MODIFIED TOMATO MOTTLE GEMINIVIRUS COAT PROTEIN GENE

Abstract: The subject invention pertains to modified plant virus genes which confer resistance on plants against geminivirus infection. A modified AV1 gene of tomato mottle geminivirus, was subcloned into an appropriate expression vector and transformed into tobacco plants. Expression of the modified gene conferred resistance against viral infection to the plant in which it was expressed. The present invention further concerns a method for conferring viral resistance on a plant. The invention also concerns modified coat protein genes, and fragments thereof, which confer viral resistance on a plant.

Description: DESCRIPTION MODIFIED TOMATO MOTTLE GEMINIVIRUS COAT PROTErN GENR This invention was made with government support under United States Department of Agriculture CSRS CBAG Grant No. 92-34135-7456. The government has certain rights in this invention. Cross-Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 60/022,680, filed July 26, 1996. Backj-Tound of the Invention Geminiviruses are a limiting factor in the production of tomatoes in the tropics. At least seven different geminiviruses have been identified as pathogens of tomato in this hemisphere (tomato mottle, potato yellow mosaic virus, tomato golden mosaic virus, tomato yellow mosaic virus, tomato leaf crumple virus, pepper huasteco virus, and the newly introduced from the Mediterranean, tomato yellow leaf curl virus). These viruses reduce yields directly by reducing the number and size of fruit. Their presence reduces profits by increasing the costs of management through increased use of insecticides to limit vector populations. In many parts of this hemisphere, there are no reliable means of control for these viruses. In the export tomato industry, which uses hybrid tomato cultivars produced in the U.S. and Europe, chemical controls are applied regularly to reduce the populations of virus-carrying whitefiies. The efficacy of this approach varies greatly, depending on a number of factors. Though, in general, insecticides may reduce virus incidence, frequently this reduction is to an insufficient level. It is not uncommon to find fields with high levels of virus-infected plants and to which large amounts of insecticides had been applied. A case in point is tomato mottle geminivinis (TMoV) which is a significant concern to tomato production in Florida (Polston, et al., 1996, Plant Disease 80: in press). This virus can occur at high rates of infection in tomato fields in Florida, but has also been responsible for epidemics in three other states and Puerto Rico (Polston et al, 1995). The virus has a narrow host range and is transmitted from tomato to tomato, primarily from plants in old fields to those in younger fields. The current method of control is the use of a systemic insecticide for approximately 8 weeks followed by foliar applications of various insecticides until the end of the season (15-18 week season). The costs of frequent pesticide use and the dependence on only one means of control could be reduced significantly through the use of tomato cultivars resistant to geminiviruses. There are a number of cultivars with tolerance to TYLCV but this resistance is narrow and has little to no effect on other geminiviruses. Ideally, what is needed is a gene or genes that give immunity to a number of geminiviruses. This would allow seed companies to put the gene or genes into cultivars with different horticultural attributes which could be grown in a wide variety of locations. The coat protein of plant viruses have a multi-functional role in viral pathogenesis and in many cases are involved in vector transmission. The coat protein of bipartite geminiviruses like TMoV is necessary for whitefly transmission of the virus and for virus epidemiology. Geminivinis coat protein also has a role in systemic virus movement (Pooma et al., 1996) and has nuclear localization signals. The coat protein domains involved with these functions have not been mapped. Based on other studies (for example, with the vector transmission for coat protein of potyviruses, mapped to a conserved Asp-Ala-Gly residues near the amino terminus), many of the functions of viral coat protein or other viral proteins are mapped to amino acid residues near the exposed surface of the protein. Terminal residues of a protein either amino or carboxy are often found near the exposed protein surface and thus implicated to have functional domains. Until now, no conventionally-derived gene has been found to give broad spectrum resistance to geminiviruses. The use of pathogen-derived resistance has been a successful approach in identifying genes which give resistance to viruses. Viral sequences or modifications of viral sequences have been shown to interfere with the process of virus infection when inserted into the plant chromosomes. This has been demonstrated for a number of plant virus families. Modified coat protein genes from viruses in the Potyviridae family, especially truncations at the carboxy terminus, have been found to give good resistance to potyviruses. This resistance appears to be at the level of mRNA, which when constitutively produced causes the plant to develop some specific responses to those sequences. Those specific responses then work to give resistance to potyviruses when inoculated to those plants. However, less is known about which genes from geminiviruses can be used to create resistance. Other reports on the use of geminivinis genes for resistance or tolerance are the movement protein gene from our laboratories (Duan et al, 1996, The Plant Cell, in submission), specific mutations to the rep gene of TMoV (pers. comm. J. Stout, Seminis Seed Co.), antisense rep gene of tomato golden mosaic virus (Day et al, 1991), subgenomic sequence of the A component of African cassava mosaic virus (Stanley et al. , 1990). Only one report has been published on the use of the geminivinis coat protein. The unmodified coat protein gene from tomato yellow leaf curl virus, a monopartite whitefly-transmitted virus from Israel, was transformed into tomato (Kunik et al, 1994). These transformed plants were reported to respond in one of two ways, a susceptible response or a delayed expression of symptoms followed by a recovery. Recovered plants exhibited some increased resistance upon repeated inoculations, but never achieved an effective level of resistance. Brief Summary of Invention The subject invention pertains to, in part, a method of modifying genes of TMoV to yield a modified gene useful for inducing resistance to TMoV in plants transformed with the modified gene. One object of this invention is to identify genes of TMoV which can be used to induce resistance to TMoV and other geminiviruses. Exemplified is the evaluation of a modified coat protein sequence of TMoV to elicit resistance to infection by TMoV in tobacco (Nicotiana tabacwri). The subject invention also pertains to modified plant virus genes which confer resistance on tobacco and tomato plants against tobacco mosaic tobamovirus and tomato mottle geminivinis infections, as well as resistance to infections of other related geminiviruses. A modified AVI gene of tomato mottle geminivinis, was subcloned into an appropriate expression vector and transformed into tobacco plants. Expression of the modified gene conferred resistance against viral infection to the plant in which it is expressed. The present invention further concerns a method for conferring viral resistance on a plant. The invention also concerns modified coat protein genes, and fragments thereof, which confer viral resistance on a plant. A further aspect of the present invention concerns novel transgenic plants with enhanced viral resistance. Other objects and advantages of this invention will become apparent from a review of the complete invention disclosure and the appended claims. Brief Description of the Drawings Figure 1 is a schematic representation of TMoV modified coat protein (AVI) construction into the binary vector pB1121. The modified coat protein gene of TMoV was cut out by using restriction enzymes. The coat protein sequence fragment was ligated to the cucumber mosaic virus (CMV) coat protein 5 '-untranslated region (leader sequence). The resulting CMV-coat protein (AV 1 ) was flanked by cauliflower mosaic virus (CaMV) 35S promoter and the polyadenylation signal from the nopaline synthase gene (NOS-ter) of the Agrobacterium Ti plasmid in Promega pGEMEX vector. The resultant cassette was cut out and inserted into the binary vector of pB1121, which contains the neomycinphosphotransferase II (NTP II) gene for selection and the reporter gene β-glucuronidase (GUS). Figure 2 is the determination of the presence of the transgene in tobacco transformants (T 0 ) by PCR amplification and agarose gel electrophoresis. The amplified product of CMV: TMoV AVI sequence from transgenic tobacco lines had an estimated size of 275 bp. Lanes from left to right: Lane 1 : M = 1 KB ladder size marker. Lanes 2-11 : T 0 tobacco plants transformed with pB1121 including the CMV: AVI sequence. Lane 12: T 0 tobacco plant transformed with pB1121 alone. Figure 3 is Northern gel blot analysis of transcripts derived from transformants (T, generation), containing tomato mottle virus (TMoV) modified coat protein (AVI) sequence. Total RNA was separated by electrophoresis in 1% agarose gel containing formaldehyde. The RNA was transferred to nylon membrane and hybridized with a 32 P- labeled DNA probe made from the TMoV coat protein sequence. The position of plant ribosomal RNA 26S and 18S in the gel is indicated. The arrow indicates the position of the - 1.0 kb transcript. Lanes from left to right are total RNA extracts from tobacco plants transformed: Lane 1 : line T,-cp0, with binary vector pB1121 only. Lanes 2-7: line T,-cp7, with pB1121 -AVI. Lanes 8-9: line T,-cp9, with pB1121-AVl . Lanes 10-1 1 : line T,-cp6, with pB1121-AVl. Lanes 12-13: line T,cp2, with pB1121-AVl. Figure 4 is the nucleotide sequence encoding a modified TMoV coat protein. Nucleotides 1-55 are the cucumber mosaic virus leader sequence, and nucleotides 56-890 encode the modified coat protein. Figure 5 is the deduced amino acid sequence encoded by the nucleotide sequence of Figure 4. Detailed Description of the Invention The subject invention concerns a modified plant virus gene that when expressed in a plant confers on that plant resistance to infection from plant pathogens. In one embodiment, the modified virus gene is an AVI gene of geminivinis which encodes a coat protein. One of the functions of movement proteins, such as the viral coat protein, involves interaction(s) with host components. Presumably, different viruses utilize the same host components for cell-to-cell movement. Therefore, interference with normal virus-host component interaction will result in a broad spectrum of virus resistance. Other viral genes, especially those involved in viral replication, tend to be very virus specific; thus, any interference at this level results in narrow spectrum virus resistance. Application of the present invention advantageously provides for a broad-based spectrum of virus resistance in plants. The subject invention also concerns polynucleotide molecules encoding the modified coat protein, or a variant or fragment thereof, of the present invention. In an exemplified embodiment of the invention, ten amino acid residues at the amino terminus of the TMoV coat protein were eliminated. The polynucleotide molecule comprises the nucleotide sequence, or a degenerate variant thereof, shown in Figure 4. The modification provides a coat protein whose core will remain intact and still be able to form virions. The subject invention also concerns a method for conferring resistance in a plant to infection by a plant pathogen such as, for example, a geminivinis. The method comprises transforming a plant with a modified plant virus coat protein such that when expressed in a plant confers on that plant resistance to viral infection. In one embodiment, the modified virus gene comprises a polynucleotide sequence that encodes a protein comprising the amino acid sequence shown in Figure 5, or a fragment thereof. In one embodiment, the methods of the subject invention can be used to confer resistance in plants to virus such as tobamoviruses and geminiviruses, including, for example, tomato mottle geminivinis, cabbage leaf curl geminivinis, potato yellow mosaic vims, tomato golden mosaic vims, tomato yellow mosaic vims, tomato leaf cmmple vims, pepper huasteco vims, tomato yellow leaf curl vims and others. The invention further concerns a recombinant polynucleotide sequence comprising a vector in which a polynucleotide sequence encoding the subject modified coat protein, or a fragment thereof, expressible in a suitable host has been inserted. Thus, the vector encodes the novel coat protein and/or a fragment of this protein with substantially the same properties. Specifically, the vector may be chosen from plasmids, phage DNA, or derivates or fragments thereof, or combinations of plasmids and phage DNA and yeast plasmids. The polynucleotide encoding the modified coat protein can be inserted into the multiple cloning site of a vector, such as the commercially available pUC vectors or the pGEM vectors, which allow for excision of the polynucleotide having restriction termini adapted for insertion into any desirable plant expression or integration vector. For this purpose any vector in which a strong promoter, such as a viral gene promoter, is operatively linked to the coding sequence of the polynucleotide of this invention could be used. For example, the powerful 35S promoter of cauliflower mosaic vims could be used for this purpose. Other plant expression vectors known can be used for this purpose. The invention also concerns a host infected, transformed, or transfected with a recombinant polynucleotide molecule comprising a vector in which a polynucleotide sequence coding for the desired protein, or fragment thereof, expressible in a suitable host has been inserted. Preferably, the polynucleotide is inserted into a suitable vector, and the recombinant vector can transformed into a bacterium or other host which is able to introduce the gene into a plant cell. The inserted polynucleotide is characterized in that the nucleotide sequence codes for the modified coat protein and/or a fragment of this protein with substantially the same properties. Among the many suitable hosts that can be infected, transformed, or transfected with the recombinant polynucleotide molecule according to the invention and thereby express this protein or fragments thereof are gram positive or negative bacteria such as E. coli, Bacillus subtilis, insect cells, plant cells, and yeast cells. In an exemplified embodiment, competent Agrobacterium cells are used for this purpose, and plant sections are exposed to the Agrobacterium harboring the recombinant vector. Regeneration of the plant cells in a selective medium to ensure the efficient uptake of the gene is preferred, following which the regenerated plants are grown under optimized conditions for survival. Alternatively, the recombinant vector can be introduced into plant cells by a biolistic method (Carrer, 1995). The subject invention also concerns transgenic plants and plant tissue comprising a polynucleotide encoding a modified coat protein of the present invention. The invention also concerns the modified coat protein, and fragments and variants thereof, that are encoded by the polynucleotide molecules described herein. For example, any modification of the N-terminus of the coat protein, including site directed mutagenesis, Bal 31 digestion, and deletions up to about 50 amino acid residues in from the N-terminus will have a similar effect in that the core of the coat protein is left intact. This invention is applicable to other geminiviruses, as similar modifications to the coat protein gene of any gemini vims will produce a modified gene useful for conferring resistance according to the teachings herein and using standard plant transformation techniques well known to those of ordinary skill in the art. As those of ordinary skill in the art will appreciate, any of a number of different nucleotide sequences can be used, based on the degeneracy of the genetic code, to produce the coat proteins described herein. Accordingly, any nucleotide sequence which encodes the modified coat protein described herein comes within the scope of this invention and the claims appended hereto. Also, as described herein, polynucleotide fragments of the gene encoding the modified coat proteins are an aspect of the subject invention so long as such fragments retain activity so that such fragments can enhance plant resistance to viral infection in methods described herein. Such polynucleotide fragments can easily and routinely be produced by techniques well known in the art. For example, time-controlled Ba 1 exonuclease digestion of the full-length DNA followed by expression of the resulting fragments and routine screening (Wei et al, 1983) can be used to readily identify polynucleotide fragments of the modified coat protein gene of the present invention capable of conferring resistance to viral infection on a plant transformed with the polynucleotide. The polynucleotides of the subject invention also encompass variant sequences containing mutations in the exemplified sequences. These mutations can include, for example, nucleotide substitutions, insertions, and deletions as long as the variant sequence encodes a modified coat protein capable of inducing resistance to viral infection in a plant transformed the variant sequence. A person skilled in the pertinent art can readily prepare and use variant polynucleotide sequences of the present invention. The amino acid sequences disclosed herein are based on standard single letter abbreviations for amino acid residues. References cited in this disclosure are incorporated herein by reference. Following are examples which illustrate materials, methods and procedures, including the best mode, for practicing the present invention. These examples are for illustrative purposes only and should not be construed as limiting. Example 1 : Construction of the Modified TMoV Coat Protein Gene Transformation Cassette. The coat protein gene of TMoV was modified in the region that encodes the N- terminus of the protein. Thirty nucleotides (nt) from the 5' end of the TMoV were deleted from a clone of the TMoV A component (Abouzid et al, 1992) using the restriction enzyme Nco 1 at the 5' end (nt 355) and Xba 1 at the 3' end (nt 1192). The cucumber mosaic vims (CMV) coat protein 5 '-untranslated region (leader sequence) which terminates with a Nco 1 restriction enzyme compatible sequence (obtained from a clone prepared by D. Gonsalves, Cornell University) was amplified and ligated to the TMoV coat protein gene sequence at the Nco 1 site. The construct is shown in Figure 1. The ligated construct consisting of the CMV leader sequence with the modified TMoV coat protein sequence was ligated into plasmid pB1121 (CLONTECH Laboratories, Palo Alto, CA), which served as a plant constitutive expression vector, and contained the cauliflower mosaic vims 35 S gene regulatory elements, the neomycinphosphotransferase 11 gene for selection, and the reporter gene β-glucuronidase (GUS). The binary expression plasmid with the modified coat protein sequence was transfened from E. coli to Agrobacterium strain LB4404 (CLONTECH Laboratories) to facilitate transformation. Example 2: Transformation of tobacco. Leaf disks of N tabacum "Xanthi" were transformed using A. tumefaciens (LB4404) carrying the modified TMoV coat protein, and shoots were regenerated according to the method of Horsch et al. (1985) on medium consisting of MS salts, sucrose (30 g/1), B5 vitamin (1 ml/1), indole-3-acetic acid (0.3 mg/1), 2iP, Ν6-[2- Isopentenyl] adenine (10 mg/1), kanamycin (100 mg/1), and mefoxin (200 mg/1). Transformed shoots were subsequently rooted on phytohormone-free medium containing 100 μg/ml of kanamycin and were transformed to soil. Example 3: Detection of transgene hv PCR amplification. The primers selected for the transgene (CMV: TMoV-AVl) amplification were, a specific primer for the cucumber mosaic vims (CMV) coat protein 5 '-untranslated region (EH 55, 5 '-GTTAGTTGTTCACCT-3 ') and an internal specific primer for tomato mottle vims (TMoV) coat protein (AVI) (EH 82, 5'-CCTTACCGATATGTGA-3'). PCR reaction was done in a Biometra thermocycler as follows: 94° C, 2 min.; 45 ° C, 2 min.; and 72° C, 3 min. for 35 cycles. The amplification was completed with a final cycle of DNA extention for 10 min. at 72° C. Example 4: Southern and Northern Blot Analysis. DNA Extraction from Tobacco Leaf Tissue DNA was isolated by modification of a method according to Soni and Murray (1994). Tissue (0.2-0.5 g) from transfoimant leaves or from transformant leaf disks agro-inoculated with sida mosaic geminivinis DNAs was ground in liquid nitrogen to a fine powder in a mortar and pestle. The powder was homogenized with 1.5 ml extraction buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 2% SDS, 100 mM LiCl, and 10 μg/ml proteinase K). The mixture was agitated for 15-30 min. and then centrifuged at 600 φm for 5 min. An equal volume of isopropanol was added to the supernatant and the nucleic acid was precipitated by centrifugation at 1000 φm for 5 min. and resuspended in 0.5 ml of TE (10 mM Tris, pH 8.0, 1 mM EDTA) with 20 μg/ml RNase. After incubation at 37° C for 30 min., the mixture was extracted once with phenol: chloroform-isoamyl alcohol (25:24:1), and once with chloroform. The nucleic acid was precipitated with a 1/3 volume of 7.5 M ammonium acetate and an equal volume of isopropanol and then resuspended in H 2 O. The resuspended DNA was used for PCR and Southern blot analysis. Southern Blots Standard protocols were followed to produce Southern blots. RNA Extraction from Tobacco Leaf Tissue Total RNA was isolated by a method according to Cocciolone and Cone (1993) and modified by Duan et al. (1996). Young tobacco leaf tissue (0.2 g) was ground in liquid nitrogen to a fine powder in a mortar and pestle. The powder was homogenized with 1.0 ml extraction buffer (7 M urea, 0.35 NaCl, 50mM Tris, pH 8.0, 20 mM EDTA, 1% sarkosyl). The mixture was extracted once with phenol: chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol (24:1) and then centrifuged at 600 φm for 5 min. An equal volume of isopropanol was added to the aqueous phase and the heavy nucleic acid was precipitated by chilling the mixture on ice for 5 min. RNA was obtained from the soluble fraction by centrifugation at 14,000 φm for 10 min. after incubation at -20° C for one hr. The pellet was washed with 70% ethanol and resuspended in 0.4 ml TE (10 mM Tris, pH 8.0, 1 mM EDTA) and extracted with chloroform-isoamyl alcohol. RNA was precipitated from the aqueous phase by adding 0.1 volume of 3 M sodium acetate and 2 volumes of 100% ethanol. Northern Blots Total DNA was electrophoresed in 0.9% agarose gel in TAE buffer and transferred to nylon membrane with alkaline transfer solution (0.4 M NaOH, 0.6 NaCl). Total RNA was electrophoresed in 1.0% agarose gel in MOPS buffer containing formaldehyde and transferred to nylon membrane with 10 x SSC solution. Probes were made by random primed 32 P-labeling of TMoV AVI DNA and SiMV DNA A component, with Prime-a-Gene™ labeling kit (Promega). Hybridization (overnight at 65 ° C) of blots were done according to Sambrook et al (1989) in 6 x SSC, 5 x Denhardt's solution and 0.5% SDS, 0.1 mg/ml salmon sperm DNA. The blots were washed according to Blair et al. (1995). Example 5: Western Immunoblot Analysis. Expression of TMoV modified coat protein in transgenic tobacco was examined by grinding young leaf tissue in 1.5% SDS in sterile water and then mixing one volume of tissue extract with one volume of Laemmli dissociation buffer (Laemmli, 1970). The mixture was boiled for 2 min. and the samples were subjected to 10% SDS-PAGE. The proteins were electrophoresed in a Bio-Rad Mini-PROTEAN II and the separated proteins were transferred to nitrocellulose membranes using Bio-Rad Trans-Blot™ electrophoretic transfer cell (Bio-Rad Laboratories, Richmond, CA). Immunoblots were labeled with polyclonal antiserum prepared to the coat protein of TMoV expressed in E. coli (Abouzid et al, unpublished) and goat antirabbit IgG alkaline phosphatase- conjugate, using chemi luminescent detection system, Western-Light (Tropix, Inc., Bedford, MA), and the protocol previously described (Cancino et al. , 1995). Example 6: Inoculation of Tobacco Leaf Disks with SiMV. The seeds of T 0 lines were germinated individually on MS rooting media containing 100 μg/ml kanamycin and the leaf disks of the progeny (T,) were agro- inoculated (Klinkenberg et al. , 1989) with SiMV DNA (Abouzid et al. , 1995). Example 7: Inoculation of Tobacco Plants with TMoV Using Whiteflies. The seeds of T 0 lines were germinated individually on MS rooting media containing 100 (μg/ml kanamycin) and the resulting T, seedlings were transplanted in soil for whitefly inoculation. Seedlings in the 4 to 6 leaf stage were inoculated with whiteflies which had been reared on TMoV-infected tomato plants maintained in a 30° C growth room. Adult whiteflies were added at a concentration of 20 whiteflies per plant in a 0.25m 3 screened cage in a growth room at 30° C. Whiteflies were given a 5-day inoculation access period on tobacco seedlings. The inoculation access period was terminated by the spraying of insecticidal soap on the inoculated plants. Example 8: Evaluation of TMoV-inoculated Tobacco Plants. Symptoms were evaluated approximately every 30 days after inoculation. Samples from the youngest leaves were collected for analysis for the systemic infection of TMoV at 70 days post inoculation by dot spot hybridization and/or by PCR primers to the B component. Samples were collected again at 100 days post inoculation and tested for systemic infection by TMoV using PCR. Dot spot hybridization using a probe composed of cloned TMoV B component under conditions of high stringency (Polston et al, 1993). Amplification of viral DNA was accomplished using degenerate primers (pBLlv2042 and pCRcl54) which amplify the B but not the A component. (Rogas et al, 1993). The amplification protocol used began with denaturation at 94° C for 5 min., followed by 35 cycles of the following: annealing at 50° C for 1 min., elongation at 72° C for 3 min., and denaturation at 94° C for 1 min. Example 9: Analysis of Tobacco Transformants. PCR amplification of the T 0 tobacco transformants with primers to the CMV leader sequence and the coat protein indicated that there were 10 plants of tobacco which had been transformed with both the CMV (leader sequence) and the modified TMoV AVI sequence (Fig. 2). In addition, as a control, one line was successfully transformed with pB1121 alone. Before transformed plants were transferred to the soil, leaf samples were collected from each plant and tested by GUS assays for transformation efficiency, Southern blots for transgene presence, and Western blots for expression of the modified coat protein. All transformed plants tested were determined to have the GUS gene. Southern blot analysis demonstrated the presence of multicopies of the transgene in the plant genome. Example 10: Presence of Modified Coat Protein mRNA. Northern blot analysis of selected tobacco transformants (T, generation), which had not been inoculated with vims, showed only one transcript approximately 1.0 kb in size (Fig. 3). The larger than expected CMV leader-coat protein transcript (1.0 kb vs. 0.9 kb) may be the result of the NOS-ter sequence (260 bp) of pB1121 in the transcript. Example 1 1 : Expression of Modified Coat Protein. Expression of the modified coat protein was not detectable in any of the transformants by standard Western blot protocols with the chemi luminescent detection system. This may have been due to instability of the amino-terminus modified coat protein, or due to levels of expression below the detection sensitivity. Example 12: Inoculation of Tobacco Plants with TMoV bv Whiteflies. The T, progeny from eight out of 10 lines T 0 tobacco plants were inoculated with TMoV by whiteflies. Analysis of the plants at 70 days post inoculation (Table 1) revealed that even though there was segregation within the lines, all lines tested had lower rates of infection than the control (T,-cp0). In general, there was good agreement between symptom expression and the presence of viral DNA as determined by either assay, since there was good agreement between the results of dot spot hybridization and PCR analyses. We did not find any cases where TMoV was replicating at levels so low as to be detected by PCR but not by dot spot hybridization. Rates of resistance to TMoV varied among the lines. In two lines, T,-cpl0 and T,-cpl 1, no plants became infected even 100 days after the initial inoculation. The variation among the lines could be due to differences in transgene copy number, location of transgenes, and/or their effects on the segregation ratio of resistant and susceptible plants. The rates of infection generally increased gradually over time. This may have been due in part to continual inoculation by whiteflies which emerged from the eggs of the whiteflies used in the initial inoculation. Overall, there was a good agreement between the presence of the transgene, as determined by PCR, and immunity to systemic infection by TMoV at 100 days after the initial inoculation (Table 2). This varied among the lines, with some lines (T,-cp4, T,-cpl0, and T r cpl l) showing a 90 to 100% correlation. Example 13: Evaluation of Transgenic Tobacco for Resistance bv Agro-inoculation. In a preliminary study we examined the effect of the TMoV resistant transgenic tobacco on the DNA replication of Sida mosaic vims (SiMV), a geminivims, which is closely related to TMoV (Abouzid et al, 1995). We conducted in vitro leaf disk agro- inoculation assays (Klinkenberg et al, 1989). Leaf disks from 10-15 T, progeny plants of transgenic T 0 plants were inoculated with mixed cultures oi Agrobacterium carrying head-tail dimer of the cloned genomes (DNA A and DNA B) of SiMV (Abouzid et al, unpublished) and assayed for their ability to suppress viral DNA replication. Ten days after agro-inoculation, assays for viral DNA were conducted with DNA extracts of the transformed leaf disks. Following hybridization of the Southern blots with SiMV DNA A probe (full-length), the presence of single-stranded (ss) SiMV genomic DNA and its replicative intermediate forms oc and sc in the inoculated vims-susceptible leaf disks indicated that SiMV DNA was released from the Agrobacterium Ti plasmid and replicated in inoculated tissues. The amounts of viral DNAs were variable in tobacco transgenic lines. Very low amounts of SiMV DNAs were detected in one transgenic line. Tobacco transgenic for a modified TMoV coat protein gene showed resistance to systemic infection by TMoV in whole plants. The presence of the transgene was correlated with symptom suppression and in inability to detect viral DNA in leaves produced 60+ days after initial inoculation with TMoV. One tobacco line transgenic for a modified TMoV coat protein gene suppressed the replication of sida mosaic geminivims in a leaf disk assay. Plants transformed with the modified TMoV coat protein gene were shown to produce mRNA transcripts, but expression of the modified coat protein was not detected in Western blots. The modified TMoV coat protein gene is useful in developing resistance in plants to geminiviruses according to the teachings herein. 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Claims: Claims We claim: 1. A method for conferring on a plant enhanced resistance against plant viral infection which comprises transforming a plant with a polynucleotide encoding a modified plant vims coat protein, wherein said polynucleotide confers on said plant resistance to plant viral infection. 2. The method according to claim 1 , wherein said polynucleotide comprises the nucleotide sequence shown in Figure 4, or a degenerate fragment or variant thereof. 3. The method according to claim 1, wherein said polynucleotide comprises nucleotides 56 to 890 of the nucleotide sequence shown in Figure 4, or a degenerate fragment or variant thereof. 4. A polynucleotide encoding a modified plant vims coat protein, wherein said polynucleotide confers enhanced viral resistance to plants harboring said polynucleotide. 5. The polynucleotide according to claim 4, wherein said polynucleotide comprises a nucleotide sequence shown in Figure 4, or a degenerate fragment or variant thereof. 6. The polynucleotide according to claim 4, wherein said polynucleotide comprises nucleotides 56 to 890 of the nucleotide sequence shown in Figure 4, or a degenerate fragment or variant thereof. 7. A modified plant vims coat protein, said modified coat protein capable of conferring enhanced viral resistance in plants expressing said protein. 8. The modified plant vims coat protein according to claim 7, wherein said protein comprises an amino acid sequence shown in Figure 5, or a fragment or variant thereof. 9. The modified plant vims coat protein according to claim 7, wherein said protein comprises an amino acid sequence, or a fragment or variant thereof, encoded by nucleotides 56 to 890 of the nucleotide sequence shown in Figure 4. 10. A transgenic plant or plant tissue having increased resistance to viral infection, said plant being transgenic for a polynucleotide encoding a modified plant vims coat protein according to claim 7. 1 1. A cell transformed with a polynucleotide according to claim 4.

Inventors: ABOUZID AHMED, HIEBERT ERNEST, POLSTON JANE E, ABOUZID, AHMED, HIEBERT, ERNEST, POLSTON, JANE, E.
Applicants: UNIV FLORIDA, UNIVERSITY OF FLORIDA
Application year: 1997
Main classification: C12N15
IPC codes: A01H5, C07K14, C12N5, C12N15, C12P21