WO1997007217A1 - Methods and materials for producing pathogen-resistant plants - Google Patents

Abstract

This invention comprises a mutant plant virus gene which confers resistance on tobacco and tomato plants against tobacco mosaic tobamovirus and tomato mottle geminivirus infections and infection by other related geminiviruses. A gene was initially isolated from the known BC1 gene, between nucleotides 1278 and 2311 of the B component of tomato mottle geminivirus. Upon subcloning of this DNA fragment into an appropriate expression vector and transformation of the gene into tobacco plants, a truncated gene product was produced which confers resistance against viral infection to the recombinant plant in which it is expressed.

Description

DESCRIPTION

METHODS AND MATERIALS FOR PRODUCING PATHOGEN-RESISTANT PLANTS

Cross Reference to Related Application This application claims the benefit of U.S. Provisional Application No. 60/015,051, filed April 9, 1996, and U.S. Provisional Application No. 60/002,158, filed August 11, 1995.

Acknowledgement of Government Support

This invention was made with government support under USDA/DSRS CBAG Special

Grants Program, grant Nos. 93-34135-8607; 92-34135-7456; and the Florida Tomato Committee

Grant No. 90153-C. The government has certain rights in this invention.

Background ofthe Invention (i). Field of the Invention

This invention pertains to the field of conferring pathogen resistance to plants. More particularly, the invention is directed to virus-resistant transgenic plants.

(ii). Background ofthe Invention

Tomato producers suffer significant losses due to tomato mottle geminivirus infection. Currently farmers must purchase chemicals in order to control tomato mottle virus in their tomato fields. Similarly, losses are experienced by farmers producing tobacco as a result of tobacco crop infection by tobacco mosaic tobamoviruses. Accordingly, there is a need for a solution to this problem which is less costly and less damaging to the environment than the chemical controls currently employed.

Production of transgenic plants with enhanced phenotypic characteristics is a relatively recent development in the arsenal available to farmers. Nevertheless, the value of this technology has been demonstrated repeatedly in recent years. However, what is required is the identification of appropriate genes to confer the desired phenotype, in this case, pathogen resistance.

Transformation of plants with portions of viral genomes may result in plants with virus resistance (Beachy, 1993). This phenomenon is known as "pathogen-derived resistance" (Sanford and Johnson, 1985). The level of resistance obtained is variable. This variability has been attributed to the random nature ofthe transformation process (Lomonossoff, 1995). Independent lines of plants generated from a single transformation experiment may contain different transgene copy numbers inserted in various chromosomes. Phenotypic differences have been noted among plant lines containing a single copy ofthe transgene. Some ofthe variability in transgene expression has also been attributed to tissue culture-induced changes (Phillips et al., 1994). This variability in the phenotype is also observed in subsequent progeny derived from the R<, plants.

Introduction of a mutation (defective) in one motif of a multimotif protein has been proposed as a strategy for interfering with viral replication. This interference with the function of wild-type genes has been referred to as a dominant negative mutation. Maxwell and his co-workers have constructed transgenic plants expressing a modified tomato mottle geminivirus replication- associated protein (RAP), mutated in a NTP-binding motif, which appears to interfere with viral replication (Hanson, et al., 1991). This dominant negative mutant for the tomato mottle gemimvirus RAP gene has been tested for tomato mottle geπ-inivirus resistance in tomatoes. Noris et al. (1994, First International Symposium on Geminiviruses, Almeria, Spain) found inhibition of Tomato

Yellow Leaf Curl Virus (TYLCV) DNA replication in tobacco protoplasts co-transfected with TYLCV and a construct ofa truncated RAP expressed under control ofa CaMV 35S promoter.

This control strategy is likely to be very virus specific since the RAP binding sites essential for function have been shown to require a sequence-specific interaction between RAP and the origin of replication (Fontes et al, 1994). This enables the transacting factors of RAP to ώscriminate between the replication origins of closely related geminiviruses. Because of Geniinivirus diversity and adaptability, virus-specific control strategies are of limited value under field conditions.

There have been several reports in recent years relating to the inhibition of infection of certain plants by specific viral pathogens. For example, Von Arnim and Stanley (1992) reported on the inhibition of systemic infection by African Casava Mosaic Virus (ACMV) by a movement protein from the related Geminivirus, Tomato Golden Mosaic Virus (TGMV). This was accomplished by replacing the ACMV coat protein coding sequence with the BL 1 or BRI movement gene sequences from TGMV and then testing the ability ofthe recombinant ACMV to infect its host, Nicotiana benthamiana (which is also the host of TGMV). The authors found that the TGMV gene did not complement the ACMV recombinant, and hypothesized that direct genomic expression of a dominant negative mutant might produce plants resistant against geminiviruses.

Cooper et al. (1995) disclosed that transgenic tobacco plants expressing a defective tobacco mosaic virus (TMV) movement protein were resistant to infection by multiple viruses, while transgenic plants expressing the natural movement protein had increased susceptibility to infection by TMV and other viruses.

Nejidat and Beachy (1990) disclosed that transgenic tobacco plants expressing a TMV coat protein have increased resistance against several of the tobamoviruses. Gilbertson et al. (1993) disclosed the reduced pathogenicity of pseudorecombinants of two bipartite geminiviruses, tomato mottle (ToMoV) and TGV-MX1.

Brief Summary ofthe Invention

We have discovered a mutated plant virus gene which protects tobacco plants against tomato mottle geminivirus and tobacco mosaic tobamovirus infections. This resistance gene has been introduced into tobacco chromosomal DNA by genetic engineering. The transgenic tobacco plants expressing this gene show resistance to tomato mottle gemiriivirus and tobacco mosaic tobamovirus infections (lack of or reduction of disease symptoms when inoculated with the viruses). The mutated gene can be introduced into chromosomes of desirable tomato and tobacco lines to develop commercially improved tomato and tobacco cultivars/hybrids.

Accordingly, this invention comprises a mutant plant virus gene which confers resistance on tobacco and tomato plants against tobacco mosaic tobamovirus and tomato mottle geminivirus infections, as well as resistance to infections of other related geminiviruses. The known BCl gene, between nucleotides 1278 and 2311 of the B component of tomato mottle geminivirus, was subcloned into an appropriate expression vector and transformed into tobacco plants. A mutated gene product was produced which confers resistance against viral infection to the recombinant plant in which it is expressed.

One object of this invention is to provide a memod for conferring viral resistance on a plant. Another object of this invention is to provide a mutated BC 1 gene and any fragment thereof which confers viral resistance on a plant.

Another object of this invention is to provide novel transgenic plants with enhanced viral resistance.

Other objects and advantages of this invention will become apparent from a review ofthe complete invention disclosure and the appended claims.

Brief Description ofthe Figures Figure 1 is the sequence ofthe single stranded mutated tomato mottle geminivirus BCl gene except for positions 1742-1766 which initially were not identified; wild-type nucleotides which are different in the mutant gene are shown in lower case text above the mutant gene sequence. Figure 2 is the sequence shown in Figure 1 along with its complementary strand; the translational start and stop codons are underlined; the terrnini are Hindlll restriction sites.

Figure 3 is the deduced amino acid sequence ofthe mutated gene product encoded by the nucleotide sequence of Figure 1, except for positions 151-159 which in initial sequencing efforts were not identified.

Figure 4 shows a comparison ofthe mutant and wild-type gene products (the mutant protein is the lower sequence).

Figure 5 shows phenotypic comparison of transgenic R, tobacco plants expressing BCl protein of TMoV. Transgenic plants were derived from a R_Q plant which contained two copies of BCl gene (see Fig. 6) and which did not show any stunting. (A) Plants from left to right: a. transgenic plant (BCl -3-11-5) expressing symptomatic BCl protein, showing stunting, mottling, and curling on the leaves. Symptoms are more sever than those induced by TMoV infection; b. transgenic plant (BCl -3- 11-2) which contains one copy of the non-symptomatic BCl and the symptomatic BCl transgene, showing mottling with no stunting; c. transgenic plant (BCl -3-11-6) which contains one copy of non-symptomatic BCl transgene; and d. non-transgenic tobacco. (B)

Plant on the left as in b, Fig. 5A and on the right as in c, Fig. 5A. The plants in A were photographed 45 days after transplanting, and in B 90 days.

Figure 6 shows Southern blot analysis ofthe R, transgenic plant with different phenotypes. Segregation ofthe BCl transgene in R, generation of transgenic tobacco plants which displayed different phenotypes in Fig. 5 (BC1-3-11-1 and -2, mottling only, -4 and -5, severe stunting and mottling, -6 and -7, no visible symptoms). Blots from BCl -3- 16-2 showing stunting and mottling, and BCl -3-6-3 and -4, no visible symptoms are shown for comparative purposes; NT- nontransformed plant; and pKYsBC 1, vector construct used for transformation. Genomic DNA of the transgenic plants was extracted and digested with Xbal. Southern blots were subjected to hybridization with ³²P-labeled BC 1 DNA fragment.

Figure 7 shows Western blot analysis ofthe P30 fraction of tissue extracts from transgenic R, tobacco plants expressing the BCl gene. Lanes represent extracts from plants described in Fig. 6 except for TMoV-infect. extract from TMoV infected tissue). The subcellular fractions, P 1, P30 and S30 were prepared (Pascal et al, 1993) and subjected to SDS-PAGE (Schagger) with some modification and immunoblots using the polyclonal antiserum against expressed BC 1 protein. The results of the P 1 and S30 fractions are not shown here.

Figure 8 shows Northern blot analysis of transgenic plants which express the BCl gene, probed with labeled-BC 1 DNA. Two BC 1 related transcripts were found in the transgenic plants which expressed the full-length BCl gene, while only one transcript was found in the transgenic plant which expressed a 3 '-truncated form ofthe BCl gene (BCl -3-11-6). The samples indicated are as in Fig. 6.

Figures 9A-1 thru 9A-5 and 9B show nucleotide sequences (A) and predicted amino acid sequences (B) ofthe TMoV BC 1 and its transgene mutants. The nucleotide sequence of TMoV BC 1 gene from GenBank Accession U14461. The sequence ofthe PCR amplified BC 1 ORF was verified before and after cloning into pGEM-T vector. BCl A sequence determined from an asymptomatic, multicopy transgenic plant which expressed full length BCl protein. The sequence was analyzed from the PCR product derived from genomic DNA (BC 1-3-6-3 A). BC 1 At r sequence determined from the cDNA, the RT-PCR products, amplified from the total RNA (BC1-3-11-6A). The sequence was also verified by sequencing the PCR product from the genomic DNA and from cloned

PCR product. BC 1 S sequence determined from a symptomatic transgenic plant which expressed full length BCl protein The sequence was analyzed after RT-PCR of total RNA (BC1-3-11-5S), after PCR amplification of genomic DNA (BC1-3-11-5S) and after PCR amplification from 3 different lines with a similar phenotype. Note that identical nucleotides and amino acid residues are indicated by (.).

Detailed Description ofthe Invention

The subject invention concerns a mutated plant virus gene that when expressed in a plant confers on that plant a resistance to infection from plant pathogens. In one embodiment, the mutated virus gene is a BC 1 gene of geminivirus. The mutated gene of the present invention can be prepared by inserting the wild-type gene into the genome of a plant and identifying those plants transformed with the gene that exhibit increased resistance to viral infection.

The subject invention also concerns a method for conferring resistance on a plant to infection by plant pathogens. The subject method comprises inserting a wild-type viral movement gene, such as BC 1 , into the genome of a plant and then identifying those plants that do not exhibit pathogenic symptoms when the inserted gene is expressed but which have enhanced resistance to infection by pathogens.

The subject invention also concerns transgenic plants and plant tissue having a mutated gene ofthe present invention incoφorated into their genome. The following is a specific example ofthe subject invention, a method for creating a virus- resistant plant, using the BCl gene of tomato mottle geminivirus to illustrate the invention. The method is generally and broadly applicable to other plant viruses.

The complete sequence ofthe BCl gene of tomato mottle geminivirus is known (Abouzid et ai, 1992, herein incoφorated by reference). The BC 1 gene of tomato mottle geminivirus ofthe B component ofthe genome is isolated in sufficient quantity for subcloning in an expression vector. This may be accomplished by any of several methods well-known in the art. A simple method is to use a pair of specific primers to amplify the desired segment according to the well known polymerase chain reaction (PCR) technique. For this puφose, a useful primer pair such as: 5 '-CCCAAGCTTCGAGTTCGAAACTGC-3 ' (SEQ ID NO. 1) and

5'-CCCAAGCTTAACGAAGTGTGTTTGAC-3' (SEQ ID NO. 2) may be used. All or portions ofthe BCl gene may be used for this puφose.

Once sufficient quantities of the gene are obtained, the gene is cloned into a vector for production of a stable source for mass production of the gene. Any vector known in the art can be used for this puφose, and mass quantities of the vector may be cultured, for example, by transformation of competent bacterial cells such as E. coli followed by harvesting ofthe plasmid DNA. Preferably, the gene is 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 gene having restriction termini adapted for insertion into any desirable plant expression or integration vector. For this puφose any vector in which a strong promoter, such as a viral gene promoter, is operatively linked to the coding sequence ofthe mutant gene of this invention could be used. For example, the powerful 35S promoter of cauliflower mosaic virus could be used for this puφose. In one embodiment of this invention, this promoter is duplicated in a vector known in the art as pKYLX 71:35S² (Morgan et ai, 1990). However, other plant expression vectors could be used for this puφose.

Once the gene is excised and re-subcloned into a desirable expression vector, the gene is transformed into a bacterium or other vector which is able to introduce the gene into a plant cell. Alternatively, the gene may be introduced into plant cells by a biolistic method (Carrer, 1995). Preferably, competent Agrobacterium cells are used for this piupose, and plant sections are exposed to the Agrobacterium harboring the BC 1 gene. Regeneration of the plant cells in a selective medium to ensure the efficient uptake ofthe gene is preferred, followmg which the regenerated plants are grown under optimized conditions for survival.

As a result of this process, it has been discovered that a large proportion of regenerated tobacco plants which were transgenic for the BCl gene had a spontaneously mutated gene which expressed a mutated gene product. Unexpectedly, the plants harboring the mutated gene had increased resistance to viral infection by both DNA and RNA plant viruses, without any observed deleterious effects resulting from expression ofthe mutated BC 1 gene (in contrast, expression ofthe wild-type gene produces disease symptoms). While those skilled in molecular biology are able to clone the known BCl gene into a plant expression vector to obtain the mutated gene of the present invention, the mutant gene of this invention has also been deposited prior to filing the instant patent application with the American Type Culture Collection (ATCC), 12301 Parkiawn Drive, Rockville, Maryland 20852 USA. The mutant gene was cloned in a bacterial vector (pGEM-T) and the construct is named TMBC lm. The deposit has been assigned accession number ATCC No. 97244 by the repository.

The subject deposit was deposited under conditions that assure that access to the deposit will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1J4 and 35 U.S.C. 122. The deposit will be available as required by foreign patent laws in countries wherein counteφarts of the subject apphcation, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject deposit will be stored and made available to the public in accord with the provisions ofthe Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample ofthe deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition ofthe deposit. All restrictions on the availability to the pubhc ofthe subject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

To use the biological materials deposited, all that is necessary is for the DNA to be solubilized in an appropriate transformation buffer for the cell type into which the gene is to be transformed. For E. coli, competent cells are prepared and transformed according to methods well known in the art (see Maniatis et al. , 1982), and transformed cells selected in an ampicillin growth medium. The plasmid is then isolated from the E. coli and excised from the pGEM-T vector using, for example, HindHI restriction enzyme. The excised gene fragment has a size of about 1100 bp. The Hindlll fragment is then cloned into the Hindlll site of an appropriate expression vector as described below. In addition to the above, Figure 1 provides the sequence of the mutant gene of this invention, except for a stretch of 25 nucleotides corresponding to positions 1742-1766, which were not identified in initial sequencing efforts. There are several mutations in the polynucleotide of Figure 1. Those mutations are part of the instant invention. Further, Figure 2 provides the complementary strand of the mutant polynucleotide and shows the HindHI termini. Figure 3 provides the deduced amino acid sequence ofthe mutated gene product, except for amino acids 151- 159 which were not identified in the initial sequencing. The differences in amino acid sequence between the wild-type BCl product and mutant BCl products are shown in Figure 9B. Figure 4 shows a comparison between the wild-type (upper sequence) and mutant protein (lower sequence) based on initial sequencing efforts.

While this description provides a specific gene and fragments thereof which confer resistance on plants to geminivirus and tobamovirus infection, those skilled in the art will recognize that mutations other than or in addition to the specific mutations shown herein could achieve similar results. In fact, the method taught herein, by which the mutant gene disclosed herein was obtained, is broadly applicable to the obtention of similarly useful mutated movement genes of any virus.

Furthermore, it is predictable, based on the instant disclosure, that the instant genes and polynucleotide molecules described herein, as well as likewise-derived genes, can confer resistance on a plant against infection by a wide variety of plant pathogens which depend on movement gene or other gene products for their pathogenesis, including both DNA and RNA viruses.

Example 1 - Development of transgenic tobacco plants

A. Construction of BCl gene into an expression vector. The BCl gene (nucleotides between 1278 and 2311 of the B component of tomato mottle geminivirus; Abouzid et al. , 1992) was amplified from the extracts of tomato mottle geminivirus infected tomato plants by polymerase chain reaction (PCR) technology. The primers used to amplify viral BCl were

5'-CCCAAGCTTCGAGTTCGAAACTGC-3' (SEQ ID NO. 1) and 5'-CCCAAGCTTAACGAAGTGTGTTTGAC-3' (SEQ ID NO. 2).

The amplified BCl segment was cloned into a pGEM-T vector and then digested with Hind HI. The excised BCl segment was ligated into the unique Hind IH site ofthe binary pKYLX 71:35 S² vector.

B. Agrobacterium transformation. Competent cells oi Agrobacterium tumefaciences LBA 4404 were prepared as described by An, et al, (1985). The BCl gene in the pKYLX 71:35 S² vector was directly transferred into the Agrobacterium. The clone was kept in a -80 ° C freezer for further use.

C. Plant transformation The Agrobacterium carrying the BCl gene in the pKYLX 71:35 S² vector was used to transform the leaf discs of Nicotiana tobacum cv. Xanthi. The Agrobacterium cells were cultured in YEP broth containing 50 μg/ml kanamycin and 10 μg/ml tetracycline and 25 μg/ml streptomycin for 24-30 hours. Agrobacterium cells were collected and resuspended in YEP broth. Leaf discs cut from expanded young sterile seedlings were dipped into the Agrobacterium suspension and then placed on a selective medium containing 200 μg/ml Mefoxin and 100 μg/ml kanamycin. Regeneration and selection were carried out with the media, and took 6-8 weeks. The kanamycin resistant plants were individually grown in soil under sterile condition for a week, and then transplanted to pots in a growth room and/or greenhouse.

Example 2 - PCR, Southern blot and ELISA analysis

Transformation of the tobacco plants was confirmed by PCR analysis for BCl gene in chromosomal DNA extracts, by Southern blotting with a BCl probe, and by ELISA analysis for NPT II (Neomycin Phosphotransferase II). Twenty-three plants were transgenic for BC 1.

Example 3 - Western blot analysis

Infected leaves of tomato plants were powdered after freezing in hquid nitrogen and extensively ground with a mortar and pestle in two volumes of ice-cold grinding buffer (GB: 100 mM Tris-HCl, pH 8.0, 10 mM EDTA and 5 ml dithiothreitol)(Deom, et al, 1990). Membrane and cell-wall fractions were prepared as described by Pascal, et al., (\ 993). The blotting procedure was conducted essentially as described by Towbin, et al, (1979) using a Bio-Rad Mini-Protein Electrophoresis Cell and Bio-Rad Trans-Blot Electrophoretic Transfer Cell. The separator gel for small proteins was prepared with 12.5% polyacrylamide in gel buffer (Laemmli, 1970). The protein gels were transferred to nitrocellulose membrane (Bio-Rad Trans-Blot, 0.4 μm). The detection of expressed BCl protein in transgenic tobacco plants was conducted with Western- Light- Chemiluminescent Detection System (TROPIX, Inc.). The BCl protein was detected at a relatively high level and extracts from about 50% ofthe plants showed a smaller (truncated) BCl protein (28k Da) than the wild-type (33k Da).

Example 4 - Evaluation of transgenic tobacco plants for symptoms due to the expression ofthe BCl

The BC 1 gene has been implicated as a symptom inducing element of a bipartite geminivirus during infection. Eleven transgenic tobacco plants which expressed the full length BCl protein showed disease symptoms. Twelve plants expressing the truncated BCl protein did not show disease symptoms. Example 5 - Resistance to tomato mottle geminivirus and tobacco mosaic tobamovirus

Transformed tobacco plants (R, generation) expressing BCl were tested for susceptibility to tomato mottle geminivirus infection by natural transmission with the whitefly vector and by mechanical inoculation with extracts from infected plants. The inoculated plants were evaluated for resistance to tomato mottle gemimvirus by symptom development, and by enzyme linked immuno¬ assays (ELISA) using antiserum reactive to tomato mottle geminivirus coat protein. The transgenic plants expressing the truncated BCl protein were free of symptoms and had very low ELISA readings. Transgenic tobacco plants subjected to mechanical inoculation with tobacco mosaic tobamovirus showed reduced disease symptoms compared to inoculated non-transgenic plants.

Example 6 - Analysis ofthe BCl gene expressing truncated protein

The BCl gene from the tobacco plants expressing the truncated BCl protein was PCR- amplified and sequenced. This data indicates that the BCl gene has undergone spontaneous mutation(s) in about 50% ofthe transgenic BCl tobacco plants. During the tissue culture phase, plant cells containing the mutated BCl gene may have a selective advantage over the wild-type BCl expressing cells.

Example 7 - Production of transgenic tomato plants

The mutated BCl gene in the pKYLX 71:35 S² vector is suitable for the production of tomatoes transgenic for the gene via Agrobacterium transformation as described above for tobacco.

The mutated BCl gene provides similar resistance to tomato mottle geminivirus in tomato as seen in transgenic tobacco. The introduction of this mutated BCl gene into the chromosome of desirable tomato lines leads to tomato mottle geininivirus resistance in commercially acceptable tomato cultivars hybrids. In addition, it is predictable that this resistance is active against other geminivirus infections. Resistance to tobacco mosaic virus was also detected in the transgenic tobacco expressing the mutated BCl gene, indicating that resistance to RNA viruses also is possible with the expression of this mutated gene from a DNA plant virus. The mutated gene in tomato offers resistance to tomato mosaic tobamovirus, a virus related to tobacco mosaic tobamovirus.

Example 8 - Production of BC 1 gene fragments useful for conferring virus resistance to plants.

Fragments ofthe mutant BC 1 gene which are useful for conferring virus resistance to plants can be produced by use of BAL31 exonuclease for time-controlled limited digestion ofthe mutant

BC 1 gene. Methods of using BAL31 exonuclease for this puφose are well known in the art, and have been widely, used for over a decade (Wei et al. , 1983). By using BAL31 exonuclease, one can easily remove nucleotides from either or both ends ofthe mutant BCl gene to systematically and certainly generate a wide spectrum of DNA fragments which have controlled lengths and are from controlled locations along the entire length of the mutant BCl gene. Hundreds of such fragments from various points along the entire mutant BCl gene DNA sequence can be systematically generated in one afternoon. These gene fragments are then cloned into appropriate vectors and ultimately transferred into plant cells according to the methods disclosed above. Plant cells transformed with these fragments are routinely cultured and regenerated into plants, which are then tested for resistance to viruses. In this manner, fragments of the mutant BCl gene which are sufficient to confer viral resistance are routinely and predictably identified.

Example 9 — Production of additional mutants conferring virus resistance to plants.

Tobacco was transformed with the movement protein (pathogenicity) gene (BCl) from tomato mottle geminivirus (TMoV) using Agrobacterium-mediated transformation. Different transgenic tobacco lines expressing the BCl protein had phenotypes ranging from plants with severe stunting and leaf mottling to plants with no visible symptoms. The sequence data for the BCl transgene for the different phenotypes indicated unexpected mutation(s). A mutated BC 1 transgene suppressed the phenotypic expression ofthe symptomatic BCl gene in tobacco lines containing both copies of the BCl gene. The present invention shows spontaneous mutations in the transgene to be common Agrobαcterium-msdiated transformations, and this phenomenon can be utilized in the creation and selection of pathogen-resistant plants using pathogenicity genes during transformation.

The expression of tomato mottle gemini virus (TMoV) (Abouzid et αl, 1992) movement protein gene (BCl) was examined in transgenic tobacco plants for evaluation of function and for possible utilization in pathogen-derived resistance. The BC 1 gene has been implicated as a symptom inducing element of a bipartite geminivirus during expression in transgenic plants (Pascal et αl, 1993 ; von Arnim and Stanley, 1992). Transgenic tobacco expressing the BC 1 gene was constructed utilizing standard Agrobαcterium-medisiod transformation. Suφrisingly, a number of plants expressing TMoV BCl protein based on Western blot analysis, did not show the expected virus- symptom phenotype. Only 11 of the 19 transgenic R_Q tobacco plants which expressed the BCl protein showed disease symptoms ranging from mild to severe. The observation that eight plants expressing the BC 1 protein did not show symptoms was unexpected.

From further analysis, the three phenotypes were observed in the R, generation derived from a R_Q plant which did not show any apparent stunting (Fig. 5). The three observed phenotypes were: 1) Severe stunting and mottling, more severe than the typical symptoms associated with TMoV infections in tobacco; 2) Mottling with no stunting of growth; and 3) No visible symptoms, plants indistinguishable from nontransformed plants.

These transgenic plants were analyzed by Southern blots to identify gene copy number (Fig.

6). The transgenic plant showing slight mottling with no stunting had two copies ofthe BC 1 gene. Other progeny from this line which had a severe symptom phenotype or a non-symptomatic phenotype only had one copy. Progeny from three other lines examined, one with symptomatic and two with non-symptomatic phenotypes, had 3, 3, and 5 copies ofthe BCl gene, respectively.

High levels of the BCl protem expression were indicated in the young tissues in all transgenic plants by Western blot analysis except for one non-symptomatic line which showed low levels ofa truncated BCl protein. Non-symptomatic plant (BC 1 -3-6-4; phenotype not shown) had a similar level of BCl protein as the symptomatic plant (BC1-3-11-5). Extracts from the attenuated- symptom plant (Fig. 5A and B; BC1-3-11-2) showed both full-length and truncated BCl proteins. The low level ofthe truncated BCl protein detection may be due to the loss of epitopes since 121 amino acid residues were lost at the carboxy end (see below). BCl proteins (full-length or tmncated form) from the non-symptomatic, transgenic plants were not detected in older tissue, unlike that seen for the transgenic plants expressing the severe symptom type BCl protein. This indicated that certain mutations in the BC 1 protein may affect its stability in planta.

Northern blots indicated a high transcript number for all the transgenic lines (Fig. 8). The non-symptomatic plant shown in Fig. 5A had a smaller than expected transcript. This apparent deletion in the transcript is consistent with the truncated BCl protein seen in Western blots (Fig. 7).

The transcript level for the plants expressing the truncated BCl protein was high and therefore the low level of truncated BCl protein detected in Western blots (Fig. 7) is not due to transcript activity. The larger than expected transcript is the result of a readthrough of BC 1 termination signals into the vector rbcS termination sequences ofthe pKYLX vector. The BCl gene from the transgenic tobacco plants showing the different phenotypes was amplified by polymerase chain reaction (PCR) and sequenced. The sequence data revealed mutations (amino acid residue 215 G-S, 219 S-L, and 247 E-G) near the carboxyl terminus ofthe BC 1 protein (Fig. 9) for the severe stunting phenotype (Fig. 5A). Two mutants were associated with non- symptomatic, transgenic tobacco. One mutant (resolved from BC 1-3-6-3) showed several changes near the amino terminus (amino acid residue 6 V-F, 7 N-S, and 35 F-L) while the other (resolved from BCl -3-11-6, Fig.5 A) showed a change in amino acid residue 12 F-C, a deletion of amino acid residues 174-293, and an unidentified fusion sequence of 26 amino acid residues starting after amino acid residue 173 (Fig. 9). This was consistent with the detection of a truncated BCl protein (- 10 kDa smaller in size compared to the wild type) in Western blots from extracts from these transgenic plants. The transcript for the truncated, BCl protein was sequenced after reverse transcription of extracted total RNA using oligo dT primer followed by PCR amplification using a BCl specific primer.

The non-symptomatic, transgenic Rø tobacco plants revealed segregation in the R, generation as indicated by the appearance of several symptomatic plants in this generation. Some lines with symptom attenuation (Fig. 5B) continued to segregate in the R₂ generation but the non- symptomatic plants did not. Southern blot analysis (Fig. 6) indicated multiple copies ofthe BCl gene in the RQ tobacco. Apparently some of the R_Q tobacco lines contained copies of both the symptomatic and non-symptomatic forms of BCl. This was confirmed by Southern blot and Western blot analyses of selected R, tobacco plants which were associated with the different phenotypes (Fig. 5). The mottling phenotype with no stunting described above (Fig. 5) had one copy each ofthe symptomatic and non-symptomatic forms of BCl. Transgenic tobacco containing copies of both symptomatic and non-symptomatic forms of the BCl gene (Fig. 5B) resulted in a nonstunting with mild mottling phenotype. This indicated that the non-symptomatic BCl gene suppressed (trans-dominant negative interference) the symptom inducing elements) of symptomatic

BCl gene in transgenic plants containing both forms. Transgene silencing (Meins Jr. and Kunz, 1995) was not evident in these plants since both proteins were detected in Western blots (Fig. 7, BC1-3-11-2). Furthermore, the expression ofthe symptomatic phenotype in subsequent generations indicated that the symptomatic BC 1 gene was not in an inactive form in the phenotype suppressed RQ tobacco. The symptom suppression was also effective against vims infection since tobacco plants with the mutated, non-symptomatic BCl transgene remained free of TMoV symptoms under high disease pressure from viruliferous whiteflies over a 3 -month period.

All of the BCl transgenic plants that were analyzed revealed spontaneous/unexpected mutations in the BCl gene. Point mutations were found in all transgenes analyzed, and one transgene showed a major deletion at the 3 ' end and with a fusion of an unidentified sequence of

-250 nucleotides (no close relationship with sequences in GenBank using BLAST). This may have occurred by a chromosomal cross-over event during plant cell division after the BCl gene was integrated into the tobacco chromosome. In the latter case, a truncated BCl protein (~ 10 kDa smaller in size compared to the wild type) was detected in Western blots and a smaller transcript was detected in Northern blots. This indicated that a deletion in the transgene as well as point mutations

(described previously) are sources of variation in transgene expression.

Other studies with the expression of foreign genes in transgenic plants show varying levels of expressivity in the different generated lines or in siblings in a transgenic line (Hull, 1994). Varying levels of resistance in different transgenic plant lines transformed with the same gene appear to be the norm in pathogen-derived resistance studies. These variations are not adequately explained by positional effects due to the random integration in the plant chromosome during transformation. Silencing of genes in transgenic plants is considered a general phenomenon when multiple copies of transgenes are introduced into plant cells (Meins Jr. and Kunz, 1995). All of our R_Q transgenic plants analyzed contained multiple copies of the BC 1 transgene with no apparent suppression of transgene expression.

Because the teachings herein used the classical methods of Agrobacterium-mediated transformation commonly used by others in the art, some of the variation in the expected phenotype reported in the literature can be explained by spontaneous mutations occurring during Agrobacterium-mediated transformation and during chromosomal rearrangements as reported here for TMoV BCl. Thus it is shown that spontaneous point mutations in the transgene during Agrobacterium-mediated transformation and other modifications in the transgene by chromosomal rearrangements affect gene function and regulation with transgenes. The subject invention also concerns the polynucleotide molecules shown in Figure 9A and the polypeptides encoded thereby shown in Figure 9B, as well as other mutated polynucleotides conferring viral resistance that can be produced using the teachings ofthe present invention.

The spontaneous mutations that can be produced in viral movement genes using the methods and materials of the present invention during Agrobacterium-mediated transformation provide a simple way to develop pathogen-resistant plants. For geminiviruses, the introduction of the pathogenicity gene (BC 1 for the bipartite geminiviruses, AC4 for the monopartite like tomato yellow leaf curl virus) into plant cells by Agrobacterium-mediated transformation will result in selection since transformed cells which express the non-mutated pathogenicity genes will not grow as well as those cells which express the mutated pathogenicity gene. After transformation, visual evaluation for the non-symptomatic phenotype and Western blot analysis for pathogenicity gene protein expression is all that is needed to select geminivirus-resistant plants for further screening and evaluation AU transgenic tobacco with a non-symptomatic phenotype and with TMoV BC 1 protein expression showed virus resistance. Similarly, certain pathogenicity genes from bacterial or fungal plant pathogens can be introduced into transgenic plants according to these teachings and the selection pressure will result in pathogen-resistant plants. The amino acid sequences disclosed herein are based on standard single letter abbreviations for amino acid residues.

While the foregoing description and examples provide details regarding the methods of making and using the invention, including its best mode, it is to be understood that obvious variations and functional equivalents thereof are to be considered part of this invention and therefore fall within the scope ofthe claims which follow.

References

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Claims

Claims 1. A mutated plant virus BCl gene which confers enhanced viral resistance to plants harboring said mutated gene.

2. The gene of claim 1 wherein the viral resistance is against a virus selected from the group consisting of tobamovirus and geminivims.

3. The mutated gene of claim 1 prepared by the process of isolating the natural gene, inserting the natural gene into the genome of a plant, and identifying plants that have increased resistance to viral infection.

4. The mutated gene of claim 1 which encodes a gene product of about 28 kDa.

S. A method for conferring on a plant enhanced resistance against plant viral infection which comprises insertion of a viral movement gene into said plant and identifying a plant spontaneously expressing a mutant of said gene which confers said enhanced resistance against plant viral infection on said plant while at the same time not inducing pathogenic symptoms in the plant.

6. The method of claim 5 in which the viral movement gene is a plant virus BC 1 gene.

7. A transgenic plant having increased resistance to viral infection, said plant being transgenic for a mutated plant virus BC 1 gene.

8. The plant of claim 7 which is a transgenic tomato or tobacco plant.

9. Tbe plant of claim 8 in which the plant has enhanced resistance against infection by tomato mottle geminivims or tobacco mosaic tobamovirus.

10. The mutated gene of claim 1 which comprises any or all of the mutations as compared to the wild-type gene, shown in Figure 1 or Figure 9A.

11. The mutated gene of claim 1 comprising all or a portion of the sequence shown in Figure 1.

12. A mutant BCl protein comprising any or all of the wild-type to mutant amino acid substitutions shown in Figure 4 or Figure 9B.