pth proteins. The avr/pth gene products encompassed withm the scope ofthe invention include avr/pth genes from all viral, microbial, insect, fungal and plant sources. Anti-Avr/Pth or anti-NLS antibodies can be generated by immunizing a suitable host animal with Avr/Pth proteins, or fragments thereof, or with NLS peptides In a preferred embodiment, animals are immunized with punfied, full-length Avr/Pth protein Alternatively, animals can be immunized with peptide fragments that correspond to the amino-, carboxyl-, or middle portions ofthe full-length protein The proteins or peptides can be punfied or cloned from suitable pathogens, such as Xanthomonas, or the they can be prepared synthetically using a peptide synthesizer or equivalent means. Antibodies can also be generated against peptides that compπse an NLS ammo acid motif such as: K-R/K-X-R/K (Yang, Y. and Gabπel, D W., 1995). Hybπdomas are generated from antibody-secreting cells of immunized animals according to standard methods. Methods for the production of hybπdomas and monoclonal antibodies are well-known in the art. (Kohler and Milstein, 1975). The hybπdomas are screened for the production of antibodies that react with the Avr/Pth proteins and/or NLS peptides. In order to determine what region ofthe protem an antibody binds to, a cloned avr/pth gene can be treated with restπction enzymes to generate vaπous fragments of the gene. These fragments, which may correspond to the 5' , 3' , or middle regions ofthe gene, can be subcloned, expressed and used for antibody screening procedures. Preferably, antibodies having the highest binding affinity for the protem are selected. The polynucleotide sequences encoding the anti- Avr/Pth antibodies ofthe mvention are cloned from the selected hybπdomas using standard molecular biology techniques Typically, mRNA is isolated from a selected hybπdoma and reverse transcπbed mto cDNA In a preferred embodiment, polynucleotide sequences encodmg antibody are amplified from cDNA sequences of selected hybπdomas using polymerase chain reaction (PCR) and PCR pπmers specific to vaπable light and heavy domams of immunoglobulin. The cloned antibody gene sequences are then incorporated into a suitable expression vector and used to transform a plant or plant tissue Methods for transforming plants with heterologous genes are well known in the art. In a preferred embodiment, a plant or plant tissue is transformed with several different antibody genes, such that the plant is capable of producing antibodies that, together, bind to a combination of sites selected from the group consisting of sites m the amino-, carboxyl-, and middle regions ofthe Avr/Pth protein. The expresiion system used in the plants transformed according to the subject invention can be selected to inducibly or constitutively express the anti-Avr/Pth antibodies. Antibodies expressed in plants are also referred to herein as "plantibodies." The transformed plants are protected against invading pathogenic organisms that express avr/pth genes by the action ofthe subject antibodies produced by the plant, wherein the "plantibodies" bind to protein products of the avr/pth genes, and thereby inhibit the function of the protein products. The antibodies of the subject invention can be prepared from hybridomas using any animal for which suitable fusion partner cell lines exist. In an exemplified embodiment, the hybridoma is generated using mouse lymphocytes. Other animals such as rat, monkey, sheep, rabbit, goat and pig can also be used to generate hybridomas. The term "antibody" as used herein refers to an immunoglobulin protein, or fragment thereof, that binds to an epitope on an antigen or a hapten. In a preferred embodiment of the present invention, the antibody is bivalent and comprises at least the variable light (V L ) and variable heavy (V H ) chains of immunoglobulin. Monovalent forms of antibody are also contemplated by the subject invention. The term "aptamer" as used herein refers to a nucleic acid or protein having the ability to bind with a high degree of affinity and specificity to a target protein molecule. The predicted amino acid sequences for every member ofthe Xanthomonas avr/pth gene family sequenced to date are greater than 95% identical; thus, antibodies can be readily prepared according to the subject invention that bind with most, if not all, proteins of the avr/pth gene family. Any Xanthomonas Avr/Pth proteins, as well as any other pathogenicity proteins that enter the plant cell, can be inhibited by antibodies and are within the scope of the present invention. Avr/Pth proteins contemplated by the present invention may include, but are not limited to, AvrB4, Avrb6, Avrb7, AvrBs3, AvrXa7, AvrXalO, AvrBlOl, AvrB102, PthA, PthB, PthC, PthN, PthPC, and PthP. Another aspect of the present invention is polynucleotide molecules that comprise nucleotide sequences that encode the antibodies or aptamers of the present invention. The polynucleotide molecules may be composed of either RNA or DNA. Preferably, the polynucleotide molecules are composed of DNA. The polynucleotide molecules ofthe subject invention may be incorporated into suitable polynucleotide vectors, such as cloning or expression vectors, all of which is well known to the skilled artisan. Vectors can be selected by the skilled artisan to maximize transformation and expression of the subject polynucleotide sequences in plants. The subject invention also Concerns methods for protecting plants from organisms that are pathogenic to plants The methods provided herein compπse providing a plant with the capacity to produce antibodies or aptamers that bind to and inhibit the function of proteins produced by the invading organism that are necessary for the pathogenicity or production of disease in the plant by the organism. In a preferred embodiment, the plant is transformed with a polynucleotide molecule that encodes an antibody or an aptamer that binds to Avr/Pth proteins encoded by avr/pth genes of pathogenic organisms. In one embodiment, the plant is transformed with multiple, distinct polynucleotide molecules encoding distinct antibodies and/or aptamers that have different binding specificities on the target protein A further aspect of the subject invention is a plant or plant tissue that has been transformed with polynucleotide molecules that encode the antibodies and/or aptamers of the subject invention Any method for transforming plants or plant tissue to incorporate heterologous DNA mto the plant genome and express that DNA in the plant cell to produce the subject antibody or aptamer is withm the scope of the present invention. The plants of the subject invention include both dicotyledonous and monocotyledonous species. The ordinarily skilled artisan can readily select and utilize specific cloning and transformation methods that are most suitable for a specific type or species of plant. Plant tissue withm the scope ofthe present invention is exemplified by, but not limited to, protoplasts, plant cells, plant seeds, and seedling tissue Following are examples which illustrate matenals and methods for practicing the subject invention These examples should not be construed as limiting and are not intended to be a delineation of all possible modifications of the matenals and methods of the present invention Example 1 — Preparation of Antibodies The protein product encoded by pthA cloned mto the pET-19b expression vector (Novagen, Inc., Madison, WI) was punfied for use as an immunogen to generate antibodies that bind to Avr/Pth proteins The pET-19b vector creates a translational gene fusion product with an N-termmal leader sequence of 10 histidine residues, which serve as a affinity punfication "tag " The PthA prote was punfied by affinity chromatography usmg "HIS-BIND" resin (Novagen) which incoφorates divalent cations (Ni^) immobilized on a support resm The protein eluent from the affinity column was further punfied by electrophoresis of the protein on a preparative SDS polyacrylamide gel, excising the ca 130 kDa band from the gel and electroeluting the protein. The electroeluted protein from the preparative SDS/PAGE gel was used to raise antibodies specific to the PthA protem. The purified PthA protem was used to immunize a rabbit to generate polyclonal antibody agamst PthA For Western blots, Xanthomonas cells were grown in PYGM broth, harvested by centnfugation and boiled for 2 mmutes in sample buffer (50 mM Tπs-HCl, pH 6 8, 10% glycerol, 2% SDS, 0.1% bromophenol blue). Lysates were centrifuged at 11,600 x g for 5 minutes, eletrophoresed on 8% SDS-polyacrylamide gels and the separated proteins were transferred onto nitrocellulose. Membranes were probed with anti-PthA antibody and visualized with goat anti-rabbit antibody conjugated with alkaline phosphatase (Sigma Chemical Co.). Figure 1 shows a Western Blot oi Xanthomonas cell lysate using the rabbit polyclonal anti-PthA antibody. In another embodiment, munne monoclonal antibodies can be generated against the 5' end, the 3 ' end and the repeat units of pthA gene products using the punfied histidine-tagged PthA protein descnbed above. The mouse hybπdomas can be screened initially for reaction to different portions ofthe PthA protem. The 5' end of pthA from the native promoter to the Stul site has been cloned and expressed in pET-19b; it encodes a predicted peptide of 240 ammo acids The central portion of the pthA gene from the Stul to the HincU sites and the 3 ' end from HincR, encoding predicted peptides of 660 amino acids and 260 amino acids, respectively, can also be separately cloned into appropnate pET vectors and the translational fusion product expressed. These three histidme-tagged gene fusions can be expressed in E coh or other suitable hosts and crude protein preparations obtained therefrom. The protem preparations can then be used m ELISA assays to screen for hybπdomas that secrete antibody that lmmunoreacts with the fusion protem Alternatively, the protein preparations can be punfied over the "HIS- BIND" resin and then used for antibody screening purposes. Antibodies that exhibit a strong reaction against either the ammo-, carboxyl-, or the middle region ofthe PthA protein by ELISA are selected for isolation and cloning ofthe antibody encoding DNA sequences. Example 2 - Preparation of Antibodv-Encoding and Aptamer-Encoding DNA Sequences. Hybndomas that produce monoclonal antibodies reactive with either the amino-, the carboxyl-, or the middle region ofthe PthA protem, based on ELISA and western blot screening results, are then used as a source of antibody genes that are used to transform a plant or plant tissue. The protocols to be used follow those of Danielsson & Borrebaeck (1992), incorporated herein by reference The selected hybridoma cell lines are grown in culture and total RNA extracted using an Ultraspec RNA Isolation System (Biotecx Labs, Houston) After quantitation of the RNA by spectrophotometer, cDNA copies of mRNAs are made using reverse transcnptase and olιgo(dT) primers. The specific cDNA's corresponding to the light and heavy chains of the immunoglobulin (Ig) mRNA can then be amplified by using polymerase chain reaction (PCR) m conjunction with the following PCR pπmers that have been designed to amplify mouse- specific antibody genes (the pnmer sequences below are shown 5' to 3'; restriction sites are underlined). Pπmers for Amplifying the Vanable Heavy fV H ι chain conserved domain . 5'pnmer (SEQ ID NO. 1)- (C/G)AG GT(C/G) (A C)A(A/G) CTG CAG (C/G)AG TC(AT) GG 3' PstI 3' pnmer (SEQ ID NO. 2): TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC 3 ' BstEII Pnmers for Amplifying the Vanable light (V_ 1 chain conserved domains: 5' pnmer (SEQ ID NO. 3): GAC ATC GAG CTC ACC CAG TCT CCA 3 ' Sacl 3' pnmer (SEQ ID NO. 4): CCG TTT CAG CTC GAG CTT GGT CCC 3 ' Xhol Alternative 3' pnmers (SEQ TD NO. 5 and SEQ TD NO. ^: (to be used if no PCR product is obtained with first 3' pnmer) 5'CCCGAATTCTTAGATCTCCAGCTTGGTCCC3' (SEQIDNO.5) 5'CCCAAGCTTGACATTGTGACCCAGTCTCCA3' (SEQIDNO.6) Standard PCR protocols are used to amplify the antibody cDNA. It may be desired for the expressed antibody gene to form a bivalent molecule. Bivalent antibody fragments have been cloned and expressed in bacteria (Holliger et al. 1993). Bivalent antibody fragments are constructed so that the carboxyl terminus of the variable heavy chain region is fused directly with the amino terminus of the light chain; thus, it will be stearically impossible for these two termini to self-associate. Following the first PCR amplification ofthe V H and V L fragments, the exact DNA sequences ofthe fragment termini can be determined by PCR sequencing. A second PCR amplification is performed with primers based on the exact sequence information. The primers can also incoφorate specific restriction endonuclease sites to simplify cloning into the pET-22b (Novagen, Madison, Wisconsin) transcription expression vector, as well as provide a Kozak consensus sequence, an in frame ATG translation start site, and a termination codon: 5 ' Second round V H 5 ' primer f SEQ ID NO. 7 ,: ' TCG GAT CCG GAA ACC ATG (C/G)AG GT(C/G) (A C)A(A/G) CTG CAG (C/G)AGTC(A T) GG 3' BamHI Kozak Pstl Second round V, 3 ' primer f SEQ ID NO. 8^: 5 ' TCC CCG GGT ACC TCA CCG TTT CAG CTC GAG CTT GGT CC 3 ' Smal Kpnl *** Xhol *** Translational termination codon. The same V H 3' and V L 5' primers used for the primary PCR amplification are used for the second round of PCR amplification. The linear V H -vector-V L construct is Klenow polished and blunt-end ligated to form the final closed circular molecule. The BamHI to Xhol restriction fragment can then be recloned into a suitable expression vector, and used to transform plants according to well known techniques. For example, the ftømHI to Xhol restriction fragment can be cloned into E. coli expression vector pET-22b, forming an in frame fusion that encodes apelB leader sequence (for export from E. coli) and a poly-histidine tag at the carboxyl-terminus (for protein purification) ofthe protein. After creating the gene fusion in pET-22b, the entire construction, including the pelB leader sequence can be recloned into pQY29.2 using Ndel/Xhol, thus providing a leader sequence that allows for the export ofthe small anti-Avr/Pth antibody peptides to the plant cell wall. Vector pQY29.2 was constructed in my lab (Yuan and Gabriel, unpublished) in order to provide a plant promoter for any cloned bacterial gene. In this regard, any suitable vector comprising a plant promoter and transcription terminator and polyadenylation signal will suffice equally well. Vector pQY29.2 carries a polylinker for cloning inserted open reading frames following an ATG translational start codon and downstream of a 35S promoter derived from cauliflower mosaic virus. A nopaline synthase 3 ' transcriptional terminator and polyadenylation signal lies on the other side ofthe polylinker. The vector is designed to be fused with a second plasmid, pGNlOO (Bogusz et ai, 1990), carrying a functional GUS reporter gene, and finally with a third plasmid pGA472 (An et ai, 1985) and providing a neomycin resistance gene and the left and right T-DNA borders for plant transformation. The final construction is transferred from E. coli to Agrobacterium tumefaciens strain AGL1 (Lazo et ai, 1991) by bacterial conjugation using standard methods. Because the BamHI and Kpήl sites are uniquely present on pQY29.2, the amplified V H and V L polynucleotide molecules can be positioned in appropriate orientations relative to the promoter and nopaline synthase 3 ' transcriptional terminator on the pQY29.2 plasmid. A polynucleotide construct in the pQY29.2 vector that has the pelB leader sequence removed can also be prepared and used to transform plants. The processes for generating and selecting aptamers are well known in the art, as described by Turk & Gold (1990); Szostak (1992); Gold (1995); Colas et al. (1996); and Conrad et al. ( 1996); each of which is incoφorated herein by reference. Example 3 - Transformation of Plants with DNA encoding anti-Ayr/Pth Antibodies. The polynucleotide molecules that encode immunoreactive anti-Avr/Pth antibodies or aptamers can be used to transform plants and plant tissue using standard materials and methods. For example, A. tumefaciens has been widely used to transform plants with heterologous DNA (reviewed in Smith et ai, 1995). Heterologous DNA is incoφorated into the Ti-plasmid of A. tumefaciens and the plasmid is introduced back into the bacterium. The plant to be transformed is then infected with bacterium, typically by inoculating a wound site on the plant or plant tissue. The Ti-plasmid containing the heterologous DNA is then transferred into the nucleus ofthe plant cell where the transferred DNA is integrated into the host cell genome. Plants and plant tissue can also be transformed using methods such as protoplast uptake of heterologous DNA (Lorz et ai, 1985) and by particle bombardment using high-velocity microprojectiles that have been coated with the DNA that is to be introduced into the plant (Klein et ai, 1988), a method which is particularly well suited and widely used for monocot transformation, especially transformation ofthe Graminae. It should be understood that the examples and embodiments descπbed herein are for illustrative puφoses only and that various optimizations, modifications, or changes in light thereof will be suggested to persons skilled m the pertinent art and are included withm the spirit and purview of this application and the scope ofthe appended claims The following references are incoφorated herein by reference as if set forth in their entirety References An, G. et al. (1985) "New cloning vehicles for transformation of higher plants" Embo J. 4:277-284. Bogusz, D. et al. ( 1990) "Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants" Plant Cell 2:633-641. Bonas, U. et al. (1989) "Genetic and structural characterization of the avirulence gene, avrBs3 from Xanthomonas campestris pv ■ . vesicatoris" Mol. Gen. Genet. 218:127-136. Colas, P., etal. (1996) "Genetic Selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2" Nature 380:548-550. Conrad, R.C, et al. (1996) "In vitro selection of nucleic acid aptamers that bind proteins" Methods Enzymol. 267:336-367. Danielsson, L., C.A.K. Borrebaeck (1992) "Amplification of rearranged Ig variable region DNA from single cells" Antibody Engineering: A Practical Guide, pages 89-102. Day, P.R. (1974) Genetics of host-parasite interaction, W.H. Freeman and Co., San Francisco. Flor, H.H. (1992) "Inheritance of pathogenicity in Melampsora lini" Phytopathology 32:653-669. Gabriel, D.W. et ai (1993) "Host-specific virulence genes of Xanthomonas" Molecular mechanisms of bacterial virulence, pages 141-158. Gabriel, D.W., B.G. Rolfe (1990) "Working models of specific recognition in plant- microbe interactions" Annu. Rev. Phytopathoi 28:365-391. Gold, L. (1995) "Oligonucleotides as research, diagnostic, and therapeutic agents" J. Biol. Chem. 270:13581-13584. Holliger, P. et al. (1993) "Diabodies: Small bivalent and bispecific antibody fragments" Proc. Natl. Acad. Sci. USA 90:6444-6448. Hopkins, CM. et al. (1992) "Identification of a family of avirulence genes from Xanothomonas oryzae pv. oryzae" Mol. Plant-Microbe Interact. 5:451-459. Joosten, M.H.A.J. et al. (1994) "Host resistance to a fungal tomato pathogen lost by a single base-pair change in an avirulence gene" Nature 367:384-386. Klein, T.M. et ai (1988) "Factors Influencing Gene Delivery into Zea mays Cells by High- Velocity Microprojectiles" Bio/Technology 6:559-563. Kohler, G., C. Milstein (1975) "Continuous cultures of Fused Cells Secreting Antibody of Predefined Specificity" Nature 256:495-497. Lazo, G.R. et al. ( 1991 ) " A DNA transformation-competent Arabidopsis genomic library in Agrobacterium" Bio/Technology 9:963-967. Leach, J.E. et al (1996) "Molecular aspects of pathogenicity and host resistance requirements for signal transduction" Induction of defense responses in rice, D. Mills and H Kunoh, eds. Academic Press, New York (in press). Preston, G. et al (1995) "The HφZ proteins of Pseudomonas synngae pvs. syπngae, glycinea and tomato are encoded by an operon containing Yersinia ysc homologs and elicit the hypersensitive response in tomato but not soybean" Molec Plant-Microbe Interact 8.717-732 Smith, R.H., E.E. Hood (1995) "Agrobacterium tumefaciens Transformation of Monocotyledons" Crop Science 35(2):301-309. Swamp, S etal (1991) "A pathogenicity locus from Xanthomonas citri enables strains from several pathovars of X campestris to elicit cankerlike lesions on citrus" Phytopathology 81:802-809. Swamp, S. et al. (1992) "A Xanthomonas citri pathogenicity gene, pthA, pleiotrophically encodes gratuitous avirulence on nonhosts" Molec. Plant-Microbe Interact. 5:204-213. Szostak, J.W. (1992) "/« vitro genetics" Trends Biochem Sci 17 89-93. Tavladoraki, P. et al. (1993) "Transgenic plants expressmg a functional single-chain Fv antibody are specifically protected from vims attack" Nature 366:469-472. Tuerk, C. and Gold, L. (1990) "Systemacti evolution of hgans by exponential engnchment: RNA ligands to bacteπophage T4 DNA polymerase" Science 249:505-510. Ward, E.S. (1992) "Expression and purification of antibody fragments using Escherichia coli as a host" Antibody Engineering. A Practical Guide, pages 121-137. Wei, Z.M. et al (1992) "Haφin, elicitor ofthe hypersensitive response produced by the plant pathogen Erwima amylovora" Science 257:85-88. Yang, Y. et al. (1994) "Host-specific symptoms and increased release of Xanthomonas citri and X campestris pv. malvaceamm from leaves are determmed by the 102 bp tandem repeats of pthA and avrbβ, respectively" Molec. Plant-Microbe Interact 7:345-355. Yang, Y., D.W. Gabnel (1995) "Intragenic recombination of a single plant pathogen gene provides a mechanism for the evolution of new host specificities" J Bacteriol 177:4963- 4968. Yang, Y., D.W. Gabπel (1995) "Xanthomonas avirulence/pathogenicity gene family encodes functional plant nuclear targeting signals" Molec Plant-Microbe Interact 8:627-631 Yang, Y. et ai (1996) "Water soaking functιon(s) of XcmH1005 are redundantly encoded by members of the Xanothomonas avr/pth gene family" Molec. Plant-Microbe Interact 9:105-113. PCT Patent No. 91/15585, published October 17, 1991 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT INFORMATION: Applicant Name(s) : University of Florida Street Address: 223 Grinter Hall City: Gainesville State/Province: Florida Country: US Postal Code/Zip: 32611 Phone number: (352) 392-8929 Fax: (352) 392-6600 (ii) TITLE OF INVENTION: MATERIALS AND METHODS FOR THE INHIBITION OF PLANT PESTS AND PATHOGENS (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Saliwanchik & Saliwanchik (B) STREET: 2421 N.W. 41st Street, Suite A-l (C) CITY: Gainesville (D) STATE: Florida (E) COUNTRY: USA (F) ZIP: 32606 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) FILING DATE: 22-MAR-1996 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Lloyd, Jeff (B) REGISTRATION NUMBER: 35,589 (C) REFERENCE/DOCKET NUMBER: UF-159C1 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (352) 375-8100 (B) TELEFAX: (352) 372-5800 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 : NAGGTNNANC TGCAGNAGTC NGG 23 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : TGAGGAGACG GTGACCGTGG TCCCTTGGCC CC 32 (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : GACATCGAGC TCACCCAGTC TCCA 24 (2) INFORMATION FOR SEQ ID NO:4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCGTTTCAGC TCGAGCTTGG TCCC 24 (2) INFORMATION FOR SEQ ID NO:5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : CCCGAATTCT TAGATCTCCA GCTTGGTCCC 30 (2) INFORMATION FOR SEQ ID NO:6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 : CCCAAGCTTG ACATTGTGAC CCAGTCTCCA 30 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TCGGATCCGG AAACCATGNA GGTNNANCTG CAGNAGTCNG G 41 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TCCCCGGGTA CCTCACCGTT TCAGCTCGAG CTTGGTCC 3

Claims: Claims I claim: 1. An antibody, aptamer, or an antigen binding fragment thereof, wherein said antibody or aptamer binds to an avirulence or pathogenicity protein from a plant pathogen. 2. The antibody of claim 1, wherein said protein comprises at least one NLS. 3. The antibody or aptamer, according to claim 1, wherein said protein is encoded by a gene selected from the group consisting of avr and pth genes. 4. The antibody or aptamer of claim 3, wherein said gene is selected from the group consisting of Xanthomonas avr/pth genes. 5. The antibody or aptamer , according to claim 1 , wherein said protein is selected from the group consisting of Avr4, hφN, hφZ, AvrB4, Avrb6, Avrb7, AvrBs3, AvrXa7, AvrXalO, AvrB 101, AvrB 102 and PthA, PthB, PthC, PthN, PthPC, and PthP. 6. The antibody or aptamer, according to claim 1, wherein said antibody comprises V L and V H immunoglobulin domains. 7. The antibody or aptamer, according to claim 1, wherein said antibody is monovalent or bivalent. 8. The antibody or aptamer, according to claim 1, wherein said antibody is expressed in a plant cell. 9. A polynucleotide molecule comprising a nucleotide sequence that encodes an antibody or aptamer according to claim 1. 10. A method for protecting plants from plant pathogens or pests, which comprises incoφorating into the genome of said plant a polynucleotide molecule of claim 9. 1 1. A plant transformed with a polynucleotide molecule of claim 9. 12. A plant according to claim 11 , wherein said plant is dicotyledonous. 13. A plant according to claim 11 , wherein said plant is monocotyledonous. 14. Plant tissue transformed with a polynucleotide molecule of claim 9. 15. The plant tissue according to claim 14, wherein said plant tissue is selected from the group consisting of protoplasts, plant cells, plant seeds, and seedling tissue.

Inventors: GABRIEL DEAN W, GABRIEL, DEAN, W.
Applicants: UNIV FLORIDA, UNIVERSITY OF FLORIDA
Application year: 1997
Main classification: C12N15
IPC codes: C07K16, C12N15