COAT PROTEIN MEDIATED RESISTANCE IN PLANTS

Abstract: The invention features a method by which a transgenic plant may be generated which exhibits increased resistance to virus-mediated disease. The method may be generally applied to any plant a) having genotypes or cultivars with increased resistance to a viral pathogen, and b) able to be infected by a virus whose coat protein gene may be used in coat protein mediated resistance (CPMR).

Description: COAT PROTEIN MEDIATED RESISTANCE IN PLANTS Background of the Invention This invention relates to disease resistance in plants. Over the past decade, there have been numerous reports of pathogen derived resistance (PDR) (Fitchen and Beachy, Ann. Rev. Microbiol. 47: 739-763, 1993), including protection resulting from the integration of viral coat protein (CP) genes into transgenic plants (Beachy et al, Annu. Rev. Phytopathol. 28: 451-474, 1990). Indeed, this type of strategy employing coat protein-mediated resistance (CPMR) may be one of the most effective means at hand to limit losses to the economically important potyviruses (Lindbo and Dougherty, Virology 189: 725-733, 1992). Although CPMR has often been demonstrated in a number of plants, the effectiveness of this strategy against potyviruses is complicated by the large number of different potyviruses that can infect the plant, by strain variation within particular potyviruses, and by the difficulty in identifying CP genes that provide a high level of protection against the various potyviruses infecting tobacco. The value of CPMR is believed to depend upon whether a CP gene can confer a high level of broad protection, defined here as resistance against an array of different potyviruses or against different strains of one potyvirus. Summary of the Invention In general, the invention generally features a method for providing resistance to a first viral pathogen in a plant. The method includes the steps of: (a) selecting a plant having an endogenous level of resistance to a second viral pathogen that is at least two times that of a non-resistant plant; (b) transforming cells of the plant with a nucleic acid molecule encoding a viral coat protein gene; (c) regenerating the plant cells to provide a differentiated plant; and (d) selecting a transgenic plant which expresses the viral coat protein gene, where expression of the gene in the transformed plant provides resistance to the first viral pathogen. Preferably, the transgenic plant is a dicot (such as a member of the Solanaceae, e.g., a member of the genus Nicotiana) or a monocot. In another preferred embodiment, the transgenic plant has increased resistance to the second viral pathogen. In yet other embodiments, the plant having an endogenous level of resistance to the second viral pathogen is a Virgin A mutant and includes a viral coat protein gene from a potyvirus (for example, tobacco vein mottling virus or potato virus Y), an alfalfa mosaic virus, or both. In addition, such a plant generally has greater resistance and a broader spectrum of resistance than a transgenic plant without an endogenous level of resistance to the second viral pathogen. In another embodiment, the transgenic plant is resistant to more than two different potyviruses. In another preferred embodiment, the first and second viruses are related viruses. In another aspect, the invention features a transgenic plant (or a seed or a cell thereof) having resistance to a first viral pathogen, which includes a plant having an endogenous level of resistance to a second viral pathogen that is at least two times that of a non-resistant plant transformed with a nucleic acid molecule encoding a viral coat protein, where expression of the gene in the transformed plant provides resistance to the first viral pathogen. By "endogenous level of resistance" is meant that level of pathogen resistance possessed by a naturally occurring or cultivated plant, cultivar, genotype, or plant variant. Such a "cultivated" plant, cultivar, genotype, or plant variant includes, without limitation, one which is selectively bred to include or exhibit a particular disease resistance characteristic. By "potyvirus" is meant a member of the Polyviridae family, the largest family of RNA plant viruses. A polyvirus is a single-stranded infectious RNA molecule that is approximately 10,000 nucleotides in length. The virus particle or virion has a flexuous rod shape and is usually 700-900 nm in length and 12-15 nm in diameter. The genome of a potyvirus encodes for a protein that aggregates in the cytoplasm during infection in the form of pinwheel or scroll-shape, or other types of inclusion bodies. There may be over 100 different members of the Potyviridae family, and it is a major viral pathogen for many crops. Exemplary potyviruses include, without limitation, potato virus Y (which infect tobacco, tomato, and potato); tobacco etch virus (which infects tobacco); maize dwarf mosaic virus (which infects maize and sorghum); soybean mosaic virus (which infects soybean); and melon mosaic virus (which infects melon). By "plant cell" is meant any self-propagating cell bounded by a semi- permeable membrane and containing a plastid. A plant cell, as used herein, is obtained from, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, protoplasts, leaves, roots, shoots, somatic and zygotic embryos, as well as any part of a reproductive or vegetative tissue or organ. By "transgenic" is meant any cell or progeny thereof, which includes a DNA sequence which is inserted by artifice and becomes part of the genome of the organism (integrated or extrachromosomal) which develops from that cell. As used herein, the transgenic organisms are generally transgenic plants and the DNA (e.g., a transgene) is inserted by artifice into the nuclear or plastidic compartments of the plant cell. By "transgene" is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the organism (integrated into the genome or maintained extrachromosomally) which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. By "resistance to a viral pathogen" is meant a greater level of resistance to a pathogen (e.g., a potyvirus) in a transgenic plant than the level of resistance relative to a control plant (for example, a nontransformed plant). In preferred embodiments, the level of resistance to a pathogen in a transgenic plant is at least 5 to 10% (and preferably 20%, 30%, or 40%) greater than the resistance of a control plant. In other preferred embodiments, the level of resistance to the pathogen is 50% greater, 60% greater, and more preferably even 75% or 90% greater than a control plant; with up to 100% or greater level of resistance as compared to a control plant being most preferred. The level of resistance is measured using conventional methods. For example, the level of resistance to potyvirus may be determined by comparing physical features and characteristics (for example, plant height and weight, or by comparing disease symptoms, such as stunting, mosaicism, chlorosis, and/or etching of leaves) of a transgenic plant or by comparing the population of a pathogen surviving on the plant (e.g., by measuring (e.g., by ELISA) the systemic population of a potyvirus in transgenic and control plants). Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Detailed Description The drawings will first be described. Drawings Fig. 1 is a photograph of an immunoblot analysis showing the expression of the TVMV CP transgenic lines. Proteins were extracted from the leaf tissue of selected lines. Lane 1, BRv-neg-t3; lane 2, BRv-neg-t30; lane 3, BRv-neg-t32; lane 4, protein marker; lane 5, untransformed BY21; lane 6, TVMV infected tissue; lane 7, BRv-pos -tl4; lane 8, BRv-pos -t21 ; and lane 9, BRv-pos -t34. Designation of transgenic lines are same as in Table 2. Fig. 2 is a graph showing the response of selected lines to TEV infection. Designation of transgenic lines are the same as in Table 2. Fig. 3 is a bar graph showing the response of untransformed genotypes to different dilutions of PVY-KY1 (days when symptoms appeared are indicated). Figs. 4A and 4B are graphs showing the effect of PVY-KY1 inoculum concentrations on disease incidence in untransformed and TVMV CP transgenic tobacco lines. Fig 4A shows untransformed BRv-neg and its transgenic lines. Fig. 4B shows untransformed BRv-pos and its transgenic lines. Figs. 5 A and 5B are graphs showing the effect of PVY-NN inoculum concentrations on disease incidence in untransformed and TVMV CP transgenic tobacco lines. Fig. 5 A shows untransformed BRv-neg and its transgenic lines. Fig. 5B shows untransformed BRv-pos and its transgenic lines. Fig. 6 is a graph showing the disease symptom development in untransformed genotypes following inoculation with TEV. The disease rating scale is defined as: 0, no symptoms; 1, mild chlorosis and mosaic; 2, moderate mosaic; 3, severe mosaic and some chlorosis and etching of leaves; 4, severe chlorosis and etching of leaves; 5, severe chlorosis and leaf distortion. The mean rating of the 12 plants that were studied in each plot was used as the experimental unit for statistical analysis. Fig. 7 is a panel of bar graphs showing the comparison of the mean of 10 transgenic lines and corresponding host controls for infection scores and virus accumulation (ELISA) following TEV inoculation. The designation (*) and (**) represent the difference between 10 transgenic lines and corresponding control was significant at the P = 0.05 and P = 0.01 levels, respectively. Fig. 8 is a panel of bar graphs showing the average TEV symptom intensity in transgenic lines and their corresponding control genotypes at 2 weeks after inoculation and before topping. Score is the average of 12 plants, using a rating of 0-5, as described in the legend to Fig. 6. Fig. 9 is a panel of bar graphs showing the average PVY symptom intensity in transgenic lines and their corresponding controls at 2 weeks after inoculation and before topping. Score is the average of 12 plants, using a rating of 0- 5, as described in the legend to Fig. 6. Fig. 10 is a bar graph showing virus accumulation at different leaf positions of transgenic line BY-3 after TVMV inoculation (from the first systemic leaf to tenth systemic infected leaf). Fig. 11 is a panel of bar graphs showing the responses of B21 and BRv- neg transgenic lines to inoculation with TEV, TVMV, and PVY under field conditions. Type 1 included the lines highly resistant to TVMV, TEV, or PVY infection. Type 2 included the lines with different responses to TEV or PVY infection. Type 3 included the lines susceptible to TEV and PVY. Type 4 included the lines susceptible to all three viruses. Fig. 12 is a photograph of an immunoblot analysis of AMV CP transgenic lines. Lane designation is presented as (BY-A-5): recipient genotype (BY, BY 21), transgene (A, AMV CP), and independent line number (5). The lane designated (+) is the leaf extract from a nontransformed control plant infected with AMV. Fig. 13 is a graph showing the disease symptom development of BY 21 AMV CP transgenic lines following AMV 425 inoculation in the greenhouse. Fig. 14 is a graph showing the responses of selected BY 21 transgenic lines to inoculation with AMV-425, AMV-KY, and AMV-NC isolates. Figs. 15 A, 15B, 15C, and 15D are a series of bar graphs showing the means for infection scores and virus accumulation (ELISA) following AMV inoculation of 10 transgenic lines and nontransformed controls. Figs. 15A and 15B show the infection scores at 21 DPI (days post-inoculation) and 67 DPI, respectively. Figs. 15C and 15D show the ELISA readings at 21 DPI and 67 DPI, respectively. The designation (*) and (**) represent differences between 10 transgenic lines and controls were significant at the P=0.05 and P=0.01 levels, respectively. Fig. 16 is a panel of bar graphs showing the average disease symptom intensity and virus accumulation (ELISA) following AMV inoculation in transgenic lines and their corresponding controls at the flowering. Symptom expression score is an average of 12 plants, using a rating scale of 0-7, where 0 indicates no symptoms and 7 indicates severe symptoms hallmarked by stunting and leaf distortion. Fig. 17 is a schematic diagram showing the construction of the plasmid pKYLX71-ACP. The CP gene of alfalfa mosaic virus strain 425 was recovered from pSP65 by preparative agarose gel electrophoresis. The resulting fragment was cloned as an EcoR I - Xba I fragment into a plasmid (pBSK) and further digested with Hind III and Xba I. This Hind III - Xba I fragment was cloned into the plasmid pKYLX71:35S 2 . Fig. 18 is a panel of bar graphs showing the frequency distribution of leaf number per plant for TN 86 and leaf width for BY 21 TVMV CP transgenic lines under non-challenged field conditions. The arrow indicates the means of these traits. Fig. 19 is a panel of bar graphs showing the comparison of AMV CP transgenic lines and nontransformed controls for plant height under natural noninoculated conditions and following infection with AMV. Fig. 20 is a panel of bar graphs showing the yield performance of AMV CP transgenic lines and nontransformed controls under natural noninoculated conditions and following infection with AMV. Fig. 21 is a panel of bar graphs showing the percent yield gain of AMV CP transgenic lines over their corresponding nontransformed controls following AMV inoculation. Fig. 22 is a bar graph showing the yield performance of BY 21 transgenic lines expressing AMV CP and controls under natural noninoculated conditions and following AMV inoculation in 1994. I. Overview To further investigate the properties of CPMR and explore the possibility of combining host-encoded resistance with CPMR, a TVMV CP gene was engineered into five tobacco genotypes, some possessing and some lacking the Virgin A mutant (VAM) gene. The VAM gene, also referred to as the va gene, is derived from the Virgin A mutant and confers high resistance to TVMV and medium resistance to TEV, but has little effect on many isolates of PVY (Johnson and Pirone, Phytopathology 72: 68-71, 1982). These transgenic lines were screened for resistance against different potyviruses. The transgenic lines derived from two near-isogenic lines (NILs) to the VAM gene provided a direct way to compare the effects of host resistance and CPMR and synergistic effects of these two types of resistance. Selected transgenic NILs were also evaluated in detail for resistance against different potyviruses. In addition, to evaluate the resistance of TVMV CP transgenic lines to infection with potyviruses, we conducted a comprehensive field experiment with transgenic plants containing the TVMV CP gene in homozygous or hemizygous form. The field test was designed to study the performance of genetically different transformed lines including a comparison of transgenic plants derived from genotypes which possess or lack an endogenous resistance gene (VAM). The performance of tobacco plants that express CP was compared with the response to virus inoculation with wild-type plants. To evaluate another approach to developing resistance against different virus groups, transgenic tobacco lines containing the CP gene from Alfalfa Mosaic Virus (AMV) were generated. AMV is substantially different from potyviruses in its morphology, genome structure, strategies of viral gene expression, and early steps in replication (Bol et al, Virology 46: 73-85, 1971; Turner et al, EMBO J. 6: 1181- 1188, 1987; Dougherty et al, Annu. Rev. Phytopathol. 26: 123-143, 1988). These AMV-CP transgenic lines, generated on different genetic backgrounds, were screened for resistance against different strains of AMV both in the greenhouse and in field studies. The use of CPMR for crop protection against virus diseases is ultimately determined by overall performance of the transgenic plants under field conditions. Any variation in agronomic performance of transgenic lines would not only affect the direct utilization of CPMR, but would also increase or decrease the difficulties encountered in using these lines as breeding stock in the development of cultivars having resistance to viruses. Studies were therefore conducted to determine the impact of genetic transformation for a TVMV CP gene or AMV CP gene on agronomic performance, and to determine whether CPMR increased yield following challenge with different viruses. II. Combining a host gene and a tobacco vein mottling virus coat protein gene for broad and effective resistance to potyviruses in tobacco (Nicotiana tabacum LΛ Expression of the TVMV CP gene and virus resistance in transgenic lines The TVMV CP gene was introduced into five genotypes of Nicotiana tabacum burley tobacco, including three VAM-positive genotypes, TN86, KY8959, and BRv-pos, according to standard methods. Twenty of the kanamycin resistance tobacco lines were assayed for expression of the foreign TVMV CP gene. All 20 lines were found to express the TVMV CP gene. In addition, immunoblot analysis showed that the CP-related polypeptide in these plants was similar in size to that predicted from recombinant DNA manipulations (Fig. 1). Plants in the R, generation of 104 TVMV CP transgenic lines in five genetic backgrounds (KY 8959, BRv-neg, BRv-pos, TN 86, and BY 21 ) were assessed for resistance to PVY-O, and 93 of these 104 lines were screened for resistance to TVMV-S. Both of these potyviruses, PVY-0 and TVMV-S, were observed to overcome resistance conferred by the VAM gene (Table 1). Table 1 Resistance of TVMV CP transgenic lines to infection by PVY-O and TVMV-S Genotype Lines Highly Resιstant b Susceptible Frequency of resistant" resιstance(%) c PVY-O KY8959 26 11 5 10 61.4 BRv-neg 31 13 6 12 61.3 BRv-pos 23 11 3 9 60.9 TN 86 14 3 4 7 50.0 BY 21 10 2 4 4 60.0 Total 104 40 22 42 59.6 TVMV-S KY8959 22 5 2 15 31.8 BRv-neg 30 5 2 23 23.3 BRv-pos 17 4 1 12 29.4 TN 86 14 4 1 9 35.7 BY 21 10 0 3 7 30.0 Total 93 18 9 66 29.0 1 Symptomless at 21 DPI. b <25% of infected plants. c Frequency of lines with resistance: (High resistance lines + resistance lιnes)/total lines These strains were used because three of the five recipient genotypes possess the VAM gene, and inoculation with these VAM breaking strains allowed detection of resistance conferred by the TVMV CP gene. Sixty-two of the tested lines (60%) were resistant to PVY-O and 27 lines (29%) were resistant to TVMV-S. Two- thirds of the resistant lines were highly resistant (HR): no symptoms of infection and no virus that was detectable by ELISA. The remaining one-third of the resistant (R) lines had delayed symptoms and a low incidence (<25%) of infected plants (Table 1). ELISA results corresponded closely to visual observations of disease symptoms in transgenic plants. For instance, nearly all transgenic lines that were classified visually as HR had ELISA absorbance readings of less than 0.2 (average of infected susceptible control was 1.2); the lines classified as R had ELISA values that ranged from 0.21 to 0.5; and all susceptible lines had ELISA readings greater than 0.8. No significant differences were observed in the frequency of resistance response among the recipient genotypes. Among the 93 lines tested against both viruses, one line was HR for both viruses; two lines were HR to PVY-O and R to TVMV-S; three lines were R to PVY-O, HR to TVMV-S; and two lines were R to both viruses. Response of transgenic plants to TVMV, TEV, and five PVY strains Based on data from challenges with PVY-O, six HR transgenic lines (three lines from BRv-neg and BRv-pos, and one each from KY 8959, TN 86, and BY 21) and four control genotypes were examined in detail for their responses to challenge by a TVMV isolate that does not break the VAM resistance, an isolate of TEV, and four isolates of PVY. Control lines in this study included nontransformed TN 86, BY 21, BRv-neg and BRv-pos and one BY 21 transgenic line containing the 35S 2 KYLX71 (KYLX) construct that expresses kanamycin resistance but lacks the CP gene. Table 2 Reaction of transgenic lines and control genotypes to infection by TVMV, TEV, and five strains of PVY Transgenic line PVY Strain or control MM" N 0 KYI KY2 TVMV TEV BRv-neg-t3 b HR C R HR LR LR LR LR BRv-neg-t30 R R HR LR LR S HR BRv-neg-t32 R R HR LR LR s LR BRv-pos-tl4 HR HR HR HR LR HR HR BRv-pos-t21 HR HR HR HR LR HR HR BRv-pos-t34 HR HR HR HR LR HR HR BRv-neg S S S S S s S BRv-pos HR R S s S HR LR BY 21 S S s s S S S BY-KYLX s S s s s S S a All inoculations were made with a 1 :50 dilution of fresh sap extracted from infected Burley 21 tobacco. b Transgenic line designation is presented as (BRv-neg-t3): recipient genotypes (BRv-neg, BRv-neg), transgene (t, TVMV CP) and independent line number (3). c HR-highly resistant: no symptoms of virus infection and no virus detected (ELISA) R-resistant- very mild symptoms and very low disease incidence and no or very low virus accumulation. LR-low resistant: low disease incidence and mild symptoms, and detectable but low virus level. S-susceptible: typical potyvirus-mduced symptoms. Table 2 summarizes the responses observed in the transgenic lines and the control genotypes to virus challenge. In general, four different plant responses were observed: following inoculation with TVMV and TEV, the susceptible reaction (S) was initially apparent in BY 21, BRv-neg, and BY-KYLX as systemic vein clearing 4-5 days after inoculation with TEV and 7 days after inoculation with TVMV. This was followed by systemic mottling and vein clearing in younger leaves, and eventually the plants became stunted and necrotic. The other controls, TN 86 and BRv-pos, showed later development of symptoms to TEV challenge (Fig. 2) and no symptoms of TVMV infection (Table 2). A low resistance (LR) phenotype, observed in some lines following virus inoculation, was classified as delayed with attenuated systemic symptoms and low virus accumulation as detected by ELISA. A second resistance phenotype (R) was noted when mild symptoms were found in a low percentage (<25%) of plants at the end of the experiment (25 DPI) and little or no virus was detected by ELISA. Symptom onset for lines rated R were 8-10 days later than susceptible lines. High resistance (HR) was noted when all plants in a line were symptomless and had no detectable virus accumulation at the termination of the experiment. Only one of the three transgenic lines which lacked the VAM gene (BRv- neg background) had low resistance (LR) to TVMV. In contrast, all three of these lines had some resistance to TEV, but only one was classified as HR (Table 2 and Fig. 2). ELISA tests confirmed these ratings. For example, the ELISA value for BRv- neg-t3 was 0.625 at 25 DPI with TVMV compared to 1.919 for BRv-neg. ELISA absorbance readings for all transgenic lines were low following TEV inoculation. Although the nontransformed BRv-pos line developed severe symptoms of TEV infection at 20 DPI, none of the BRv-pos transgenic lines were observed to develop disease symptoms (Fig. 2) or accumulated detectable levels of TEV. Indeed, even after these transgenic VAM-pos lines were reinoculated with a high concentration of TEV, they did not show any symptoms of infection and had no detectable TEV accumulation. Infectivity of the five PVY isolates on tobacco was different, especially on the VAM-pos genotypes. All plants of BY 21, BRv-neg, and BY-KYLX developed symptoms 7 days after inoculation with all five PVY isolates. BRv-pos and TN 86 showed disease symptoms 14 DPI with PVY-O, PVY-KY1, and PVY-KY2. BRv-pos failed to develop symptoms following PVY-MM and PVY-NN inoculation (Table 2). Three BRv-neg transgenic lines were highly resistant or resistant to PVY- MM, PVY-NN, and PVY-O, and had low resistance to both PVY-KY isolates. Plants with symptoms were 20-30% shorter than mock inoculated controls. All VAM-pos transgenic lines were highly resistant or resistant to four of the PVY strains. Inoculation of the VAM-pos transgenic lines with PVY-KY2 resulted in LR (symptoms in less than 25% of the plants of those lines) 25 DPI, except for TN-t46, which showed obvious symptoms 14 DPI (Table 2). Sero logical tests confirmed all of these visual observations. Response of transgenic lines to inoculation with different inoculum concentrations of PVY-KY1 and PVY-NN The virus isolates PVY-KY 1 and PVY-NN were chosen as inocula for this experiment because they elicited different responses among these genotypes. Inoculation of the nontransformed plants BY 21 and BRv-neg with PVY-KY 1 resulted in disease symptoms even with the most dilute virus concentration (Fig. 3). BY 21 appeared slightly more susceptible than BRv-neg based on the earlier onset of symptoms in BY 21 at the inoculum dilutions of 1 :1000 and 1:5000. BRv-pos failed to show any symptoms of PVY-KY 1 infection when inoculated at the 1 : 1000 and 1:5000 dilutions, and symptoms in BRv-pos developed later than the other control genotypes at all of the higher inoculation concentrations (Fig. 3). Following inoculation with the three highest concentrations of virus (1:5, 1 :50, and 1 : 100), the BRv-neg transgenic lines developed symptoms at 14 DPI which was 7 days later than the nontransformed VAM-neg genotypes. One of the three BRv-neg transgenic lines had low disease incidence (Fig. 4A). After inoculation with the three low inoculum concentrations, these three transgenic lines displayed obvious protection, with none developing symptoms at the 1:5000 dilution of virus. Two of three BRv-pos transgenic lines failed to develop any symptoms following challenge with all concentrations of PVY-KY 1. The remaining BRv-pos line, BRv-pos-tl4, developed symptoms only after inoculation with the two highest PVY-KY 1 concentrations (Fig. 4B). Except for the fact that transgenic lines infected with PVY-NN showed less severe symptoms and had a longer delay in the development of disease than those observed following PVY-KY 1 challenge, the overall response to PVY-NN inoculation was similar to that with PVY-KY 1. None of the VAM-pos transgenic lines showed disease symptoms at any concentrations of PVY-NN inoculum (Fig. 5B). In contrast, the BRv-neg developed symptoms under high inoculum concentrations (Fig. 5 A). A large number of TVMV CP transgenic tobacco lines were screened against two different potyviruses, PVY-O and TVMV-S, to determine the extent of variation in expression of CPMR, to estimate the extent of protection to different potyviruses, and to determine the influence of genetic background on the level and frequency of CPMR. Although the majority of these transgenic lines possessed the endogenous VAM gene for potyvirus resistance, both PVY-O and TVMV-S overcome this type of resistance (i.e., overcome the VAM gene). The use of these isolates permitted a direct assessment of the impact of the TVMV CP gene on virus resistance. There was a considerable difference in the frequency of lines resistant to PVY-O and TVMV-S. While 60% of the 104 TVMV CP lines were classified as resistant to PVY-O, only about 29% of these same lines were resistant to TVMV-S (Table 1). Most investigations of CPMR (Powell-Abel et al, Science 232: 738-743, 1986; Stark et al, Bio/Technology 7: 1257-1262, 1989; Loesch-Fries et al, UCLA Symp. Mol. Cell Biol. 48: 221-234, 1987) have found that CPMR is most effective against infection by the virus from which the CP gene was obtained. However, there is evidence that the TVMV CP gene provides less protection against TVMV than it does against other potyviruses (Maiti et al, Proc. Natl. Acad. Sci. 90: 6110-6114, 1993). The fact that nearly 20% of the transgenic lines were classified as highly resistant was unexpected given that a severe strain of TVMV was used, and our findings suggest that the TVMV CP gene is effective in reducing disease caused by TVMV infections in tobacco. A thorough investigation using a subset of PVY-O resistant transgenic lines revealed that the genotype of the recipient host plant has an important role in determining the level of protection (Table 2). Genotypes used in these experiments differed mainly for the presence or absence of a recessively inherited gene derived from a mutant line designated Virgin A Mutant (Koelle, G., Zuchter 31 : 71-72, 1961). This VAM gene (also referred to as the va gene) confers resistance to some but not all potyviruses (Johnson, M. C, Ph. D. dissertation, University of Kentucky, Lexington, 1980). In general, TVMV CP transgenic lines possessing the VAM gene had a much higher level of protection than transgenic lines lacking the VAM gene. This became especially evident by comparing the transgenic NIL's BRv-neg and BRv-pos (Table 2). The BRv-pos transgenic lines were highly resistant to all challenge potyviruses; only against PVY-KY2 were they rated as having low resistance. In contrast, the BRv-neg transgenic lines were less resistant to most of the challenge viruses. The effect of recipient genotype on protection did not appear to be limited to the role of the VAM gene. Transgenic lines of the susceptible cultivars BY21 and BRv-neg differed in their reaction to infection by the different potyvirus strains. Indeed, the two transgenic lines in other genotypes possessing the VAM gene, KY 8959 and TN 86, appeared less resistant to most potyvirus strains than the three BRv- pos transgenic lines. Although the number of lines examined in this experiment was probably not sufficient to definitively assess the magnitude of the genetic background effect, there was sufficient evidence to indicate that genetic background impacts both the degree of resistance to a particular challenge virus and the extent of protection against related viruses. The synergistic effect of combining CPMR with an endogenous resistance gene was further established by inoculation of TVMV CP transgenic lines and control genotypes with serial dilutions of PVY strains KYI and NN (Figs. 4A, 4B, 5 A, and 5B). In each case, transgenic lines possessing the VAM gene exhibited resistance at higher inoculum concentrations than transgenic lines without the VAM gene. This synergistic effect may reflect what are most likely different mechanisms for CPMR and host resistance. Although the molecular or cellular basis of CPMR has not been clearly defined, CPMR is thought to result from interference with movement of the infectious virus particle, preventing uncoating of the infecting virus, or blocking virus replication by CP- or RNA-mediated mechanism (Beachy et al, Annu. Rev. Phytopathol. 28: 451-474, 1990; Register et al, Virology 166: 524-532, 1988; Nelson et al, Virology 158: 126-132, 1987; Lindbo et al, Virology 189: 725-733, 1992). Johnson and Pirone (Johnson, M. C, Ph. D. dissertation, University of Kentucky, Lexington, 1980; Johnson and Pirone, Phytopathology 72: 68-71, 1982) and Gibb et al (Mol. Plant-Microbe Interactions 2: 332-329, 1989) showed that the resistance of the VAM gene was primarily due to restricted virus movement. Combining two, apparently different, mechanisms for virus resistance has resulted in an enhanced level of resistance to a wider spectrum of potyviruses. The results described above have important implications for plant improvement programs using CPMR against economically important virus diseases. One of the initial steps in developing transgenic CP plants is the selection of suitable recipient genotypes. To our knowledge, this selection has been based primarily on agronomic or horticultural characteristics and product quality traits. We propose that the selection of a host or recipient genotype for a viral CP gene should also include an analysis of the host genotype's response to the target challenge viruses. Inserting viral CP genes into genotypes possessing some level of resistance or tolerance could be vitally important to achieving the level of resistance needed to reduce losses to viral diseases. The above-described results were obtained using the following materials and methods. Recipient genotypes Five genotypes of Nicotiana tabacum burley tobacco, including the cultivars Burley 21, TN 86, KY 8959, and two near isogenic lines (NIL's), BRv-pos and BRv-neg, were chosen for this study. Burley 21 (BY 21) is a commercial cultivar that is susceptible to many potyviruses. TN 86 (Miller, R. D., Crop Science. 17: 365- 366, 1987) and KY 8959 (Nielsen et al, Crop Science 33: 1103, 1993) are commercial cultivars that possess the Virgin A Mutant (VAM, also known as va) gene (Tl 1406) that confers high resistance to TVMV and medium resistance to TEV. However, these cultivars are susceptible to most strains of PVY. The two NIL's have the same parentage as KY 8959 and were derived from single plant selections in the same F 4 line. They were found in subsequent tests to differ in their reaction to infection by TVMV and TEV. BRv-neg is susceptible and BRv-pos is resistant to TVMV (i.e. , BRv-neg lacks the VAM gene and BRv-pos has the VAM gene). Plant transformation The TVMV CP gene was cloned into the plasmid pKYLX71 :35S 2 according to standard methods (Schardl et al, Gene 61: 1-11, 1987; Mogen et al, Plant Cell 2: 1261-1272, 1990; Maiti et al, Proc. Natl. Acad. Sci. 90: 6110-6114, 1993). The CP gene, from the strain TVMV- AT (aphid transmittable), was introduced into the recipient genotypes using an Agrobacterium tumefaciens mediated leaf disk transformation system (Schardl et al, Gene 61 : 1-11, 1987). Leaf tissue from 4-week-old aseptically-grown plants was cut into pieces and incubated in liquid medium containing A. tumefaciens (strain GV3850) with the desired construct. The tissue was allowed to grow on basal medium without antibiotics for two days to enhance tissue infection. On the third day, the tissue was plated on media containing kanamycin (300mg/L), for transformant selection, and Mefoxin (500mg/L) to kill the remaining A. tumefaciens. The culture medium was replaced each week until callus tissue developed. Shoots derived from the callus tissue were transferred to media containing Mefoxin for rooting, and subsequently, rooted plantlets were transplanted into 4 inch pots containing commercial soil mix. Plants were grown to maturity in a greenhouse and self-pollinated. Screening and regeneration of transgenic lines R, seeds derived from selfing the primary transformants were germinated and grown on media containing 300 mg/L kanamycin. The number of kanamycin- resistant and karamycin-sensitive seedlings was determined 2-3 weeks after germination when the sensitive seedlings were chlorotic and unable to produce true leaves. These data were used to compute χ 2 values and to identify segregation pattern indicating 3:1 or 15:1 segregation ratios and only those lines segregating for a single or double gene insertion were kept. Plants resistant to kanamycin were grown to maturity in a greenhouse and self-pollinated. Seeds were collected from each Ri plant and sown on media containing kanamycin to determine homozygosity for the CP gene. Analysis of expression of coat protein genes in transformed plants Immunoblot analyses were used to assess the level of expression of the coat protein gene in selected transgenic lines. Leaves from young 3-week-old seedlings were homogenized in extraction buffer (0.0625 M Tris-HCl, pH 6.8/10%> glycerol/2%SDS/10%> 2-mercaptoethanol), boiled for 10 min, and centrifuged at 12,000 x g for 10 min. Proteins (50 μg of total soluble leaf protein) were separated on 12.5%) polyacrylamide gels/SDS, transferred to nitrocellulose, and probed with a TVMV antiserum. Inoculation and evaluation of transgenic lines R, and R 2 plants from all independent transgenic lines were inoculated with a common strain of PVY (PVY-O) and a systemic strain of TVMV (TVMV-S). The nontransgenic recipient genotypes and nine transgenic lines, selected on the basis of their reaction to PVY-O, were tested for their response to infection by TVMV, TEV and five strains of PVY (Table 2). The five PVY isolates included PVY-MM and PVY-NN. These two isolates were identified by Gooding (Gooding et al, Plant Dis. Rep. 57: 200-214, 1973), and have often been used as standard strains in breeding programs and pathology studies. The other isolates were PVY-O, an aphid- transmissible common strain of PVY, and PVY-KYl and PVY-KY2, two isolates collected from tobacco grown in Kentucky. These two Kentucky isolates were characterized as PVY using a serological assay and by local lesion host (Chenopodium) differentiation. In separate experiments designed to determine the threshold level of resistance, these same nine transgenic lines were challenged with different concentrations of a severe strain of PVY (PVY-KYl) and PVY-NN. The source of inoculum for all experiments was fresh sap, obtained by grinding leaves of the susceptible cultivar Burley 21 infected with TEV, TVMV or PVY isolates (PVY- MM, PVY-NN, PVY-O, PVY-KYl and PVY-KY2). Inoculum dilution was 1:50 (w:v) with 0.05 M potassium phosphate buffer (pH 7.0) for all except one experiment. For dilution experiment, sap from PVY-KYl, PVY-NN infected Burley 21 plants was extracted at a 1 :5 (w:v) concentration. Dilutions of this 1 :5 concentration extract with phosphate buffer resulted in concentrations of 1:50, 1:100, 1:500, 1 :1000 and 1 :5000. In all experiments, five-week-old tobacco seedlings growing in soil were mechanically inoculated on the second leaf from the base of the plant after lightly dusting the leaf with carborundum. Each treatment contains 8 to 10 plants for all experiments. Inoculated plants were examined visually, and symptoms of disease development were recorded daily. Virus accumulation in plants was detected by protein A double-antibody sandwich ELISA (Clark et al, J. Gen. Virol. 34: 475-483, 1977) for all viruses. Five leaf discs (3 cm 2 ) from the third systemic infected leaf of each infected plant were sampled for ELISA analysis. Viruses were extracted by homogenization with 5 ml of PBS-Tween (phosphate-buffered saline, 0.05 (v/v) Tween, pH 7.3) per gram of tissue using a homogenizer. Samples were applied to microtiter plates and tested in duplicate. Polyclonal antibodies were used with dilutions of 1 :2000. 1 :3000, 1 :2000 for TEV, TVMV and PVY, respectively. Reactions were measured spectrophotometrically at 405 nm using a BIO-RAD Microplate Reader Model 450. Samples were rated as positive for the presence of the virus when the sample ELISA absorbance value was three times the mean absorbance value for non-inoculated plants. III. Field resistance of transgenic burley tobacco lines and hybrids expressing the tobacco vein mottling virus coat protein gene Response of transgenic lines to TEV infection Transgenic plants expressing TVMV CP were inoculated with a high tobacco etch virus (TEV) inoculum concentration (1 g infected tissue: 10 ml of buffer solution) early in the growing season to ensure infection and to simulate a severe natural infection. This resulted in the susceptible control genotypes, BY 21 and BRv- neg, developing symptoms 7 DPI (Fig. 6). All BY 21 and BRv-neg plants exhibited severe infections 14 DPI, and these symptoms remained severe throughout the growing season. Symptom expression in nontransformed VAM-pos genotypes, KY 8959, TN 86, and BRv-pos appeared 14 DPI, but their symptom ratings were significantly less than the VAM-neg genotypes (Fig. 6). Symptoms on the VAM-pos genotypes gradually increased in severity throughout the season, but they always remained significantly less than those observed for the VAM-neg control genotypes. Based upon these observations, results for symptom readings and ELISA samplings made 14 DPI and 46 DPI are reported. This analysis permitted the identification of both initial symptom severity (14 DPI) and the progression of disease (46 DPI). Table 3 Analysis of variance for virus infection scores, ELISA values, and plant height of transgenic lines and control genotypes following inoculation with TEV Mean squares Infection score ELISA absorbance Source df 14 DPI 46 DPI 14 DPI 47 DPI Replication 2 0.072 1.030 0.064 0.371 Genotype 4 9.789 ** 22.550 ** 3.760 ** 2.294 * Error A 8 0.187 0.321 0.111 0.348 Lines within genotype 50 2.875" 6.892 ** 1.022 ** 0.901 ** Error B 100 0.130 0.341 0.127 0.159 df : degree of freedom * ** Mean square significant at P= 0.05 and P=0.01, respectively. Analysis of variance of data on four traits in the TEV experiment revealed significant mean squares for genotype effects and for the effect of transgenic lines within genotypes (Table 3). However, the magnitude of the mean squares for genotypes was approximately three times greater than that found for the effects of transgenic lines within genotype effect. Contrasts between means of 10 transgenic lines and the corresponding control genotypes revealed the overall effects of the TVMV CP gene on disease expression (Fig. 7). Early in the season, only transgenic lines of BY 21 and BRv-neg had significantly lower average infection score and virus accumulation than their corresponding nontransformed controls (Fig. 7, 14 DPI). Most of these lines showed delayed symptom initiation that was on average 6-20 days later than the corresponding controls. At 46 DPI, mean symptom scores for the VAM-neg transgenic lines were substantially greater than they were over 4 weeks earlier, but they were still significantly less than those observed in their controls (Fig. 7, 46 DPI). Two weeks after inoculation, only a few VAM-pos transgenic lines displayed symptoms of TEV infection. Indeed, most VAM-pos transgenic lines did not develop any symptoms until 4-5 weeks after inoculation and some transgenic lines never developed symptoms of TEV infection (Fig. 8). At flowering, the nontransformed VAM-pos genotypes had developed mild symptoms, while symptoms of TEV infection of the VAM-pos transgenic lines remained significantly less than the controls (Fig. 7). Transgenic lines in the KY 8959 and BRv-pos backgrounds had significantly lower mean infection scores and mean ELISA values than the other three background lines. In fact, nearly all of BRv-pos lines escaped TEV infection entirely. The average effect of inserting the TVMV CP gene was observed to be about the same in the different backgrounds: a 47-71% decrease in infection rating and 58-65%> decrease in ELISA absorbance at 46 DPI. Variation for response to TEV infection was evident among transgenic lines of all genotypes (Fig. 8). Variation within a genetic background was greatest for transgenic lines of BY 21, followed by BRv-neg and TN 86. This was illustrated by the magnitude of the standard deviations for ELISA values 46 DPI: 0.046 for BY 21, 0.042 for BRv-neg, and 0.038 for TN 86 (Fig. 8). On the other hand, most BRv-pos lines appeared nearly immune to TEV (SD 0.0097, Fig 8). For all genotypes other than BRv-pos, 6-7 lines of each genotype showed significantly lower TEV infection 46 DPI compared with their corresponding controls. The remaining lines in these backgrounds displayed about the same level of susceptibility as the controls. Highly resistant lines were generated in the susceptible backgrounds, namely BY 21 lines 4 and 6 and BRv-neg lines 1, 2, 7, and 8 (Fig. 8). A high positive correlation was obtained between infection score and ELISA at 46 DPI (R=0.72). In general, transgenic lines with less severe symptoms had lower virus accumulation in systemic leaves. The infection level at 14 DPI was also highly correlated with that obtained 46 DPI (R=0.6893 for infection scores, 0.6655 for ELISA readings). Most lines that had low infection scores at the beginning of the season displayed mild symptoms at flowering. However, some lines, such as BY-1 and BRv-neg-6, had delayed symptom onset, but developed the same level of disease as control plants by flowering time (Fig 8). Response of transgenic lines to PVY infection Results following inoculation with PVY were similar to those obtained after inoculation with TEV Differences among genotypes as well as among transgenic lines withm genotypes were statistically significant for symptoms of PVY infection However, the VAM gene was less effective against the PVY isolate (PVY- KYl) that was used in this study Table 4 Mean infection scores and ELISA values for transgenic lines and control genotypes at two growth stages following PVY inoculation Transgem c lines Score ELISA or control 14 DPI 46 DPI 14 DPI 47 DPI KY-CPs 0 190" 0 757" 0 160 0 249 * KY 8959 1 867 3 100 0 404 0 688 TN-CPs 0 350" 1 343" 0 214 * 0 234 * TN 86 1 500 3 633 0 635 0 659 BY-CPs 0 950" 2 247 ** 0 461" 0 557" BY 21 2 700 4 567 1 205 1 162 BRv-neg- CPs 0 577" 1 303" 0 401" 0 455 * BRv-neg 2 133 4 500 1 095 0 866 BRv-pos-i CPs 0 093 * 0 160" 0 060 0 208 * BRv-pos 0 900 2 400 0 279 0 878 * "Difference between mean of transgenic lines and host genotype significant at P= 0 05 and P=0 01, respectively All five control genotypes had symptoms of PVY infection 10-12 DPI, but symptoms in the VAM-pos genotypes were less severe (Table 4) The mean delay in PVY disease development of the transgenic lines was highly significant for all genotypes through 14 DPI The reduced symptom development in the transgenic lines was still evident at 46 DPI (Table 4) Differences in symptom seventy between transgenic lines and their respective controls 46 DPI were very similar for the different genetic backgrounds, 2 24 to 2 34 for KY 8959, TN 86, BY 21 , and BRv- pos, but the mean symptom score for BRv-neg transgenic lines was 3 20 less than the susceptible BRv-neg (Table 4). All BRv-pos transgenic lines were highly resistant to PVY infection (Fig. 9). In contrast, transgenic lines of the other four genotypes varied considerably for their reaction to infection by PVY (Fig. 9), but none of the BY 21 transgenic lines had infection scores that were less than 1. In contrast, 16 of the 30 transgenic lines in the KY 8959, TN 86, and BRv-neg backgrounds were highly resistant (infection score <1.0 at 46 DPI). Response of transgenic lines to TVMV infection Analysis of variance of the TVMV symptom scores and ELISA readings indicated significant differences among genotypes and among transgenic lines within a genetic background. However, the response of the transgenic lines to TVMV infection was different from those observed following infection with TEV and PVY. Since the VAM gene is very effective against TVMV, nontransformed genotypes and transgenic lines that possess this gene failed to develop symptoms early in the growing season. Only towards the end of the season did some plants of these genotypes show mild vein clearing and mottling. Symptoms appeared in the VAM- neg transgenic lines at the same time as in BY 21 and BRv-neg, but symptoms in the transgenic lines were attenuated when compared to the controls (Table 5, ELISA 14 DPI). Table 5 Mean infection scores and ELISA values for transgenic lines and control genotypes at two growth stages following TVMV inoculation Transgenic lines Score ELISA or host co ntrols 48 DPI 14 DPI 48 DPI KY-CPs 0.427 0.018 0.183 KY 8959 0.800 0.067 0.276 TN-CPs 0.710 0.053 0.134 TN 86 1.170 0.182 0.201 BY-CPs 1.330" 0.301" 0.166" BY 21 5.000 1.533 1.695 BRv-neg- CPs 1.417" 0 132" 0.145" BRv-neg 4.830 0.866 1.286 BRv-pos- 1 CPs 0.410" 0.041 0.104 BRv-pos 1.670 0.044 0.110 " Difference between mean of transgenic lines and host genotype significant at P= 0.05 and P=0.01, respectively. Later in the season, the upper leaves of the infected VAM-neg transgenic plants were essentially symptomless. ELISA results of samples taken from leaves at different stalk positions showed no TVMV in the upper leaves (Fig. 10). The mean virus level (ELISA) in top leaves at the end of season was not different among the transgenic lines regardless of the presence or absence of the VAM gene (Table 5). This recovery phenomenon varied among transgenic lines and genotypes. Seven BY 21 transgenic lines exhibited different degrees of recovery. Only four BRv-neg lines showed this phenomenon. Correlations among TVMV, TEV, and PVY infection Correlation coefficients, using data from all three experiments, were calculated to determine the general relationship among the different virus disease ratings. High correlations between TEV and PVY infections for infection score (46 DPI) and ELISA (47 DPI) were found. Low correlations were found between TEV and TVMV infections, PVY and TVMV infections (Table 6). Table 6 Correlation coefficients between infection scores (46 DPI) and ELISA values (47 DPI) for transgenic lines infected with TEV, PVY, and TVMV PVY TVMV Score TEV 0.8517 ** 0.1738 PVY 0.3148 * ELISA TEV 0.6480" 0.1631 PVY 0.1967 *,** Correlation coefficients between infection scores and ELISA values significant at P < 0.05 and P < 0.01, respectively. The VAM-pos transgenic lines could be classified into three groups based on the responses to TEV and PVY, as all VAM-pos transgenic lines were highly resistant to TVMV inoculation. The first group included the transgenic lines highly resistant or immune to infection by both TEV and PVY. Only those lines with infection scores that were less than one and ELISA values that were not significant from zero for all three inoculation experiments were included in this group. Six lines in a KY 8959 background, 4 lines of TN 86, and all 10 lines of BRv-pos were included in this group. The second group of lines had significantly delayed and attenuated disease symptoms and had different reactions to TEV and PVY infection. Four lines of KY 8959 and 4 lines of TN 86 belong to this group. The last group included 1 KY 8959 line and 2 TN 86 lines that were susceptible to both viruses. In contrast, only one VAM-neg transgenic line (BRv-neg- 1) was resistant to all three viruses. Other VAM-neg lines were classified into four response categories. Fig. 11 summarizes these responses. The first response category included those lines with some resistance to TEV and PVY and a recovery phenotype to TVMV. Another group of lines had reduced infection to all three viruses, and the third group included those lines that were susceptible to TEV and PVY but recovered after infection with TVMV. The remainder of the lines were susceptible to all viruses (Fig. 11). Table 7 Infection scores and ELISA values of hybrids of TVMV CP transgenic lines and their respective parents following inoculation with TEV, TVMV, and PVY. TEV TVMV PVY Entry Score ELISA Score ELISA Score ELISA 1 BRv-neg 4.50 0.60 3.90 1.35 3.77 1.18 2 BRv-pos 1.80 0.00 1.10 0.08 2.20 1.22 3 BRv-neg-t3 0.13 0.00 1.03 0.02 0.83 0.31 4 BRv-neg-t30 0.47 0.00 0.87 0.28 1.27 0.46 5 BRv-pos-t21 0.20 0.00 0.67 0.00 0.00 0.00 6 BRv-pos-t34 0.00 0.00 0.43 0.04 0.00 0.00 7 BRv-neg x BRv-pos 4.50 1.34 3.03 1.17 3.57 1.20 8 BRv-neg-t3 x BRv-neg 0.73 0.00 2.33 0.15 1.47 0.43 9 BRv-neg- 130 x BRv-neg 0.63 0.00 2.10 0.61 1.30 0.38 10 BRv-neg-t3 x BRv-pos 0.07 0.00 1.33 0.35 0.43 0.15 11 BRv-neg-t30 x BRv-pos 0.20 0.00 2.50 0.21 1.67 0.30 12 BRv-pos-t21 x BRv-neg 0.00 0.00 0.80 0.07 0.63 0.58 13 BRv-pos-t34 x BRv-neg 0.00 0.00 1.77 0.06 0.57 0.20 14 BRv-pos-t21 x BRv-pos 0.00 0.00 0.10 0.01 0.03 0.00 15 BRv-pos-t34 x BRv-pos 0.00 0.00 0.43 0.04 0.00 0.00 16 BRv-neg-t3 x BRv-neg-t30 0.30 0.00 0.70 0.17 0.93 0.40 17 BRv-neg-t3 x BRv-pos-t21 0.03 0.00 0.70 0.18 0.27 0.00 18 BRv-neg-t3 x BRv-pos-t34 0.03 0.00 0.83 0.13 0.23 0.08 19 BRv-neg-t30 x BRv-pos-t21 0.00 0.00 1.13 0.05 0.03 0.03 20 BRv-neg-t30 x BRv-pos-t34 0.23 0.00 0.97 0.21 0.40 0.08 21 BRv-pos-t21 x BRv-pos-t34 0.00 0.00 0.03 0.06 0.00 0.00 LSD (0.05) 0.52 0.20 0.80 0.47 0.43 0.36 Contrast 1 BRv-neg-t x BRv-neg vs. BRv-neg ** ** ** #* ** ** 2 BRv-neg-t x BRv-neg vs. BRv-pos ** NS ** NS ** ** 3 BRv-pos-t x BRv-pos vs. BRv-neg ** ** ** ** ** ** 4 BRv-pos-t x BRv-pos vs. BRv-pos ** NS * NS ** ** 5 BRv-neg-t x BRv-neg vs. BRv-neg-t * NS ** NS * NS 6 BRv-pos-t x BRv-pos vs. BRv-pos-t NS NS NS NS NS NS 7 BRv-neg-t vs. BRv-neg ** ** ** ** ** ** 8 BRv-neg-t vs. BRv-pos ** NS NS NS ** ** 9 BRv-pos-t vs. BRv-neg ** ** ** ** ** ** 10 BRv-pos-t vs. BRv-pos ** NS NS NS ** ** 11 BRv-neg-t x BRv-neg-t vs. BRv-neg ** ** ** *=> * ** 12 BRv-pos-t x BRv-pos-t vs. BRv-pos ** NS * NS ** ** 13 BRv-neg-t x BRv-neg-t vs. BRv-neg-t NS NS NS NS NS NS 14 BRv-pos-t x BRv-pos-t vs. BRv-pos-t NS NS NS NS NS NS 15 BRv-neg-t x BRv-pos-t vs. BRv-neg ** ** ** ** ** ** 16 BRv-neg-t x BRv-pos-t vs. BRv-pos ** NS NS NS ** ** 17 BRv-neg-t x BRv-pos-t vs. BRv-neg-t NS NS NS NS ** ** 18 BRv-neg-t x BRv-pos-t vs. BRv-pos-t NS NS NS NS NS NS ** Difference of contrasts at the level of P=0.05 and P=0.01, respectively. Experiment of TVMV CP transgenic hybrids Analysis of variance revealed that genotype effects for virus infection scores were significantly different following inoculations with each virus. Only the nontransformed susceptible controls (Table 7, Entry 1) and the hybrid of the nontransformed controls (Entry 7) developed severe symptoms of TEV and TVMV infection at flowering, while the VAM-pos control (BRv-pos, Entry 2) had developed mild symptoms by the same growth stage. All homozygous transgenic lines possessing the VAM gene (Entries 5 and 6) and their hybrid (Entry 21), which has two nonallelic copies of CP, were resistant to TEV and TVMV infections. No VAM- neg transgenic lines and hybrids developed significant symptoms of TEV infection, nor did they accumulate detectable levels of TEV regardless of the number of TVMV CP copies they possessed (Table 7). In contrast, VAM-neg transgenic lines hemizygous for the TVMV CP gene (Entries 8-11) developed symptoms following TVMV infection and had some TVMV accumulation regardless of whether the hybrids had BRv-neg or BRv-pos as the other parent. Entries with two copies of the TVMV CP gene and heterozygous for the VAM gene (Entries 16-20) had symptoms of TVMV infection at end of the season. Contrasts of the hemizygous and nontransformed controls (contrasts 1-4) revealed that hemizygotes had significantly reduced TEV and TVMV infections. A contrast (contrast 5) between entries hemizygous or homozygous for the TVMV CP gene were significant for TEV and TVMV infection scores but not for ELISA for each virus. Hybrids containing two copies of CP in a nonallelic position had lower infection scores and ELISA values than their corresponding nontransformed controls regardless of whether the VAM gene was present (contrasts 11 and 12). These hybrids had the same level of resistance as their homozygous parent lines (contrast 13 and 14). The same trends were observed in the hybrids containing two copies of the TVMV CP gene and heterozygous for the VAM gene against TEV infection (contrast 15, 16, 17, and 18). However, these hybrids did not exhibit a higher level of resistance than the VAM-pos nontransformed controls following TVMV infection (contrast 16). The VAM gene is not completely effective against the isolate of PVY that we utilized in this study. All plants of BRv-pos, BRv-neg, and their hybrids displayed severe symptoms following PVY inoculation (Table 7). However, VAM-neg lines homozygous or hemizygous for the TVMV CP gene (Entries 3, 4, and 8-11) had attenuated symptoms, but symptom reduction was more evident in VAM-pos lines homozygous or hemizygous for the TVMV CP gene (Entries 5, 6, 14, and 15). Hybrids which were hemizygous for TVMV CP gene and heterozygous for the VAM gene (Entries 12 and 13) also had strong resistance to PVY infection. Contrasts revealed that VAM-neg hybrids hemizygous for CP had significantly reduced PVY infection compared with both nontransformed controls (contrasts 1 and 2). The same effect was also found in VAM-pos hemizygous lines (contrast 3 and 4). However, VAM-neg hemizygous lines were somewhat less resistant than homozygous transgenic lines (contrast 5). All homozygous transgenic lines significantly differed from the controls for PVY infection, although the VAM- neg transgenic lines were not symptomless. Hybrids between two transgenic lines were equally as effective in controlling PVY infection as homozygous lines (contrasts 11-14). Interestingly, hybrids with two single copies of the CP gene and heterozygous for the VAM gene had significantly higher resistance than VAM-neg homozygous lines (contrast 17) and the same level of resistance as VAM-pos transgenic lines (contrast 18). Table 8 Plant height and yield of hybrids of TVMV CP transgenic lines and their respective parents: no virus challenge and following inoculation with TEV, TVMV, and PVY in the field Inoculation treatment Inoculation treatment Entry NONE TEV TVMV PVY NONE TEV TVMV PVY __ ___T^1 * + l-ιe»irτlι+ ( f- n) Yield τ l * «H r 1 BRv-neg 117.2 109.1 116.8 119.0 146.5 144.5 152.8 130.6 2 BRv-pos 121.4 124.6 123.3 116.1 124.8 167.4 150.5 157.3 3 BRv-neg-t3 115.6 129.8 113.2 122.3 130.2 170.9 140.4 151.8 4 BRv-neg-t30 125.3 130.8 125.2 114.5 143.0 158.5 137.5 141.0 5 BRv-pos-t21 104.3 97.6 103.4 88.1 107.6 108.6 106.2 95.1 6 BRv-ρos-t34 117.9 124.5 119.2 116.5 142.5 156.3 138.2 148.0 7 BRv-neg x BRv-pos 112.9 115.3 123.3 116.3 141.4 136.1 152.2 157.8 8 BRv-neg-t3 x BRv-neg 113.6 120.2 117.8 117.2 131.7 156.4 150.3 156.3 9 BRv-neg-t30 x BRv-neg 122.5 126.3 119.1 112.1 140.0 163.3 125.6 147.0 10 BRv-neg-t3 x BRv-pos 117.0 136.4 121.8 109.9 129.9 163.2 158.2 151.7 11 BRv-neg-t30 x BRv-pos 122.0 130.4 1 15.6 114.0 152.1 165.1 148.5 136.8 12 BRv-pos-t21 x BRv-neg 107.7 1 11.1 118.5 97.8 116.1 135.2 131.3 130.1 13 BRv-pos-t34 x BRv-neg 120.3 131.7 118.0 121.4 128.8 160.6 151.9 146.3 14 BRv-pos-t21 x BRv-pos 112.7 107.8 112.1 107.3 118.7 124.4 130.7 108.2 15 BRv-pos-t34 x BRv-pos 122.7 121.4 117.0 122.4 145.6 156.1 155.8 149.1 16 BRv-neg- 13 x BRv-neg- t30 123.3 131.6 123.9 104.7 137.4 166.5 138.9 159.3 17 BRv-neg-t3 x BRv-pos- 121 113.7 103.7 114.2 99.8 118.6 129.6 123.9 106.7 18 BRv-neg-t3 x BRv-pos- t34 116.2 126.0 119.4 119.6 126.0 160.5 132.1 157.5 19 BRv-neg-t30 x BRv-pos- -t21 109.8 114.8 111.7 96.6 114.5 121.6 128.0 120.4 20 BRv-neg-t30 x BRv-pos- -t34 128.4 127.5 116.0 1 18.2 124.3 157.2 137.8 156.7 21 BRv-pos-t21 x BRv-pos- ■t34 108.7 106.7 108.5 98.1 120.2 124.0 125.1 107.3 LSD (0.05) - 10.8 10.4 41.7 - 18.9 21.5 34.1 Contrast 1 BRv-neg-t x BRv-neg vs. BRv-neg - ** NS NS - NS NS NS 2 BRv-neg-t x BRv-neg vs. BRv-pos - NS NS NS - NS NS NS 3 BRv-pos-t x BRv-pos vs. BRv-neg - #* NS NS - NS NS NS 4 BRv-pos-t x BRv-pos vs. BRv-pos - NS NS NS - NS NS NS 5 BRv-neg-t x BRv-neg vs. , BRv-neg-t - NS NS NS - NS NS NS 6 BRv-pos-t x BRv-pos vs. BRv-pos-t - NS NS NS - NS NS NS 7 BRv-neg-t vs. BRv-neg - ** NS NS - * NS NS 8 BRv-neg-t vs. BRv-pos - NS NS NS - NS NS NS 9 BRv-pos-t vs. BRv-neg - ** NS NS - NS NS NS 10 BRv-pos-t vs. BRv-pos - NS NS NS - NS NS NS 1 1 BRv-neg-t x BRv-neg-t vs. BRv-neg - ** NS * - * NS NS 12 BRv-neg-t x BRv-neg-t vs. BRv-neg- NS NS ** - NS NS NS 13 BRv-neg-t x BRv-pos-t vs. BRv-neg - ** NS NS - NS NS NS 14 BRv-neg-t x BRv-pos-t vs. BRv-pos - NS NS NS - NS NS NS 15 BRv-neg-t x BRv-pos-t vs. BRv-neg- NS NS NS " NS NS NS *, ** Difference of contrasts at the level of P=0.05 and P=0.01, respectively There were no significant differences among entries for plant height and yield under noninoculated conditions (Table 8). Following virus inoculation, there were significant differences among entries for both plant height and yield. Since BRv-pos-t21 showed inferior performance under all treatments, although it did not reach a statistically significant level, data for it was eliminated in contrasts for plant height and yield means (Table 8). The vast majority of contrasts for plant height and yield were not significant. The VAM-neg homozygous and hemizygous lines had significantly greater height than the BRv-neg control following TEV inoculation. The VAM-neg homozygous lines and hybrids also had greater yield after TEV infection. The effectiveness of host plant resistance in mediating potyvirus-induced disease in tobacco and other crops is complicated by the extreme variation among potyviruses for infectivity and pathogenicity. The main focus of this study was to test the effectiveness of combining two strategies, CPMR and endogenous host resistance, for resistance against potyvirus infection under field conditions. In general, TVMV CP transgenic lines possessing the VAM gene had significantly less potyvirus-induced disease than TVMV CP transgenic lines that lacked the VAM gene. This was manifested as a higher frequency of resistant transgenic lines and as a greater level of resistance. About three-fourths of the VAM- pos transgenic lines had high resistance to TEV infection and about 50%ι of these lines had high resistance to PVY infection. In contrast, only 40% or less of the VAM-neg transgenic lines had high resistance to TEV infection and 10%> or less of these lines had high resistance to PVY infection. Furthermore, the VAM-pos transgenic lines had average disease ratings and ELISA values that were significantly less than those for VAM-neg transgenic lines for all three viruses. These results suggested that combining these two strategies had synergistic effects against potyvirus infection. Two NILs, BRv-neg and BRv-pos, were used to provide a direct assessment of the impact of combining the VAM gene and CPMR. More transgenic lines in the BRv-pos background had significantly higher resistance than those in a BRv-neg background for all tested viruses. When averaged across all three virus experiments, BRv-neg transgenic lines had an average infection score and an ELISA value that was less than those of the nontransgenic BRv-pos. Across all genotypes, the TVMV CP gene appeared to cause stronger resistance than the VAM gene, but combining the two factors was undoubtedly superior to either factor alone. Genotype background effects on expression of resistance were not limited to presence or absence of the VAM gene. This was evident among the three VAM- pos genotypes. Most BRv-pos transgenic lines appeared immune to all three tested viruses, while most lines from KY 8959 displayed a lower level of resistance than BRv-pos, but a higher level of resistance than those from TN 86. These varied responses followed closely the differences in the level of the tolerance of the nontransgenic recipient genotypes. These results suggest that, in a breeding program using CPMR, selection of an appropriate recipient parent would be an important step in maximizing the level of resistance in the transgenic cultivar. The above-described results demonstrate that combining CPMR with the endogenous resistance VAM gene increased resistance to the VAM breaking strains of PVY. This study evaluated near isogenic lines for endogenous resistance gene (VAM) and their transgenic lines and hybrids. The data revealed that hybrids hemizygous for two copies of TVMV CP transgene and homozygous for the VAM gene (VAM-pos) protected against TEV, TVMV, and PVY infections at the same level as homozygous VAM-pos transgenic lines. VAM-pos hybrids hemizygous for one insertion of the TVMV CP gene had the same level of resistance to all three viruses as the VAM-pos homozygous lines. These results indicated that protection against all three potyviruses was obtained in hybrids from crosses VAM-pos transgenic line and a VAM-pos genotype. Other types of hybrid combinations were less effective in reducing potyvirus-induced disease symptoms. These combinations included TVMV CP transgenics that were heterozygous for the VAM gene and VAM- neg transgenics with two hemizygous TVMV CP genes. The effective form of the VAM gene for controlling virus infection is as a homozygous recessive (Legg et al, Can. J. Genet. Cytol. 22: 21-26, 1980; Gupton and Burk, J. Hered. 64: 289-290, 1973). Hybrids hemizygous for one and two copies of the TVMV CP gene and heterozygous for the VAM gene had higher PVY resistance than hybrids that had the same number of copies of the TVMV CP gene but lacked the VAM gene. This indicated that CPMR enhanced the effect of the VAM gene against PVY infection even when the VAM gene was heterozygous genotype. Broad spectrum protection provided by CPMR could be most important in its value to crop improvement. Several studies have reported potyvirus CPMR (Stark et al, Bio/Technology 7: 1257-1262, 1989; Maiti et al, Proc. Natl. Acad. Sci. 90: 6110-6114, 1993; Namba et al, Phytopathology 82: 940-946, 1992). These results revealed that the TVMV CP gene confers protection to three viruses, and to also 5 different PVY strains. Namba et al. (Phytopathology 82: 940-946, 1992) studied the protection in plants expressing the watermelon mosaic virus II or zucchini yellow mosaic virus CP genes to six different potyviruses, and concluded the level of cross protection depended on the challenge virus and inoculum strength. In our study, some BRv-pos TVMV CP transgenic lines are immune to a heterologous virus, PVY, and this immunity did not depend on the concentration of inoculum. The inconsistencies between results of Namba et al. (Phytopathology 82: 940-946, 1992) and the results here may be explained by the advantage by combining two resistance strategies. CPMR displays a high potential of feasibility as a new source of potyvirus resistance on tobacco breeding, since CPMR provides a source of protection against the virus or strains which the VAM gene can not protect. Our results have important implications for programs using CPMR to improve virus resistance in crops. First, we have shown the importance of selecting an appropriate recipient genotype, and second, we have provided guidelines for the development of transgenic lines with high resistance. This latter point addresses the need to generate a sufficient number (30-50) of primary transformants to obtain optimum resistance. Correlation between infection scores for PVY-O in the greenhouse experiments and PVY results in the field were significant (r=0.51), but this correlation was not sufficient to allow using greenhouse results as a very reliable prediction of field performance. Third, the CPMR approach can produce a homozygous transgenic line much faster than breeding for virus resistance using traditional methods. This has obvious advantages when new strains of a pathogenic virus may quickly become predominant. Fourth, the effectiveness of protection provides by a CP gene in the hemizygotes state makes it convenient and efficient to transfer CPMR to other genotypes by conventional plant breeding methods. The above-described results were obtained using the following materials and methods. Plant materials and transformation Five genotypes of burley tobacco (Nicotiana tabaccum L.) were transformed with a coat protein gene obtained from the TVMV. The five genotypes were: Burley 21, TN 86, KY 8959, BRv-neg, and BRv-pos. Burley 21 is susceptible to potyvirus infection, and TN 86 and KY 8959 possess the VAM gene (va gene) that provides protection against infection by some potyviruses. BRv-neg and BRv-pos are near isogenic lines (NILs) that differ for the presence or absence of the VAM gene. The transformation method and transgenic line development were performed as described above. Approximately 30 transgenic lines were produced in each genetic background. Hybrids Two BRv-neg and two BRv-pos transgenic lines, each homozygous for a single insertion of the TVMV CP gene, were selected based their expression of a high level of virus resistance in greenhouse experiments. These four lines and the two original nontransformed lines were crossed in a partial diallel design with no reciprocals. The resulting fifteen hybrids and their six parent lines provided various combinations of the endogenous VAM gene and TVMV CP gene. Two transgenic lines expressing only pKYLX vector and designated as BY-KYLX and BRv-pos- KYLX were also included in this experiment. Field experimental design Field experiments were designed in a split-plot arrangement with three replications. The five recipient genotypes were designated as main plots. Sub-plots consisted of the original nontransformed genotypes and 10 transgenic lines that were randomly selected from among all transgenic lines developed for each genotype. Therefore, each replication included 55 entries, 50 transgenic lines and the 5 nontransformed controls. The basic design was repeated three times using different randomizations to allow for separate inoculations with TEV, PVY, and TVMV. The three experiments were established in close proximity in the same field. Three weeks prior to tobacco transplanting, maize was planted between the three experiments to provide a partial barrier against the movement of aphids among these experiments. Seeds from each transgenic line in the R 2 generation and homozygous for the CP gene, and seeds of the five control genotypes, were germinated in 10 cm diameter pots containing a commercial potting mix on March 20, 1995. Four weeks after seeding, plants were transferred to trays designed for greenhouse transplant production. Plants were fertilized once a week for five weeks with a water soluble fertilizer until reaching a size (15-20 cm in height) suitable for transplanting to the field. Plants were transplanted to the field, a Maury silt loam soil (fine, mixed, mesic Typic Paleudalfs), using a mechanical transplanter on May 23 and May 24, 1995. Each plot consisted of 12 plants spaced 46 cm apart. Row width was 107 cm. All production practices used were those recommended for burley tobacco. Evaluation of hybrids The 15 hybrids and 6 parental lines were grown in a randomized complete block design with three replications. The basic design was repeated three times using different randomization to allow for separate inoculations with TEV, PVY, and TVMV. Field layout and cultural practices were the same as those for the experiments with the homozygous lines. Field inoculation and evaluation The TEV, PVY, and TVMV isolates used in these experiments were originally isolated from infected tobacco growing in Kentucky. Virus stocks were maintained in susceptible Burly 21 plants grown in a greenhouse. When virus infections reached a peak stage (2 weeks post inoculation), infected leaf tissue was triturated into 0.05 M Na 2 HPO 4 KH 2 PO 4 buffer (pH 7.0) at the rate of 1 g of tissue per 10 ml of buffer and used as inoculum. One gram of 600-mesh carborundum was added to each 100 ml of inoculum. Inoculum was kept on ice in flasks and transported to the field for inoculation the same day it was prepared. Plants were inoculated mechanically by applying inoculum to one expanding leaf per plant using a sponge. Inoculations were performed during the period between June 7 to June 9, 1995. One week after inoculation and at weekly intervals thereafter, each plant was inspected visually and rated for symptom expression. Symptom expression was rated using a scale from 0 to 5, where 0=no symptoms, l=mild chlorosis and mosaic, 2=moderate mosaic, 3-severe mosaic and some chlorosis and etching of leaves, 4=severe chlorosis and etching of leaves, and 5=severe chlorosis and leaf distortion. The mean rating of the 12 plants in each plot was used as the experimental unit for statistical analysis. Virus detection in inoculated plants was accomplished by direct ELISA (Clark et al, J. Gen. Virol. 34: 475-483, 1977; Clark et al, Methods Virol. 7: 51-85, 1984). IgG of antisera was purified by passing through a protein A column (Bio-Rad Co.). Tissue samples were taken twice, approximately 14 and 47 days post inoculation (DPI). Each sample consisted of a bulk of 12 cm 2 discs from young expanding leaves on five plants per plot. Details of the ELISA procedure were described above. Data analysis Data were analyzed by standard analysis of variance procedures (SAS/STAT, User's Guide, SAS Institute Inc., 1993) for each experiment in which TEV, PVY, or TVMV were used as inoculum. Mean comparisons for infection scores and ELISA readings were accomplished using least significant difference (LSD) test. Contrasts between the mean of the 10 transgenic lines and their corresponding non-transformed host were performed. Variation among lines for expression of resistance was described by calculating the standard deviation for each genotype group. Contrasts of CP lines versus VAM resistance and CP VAM-pos (host genotype has VAM gene) transgenic lines versus CP VAM-neg (host genotype lacks VAM gene) transgenic lines were also performed using SAS. Pearson correlation coefficients for mean symptom ratings among transgenic lines inoculated with TEV, PVY, and TVMV were determined to describe the effectiveness of resistance against the different viruses. IV. Resistance of Transgenic Burley Tobaccos Expressing Alfalfa Mosaic Virus Coat Protein Gene to AMV Infection Expression of AMV CP gene in different burley tobacco genotypes Ninety-four independent R Q lines in TN 86, BY 21, and KY 8959 background were generated by transformation with A. tumefaciens. Based on the results from the kanamycin screens, most of the lines had a single insertion and the remainder had double insertions. Expression of AMV CP in 29 of the transgenic lines was determined by immunoblotting. The blots indicated that the transgenic lines expressed the full length AMV CP, and that the insert was the same size as seen in the infective virion. The transgenic lines expressed various levels of AMV CP, ranging from 0 to 300 ng/mg of soluble leaf protein. Most transgenic lines expressed approximately 100 ng AMV CP per mg of soluble protein (Fig. 12). Determination of the level and extent of virus resistance in greenhouse experiments Plants of the R, and R 2 generation lines were screened for resistance to AMV in the greenhouse. The first experiment assessed the reaction of 11 R, AMV- CP transgenic lines in the BY 21 background to infection with three P^s isolates: AMV-425 (strain from which the AMV CP gene was cloned), AMV Kentucky (AMV-KY), and a North Carolina (AMV-NC) isolate. Symptoms typical of AMV infection (vein clearing, mottling) appeared in nontransformed BY 21 plants 7 days after inoculation with each of the three isolates. Symptoms were further observed to be expressed as a typical golden yellow mosaic pattern on infected leaves 14 DPI. In contrast, symptoms in most transgenic lines were delayed and attenuated. A few lines did not develop any symptoms after inoculation with AMV-425 (Fig. 13). Resistance was also expressed in the transgenic lines following infection with AMV-KY. However, only a slight symptom delay and a moderate attenuation of symptoms were evident in transgenic lines infected with AMV-NC (Fig. 14). Results from ELISA readings indicated that transgenic lines highly resistant to AMV-KY had barely detectable levels of AMV in the upper leaves. Reinoculation to cowpea confirmed the results since no symptoms developed in the cowpea plants. Expression levels of AMV CP in the transgenic lines were not correlated with the level of virus resistance A high concentration of a local isolate of AMV (AMV-KY) was used to ensure maximum infection in a screen of 47 R 2 AMV transgenic lines in three different genetic backgrounds. Nontransformed controls and 5 transgenic lines developed obvious symptoms 6 days after AMV inoculation. Symptoms in two control genotypes, TN 86 and KY 8959, were more severe than symptoms in BY 21. At the termination of the experiment (25 DPI), an additional eight transgenic lines (all from TN 86 and KY 8959) displayed severe symptoms including stunting and severe leaf distortion. Twenty-eight (60%) of the transgenic lines showed varying levels of resistance. One third of these lines (10) failed to develop any visible symptoms; the remaining two thirds (18) developed mild symptoms (Table 9). However, ELISA and a reinoculation assay revealed that AMV was present at low levels in samples taken from leaves above the inoculated leaf in the symptomless transgenic lines. Transgenic lines derived from KY 8959 and TN 86 appeared more resistant to AMV infection than BY 21 transgenic lines (Table 9). Table 9 Response of transgenic lines expressing AMV CP to AMV-KY infection in the greenhouse experiments Infection" 0% 1-20% 21-60% 61-100% 100% Total Index b 0 0.1-0.2 0.2-1.0 1.0-2.0 2.0-6.0 KY 8959 4 3 1 1 5 14 TN 86 2 2 4 5 3 16 BY 21 4 3 5 5 0 17 Total 10 8 10 11 8 47 % 21.3 17.0 21.3 23.4 17.0 a Percent of plants showed symptoms. b Average rating of plants, using the rating of 0-6. 1994 field experiment The objectives the initial field tests were: (1) to evaluate the response of transgenic lines under natural conditions to AMV; and (2) to compare the effects of two different inoculation times on the expression of AMV resistance. All plants of TN 86 were inoculated one day prior to transplanting and developed AMV symptoms within 14 days. TN 86 plants inoculated with AMV 14 days after transplanting showed symptoms 8 days later. All BY 21 transgenic lines displayed an AMV symptom delay of 4-10 days and a lower disease incidence than that observed for TN 86 (Table 10). Table 10 Resistance of transgenic lines expressing AMV CP to AMV-KY infection under field conditions (1994) 21 DPI 67 DPI Inoculated Inoculated Non- Inoculated Noninoculated inoculated Incidence" ELISA •-Incidence -ELISA BY-A-1 22.69 0.653 27.08 9.75 0.420 0.139 BY-A-2 30.78 0.453 36.04 4.18 0.464 0.330 BY-A-3 28.14 0.743 29.88 1.40 0.485 0.196 BY-A-4 19.74 1.056 19.39 2.78 0.446 0.186 BY-A-5 22.43 0.055 30.08 6.98 0.397 0.136 BY-A-6 26.28 0.563 28.70 6.95 0.376 0.017 BY-A-11 25.12 0.252 24.72 2.80 0.428 0.051 BY-A-16 35.09 0.624 35.68 8.35 0.621 0.323 BY-A-17 21.37 0.717 32.67 5.58 0.773 0.365 BY 21 54.56 1.688 65.55 5.55 1.358 0.238 TN 86 90.09 2.336 97.23 5.58 1.718 0.547 L D 005 12.81 0.605 15.91 15.91 0.409 0.409 ' Percent of plants showed symptoms. The incidence of infection and ELISA readings recorded for all the tested transgenic lines were significantly lower than those recorded for the nontransformed controls, BY 21 and TN 86, under both inoculation regimes (Table 10). There was no significant differences for incidence of AMV symptoms or ELISA readings between the two inoculation stages. However, because of high ambient temperatures after inoculation at the second inoculation (mid- June, 30°C), some infected plants showed deterioration of the meristem and died. During the growing season, all AMV CP lines had lower infection scores than the BY 21 and TN 86 control plants. Disease symptoms did not increase appreciably dramatically in the AMY CP lines from early in the season until the experiment was terminated (Table 10). In the noninoculated plots, only a few scattered plants were infected and there were no significant differences between transgenic lines and controls for disease incidence (Table 10). Some variation was observed among transgenic lines for their response to AMV inoculation. However, the majority of the lines were found to respond similarly to AMV inoculation. 1995 field experiment As was observed in the 1994 test, symptoms of AMV infection also appeared in the control plants 14 days after transplanting in the 1995 experiment. Disease incidence in the transgenic lines was significantly lower than in the control plants. During the growing season, there were dramatic differences in the severity of infection between transgenic lines and controls. Therefore, infection scores were used to estimate both disease incidence and severity. Analysis of variance for infection score and virus accumulation (ELISA readings) at 21 DPI indicated no significant differences among background genotypes. Differences among transgenic lines within genotypes were significant for all observed traits. Means for infection scores and ELISA values of the 10 transgenic lines in each genotype were significantly lower than those of the corresponding controls, except for BY 21 and its transgenic lines (Figs. 15A and 15C). Symptom severity increased in the transgenic lines and in the nontransformed controls during the growing season. However, all of the control plants had severe stunting and leaf distortion, and disease scores in the transgenic lines were significantly reduced (60-80%) compared to the controls (Fig. 15B). Virus levels were also significantly decreased, although the magnitude of reduction was not as great as for the visible symptoms (Fig. 15D). Virus accumulation in the transgenic lines was higher than for the same lines in the 1994 trial (Table 10). This may have resulted from the higher concentration of challenge virus in the 1995 trial. Differences among the transgenic lines in the three genetic backgrounds were evident. The KY 8959 and TN 86 transgenic lines also exhibited higher levels of AMV resistance than the BY 21 transgenic lines, even though KY 8959 and TN 86 were more susceptible to AMV than BY 21. Variation among transgenic lines in each genotype background was evident (Fig. 16). One TN 86 line was found to not develop symptoms in any of the three replications in the trial, while all other lines had some symptoms of infection. Eight KY 8959 lines, ten TN 86 lines, and nine BY 21 lines had significantly reduced symptom severity compared with nontransformed controls. Three KY 8959 lines, ten TN 86 lines, and two BY 21 lines had both reduced symptom intensity and lower virus accumulation. None of the transgenic lines developed a disease severity that was equivalent to the nontransformed controls. The main effect of the AMV CP gene in controlling AMV infection was a reduction in visible symptoms. A low correlation between infection score and ELISA value was found (Table 11). This was attributed to the fact that the reduction in virus levels in systemically infected leaves was proportionately less than the reduction in infection scores. Table 11 Correlation coefficients between infection score and ELISA reading for the transgenic lines infected with AMV under field conditions (1995) Score 67 DPI ELISA 27 DPI ELISA 67 DPI Score 27 DPI 0.7250" 0.0589 0.1285 Score 67 DPI 0.1396 0.2382" ELISA 27 DPI 0.3244" Expression of virus resistance in hybrids containing both TVMV CP and AMV CP genes and hybrids hemizygous for these genes Analysis of variance indicated significant differences among entries for all traits. BY 21 and the control BY-kylx transgenic lines that had no viral CP gene developed severe symptoms to all the tested viruses. KY 8959, TN 86, and BRv-pos- kylx developed mild symptoms following TEV and TVMV infections and severe symptoms following PVY and AMV infections (Table 12). Table 12 Infection scores and ELISA values of hybrids that combine TVMV and AMV CP and their respective parents following inoculation with TEV, TVMV, PVY, and AMV in the field. TEV TVMV PVY AMV Entry Score ELISA Score Score ELISA Score ELISA 1 KY 8959 3 50 0 85 1 03 2 90 0 39 8 00 1 52 2 TN 86 2 67 0 98 2 33 3 00 0 81 7 93 1 38 3 BY 21 5 00 1 47 4 57 4 50 1 27 5 03 1 55 4 BY-kylx 5 00 1 43 4 93 4 50 1 24 6 13 1 45 5 BRv-pos- -kylx 1 23 0 28 2 13 2 25 0 84 8 93 1 55 6 KY-t41 0 07 0 03 0 87 0 00 0 19 8 57 1 58 7 TN-t46 0 03 0 02 1 40 0 00 0 00 8 80 1 54 8 TN-tl9 0 20 0 01 1 73 0 00 0 00 9 00 1 49 9 BY-t2 1 80 0 25 2 67 3 95 0 69 4 07 1 45 10 BY-t4 0 10 0 04 2 53 1 00 0 73 4 80 1 34 1 1 KY-al2 2 90 1 20 0 93 2 05 0 70 1 43 1 30 12 KY-al 7 2 57 1 51 1 17 3 50 0 76 1 53 1 22 13 TN-a3 3 67 1 02 1 63 3 35 0 82 1 73 0 99 14 BY-a5 5 00 2 01 5 00 2 50 1 15 4 43 1 23 15 KY-t41 : x. KY 8959 0 03 0 16 0 83 2 50 0 33 7 83 1 54 16 TN-t46 x T 86 0 10 0 01 1 20 0 00 0 01 7 63 1 48 17 BY-t4 x BY 21 1 77 0 10 2 80 2 00 0 80 4 20 1 50 18 KY-al2 x KY 8959 - - - - - 2 30 1 25 19 TN-a46 : X TN 86 - - - - - 1 83 1 29 20 BY-a5 x BY 21 - - - - - 4 63 1 57 21 KY-al2 x KY-t41 3 30 1 06 0 80 2 65 0 77 2 33 1 37 22 KY-al 7 x KY-t41 2 43 1 60 0 93 3 15 0 90 2 10 1 27 23 TN-a3 x TN-tl9 2 93 0 86 1 83 2 05 0 27 1 43 1 35 24 TN-a3 x TN-t46 2 07 0 74 1 63 2 40 0 52 5 73 1 44 25 BY-a5 x BY-t2 4 67 1 78 3 97 4 00 0 88 3 13 1 00 26 BY-a5 x BY-t4 4 00 1 83 3 03 5 00 1 20 2 60 1 06 LSD (0 05) 0 65 0 42 0 93 2 62 0 65 1 04 0 38 Contrast I KY-t-a vs KY-t x KY ** ** NS NS NS ** NS 2 KY-t-a vs KY-t ** ** NS * * ** NS 3 KY-t-a vs KY-a x KY - - - - - NS ** 4 KY-t-a vs KY-a NS NS NS NS NS ** NS 5 KY-t-a vs KY * * NS NS NS ** NS 6 TN-t-a vs TN-t x TN ** ** NS NS NS ** NS 7 TN-t-a vs TN-t ** * * NS ** NS 8 TN-t-a vs TN-a x TN - - - - - NS NS 9 TN-t-a vs TN-a ** NS NS NS NS * NS 10 TN-t-a vs TN NS NS NS NS NS ** NS I I BY-t-a vs BY-t x BY ** ** NS * NS ** NS 12 BY-t-a vs BY-t ** ** ** * NS ** NS 13 BY-t-a vs BY-a x BY - - - - - ** NS 14 BY-t-a vs BY-a * NS ** NS NS ** NS 15 BY-t-a vs BY NS * NS NS ** NS 16 V AM-pos-a vs VAM-pos NS * NS NS NS ** * 17 V AM-pos-t-a vs V AM-pos-t " 2 ** ** NS NS * ** NS : VAM-pos-t vs VAM NS NS NS NS NS NS NS , * * Difference of contrasts at the level of P=0 05 and P=0 01 , respectively Three of the four lines homozygous for the TVMV CP gene except one BY 21 line (Entry 9) did not develop symptoms after TEV, TVMV, or PVY infection. Results for VAM-pos entries hemizygous for the TVMV CP gene (Entries 15 and 16) were the same for controlling potyvirus infection as homozygous lines (Entries 6-8), but not the for VAM-neg line (Entry 17 versus 10). Hybrids (Entries 21-26) combining TVMV and AMV CP genes are not highly resistant to potyviruses, especially those lines with BY 21 background. All transgenic lines expressing AMV CP gene had no resistance to potyviruses (Entries 11-14). Hybrids with both CP genes had effects against TEV and TVMV infection when compared with nontransformed controls (contrasts 5, 10, and 15) except TN hybrids. Contrasts (1, 6, and 11) between hybrids hemizygous for both CP genes and hybrids hemizygous for only the TVMV CP gene were highly significant for TEV infection data. There was little or no significant differences in the same comparison for PVY or TVMV results. Contrasts (2, 7, and 12) between hybrids expressing both CP genes and homozygous TVMV CP lines revealed significantly lower resistance in the hybrids than homozygous TVMV CP lines for all potyviruses regardless of the background genotype. However, hybrid hemizygous for both CP genes had a slightly higher level of TEV or TVMV resistance than AMV homozygous transgenic lines in the BY 21 background (contrast 14). Following AMV inoculation, all susceptible control plants, including lines containing only the vector, developed severe leaf distortion, stunting, and, in most cases, meristem necrosis. All transgenic entries expressing the TVMV CP gene showed similar symptoms as the controls regardless of the number of TVMV CP gene insertions (Entries 6-10 and 15-17). In contrast, all transgenic lines (Entries 11-14) expressing the AMV CP gene displayed significant symptom attenuation except for BY-a5. Hybrids hemizygous for the AMV CP gene also had significant symptom attenuation (Entries 18-20). However, hybrids (Entries 21-26) with both CP genes also had significant less AMV infection, except for one TN 86 hybrid. Contrasts of all combinations for AMV inoculation used in this study were significant for infection score except for the contrast of KY 8959 and TN 86 hybrids expressing two constructs versus their corresponding hemizygous hybrids (contrast 3 and 8) for infection scores. The level of virus detected was high in all lines and hybrids and no significant difference were found. Table 13 Plant height and yield of hybrids that combining TVMV and AMV CP and their respective parents no virus challenge and following inoculation withTEV, TVMV, PVY, and AMV in the field Inoculation treatment Inoculation treatments Entry NONE TEV TVMV PVY AMV NONE TEV TVMV PVY AMV pin int height (cm)- Yield Plant '(g) 1 KY 8959 1156 945 1065 910 952 1633 1623 1646 1618 1257 2TN86 1075 961 1159 940 922 1609 1495 1736 1710 1145 3 BY 21 1171 882 1133 1090 950 1588 1182 1536 1544 1119 4 BY-kylx 1150 953 1178 1044 873 1593 1326 1453 1530 1188 5 BRv-pos-kylx 1140 1102 1188 1090 516 1257 1652 1580 1502 956 6KY-t41 1133 1011 1187 931 862 1532 1589 1491 1415 890 7 TN-t46 1000 1063 1040 954 515 1607 1698 1473 1383 943 8 TN-t 19 992 999 1091 844 671 1421 1699 1517 1646 881 9 BY-t2 1138 976 1148 1091 998 1601 1721 1439 1525 1607 10BY-t4 1102 1065 1123 1083 989 1296 1687 1448 1442 1303 HKY-al2 1069 876 1139 961 889 1546 1563 1597 1771 1735 12KY-al7 1112 882 1077 987 974 1558 1505 1557 1707 1928 13TN-a3 1072 960 1166 1020 950 1291 1474 1614 1652 1435 14BY-a5 1125 781 1098 969 1014 1364 1001 1302 1327 1255 15KY-t41 XKY8959 1042 912 1112 959 954 1409 1549 1506 1514 1156 16TN-t46xTN86 1167 999 1148 1002 824 1630 1557 1751 1762 1118 17BY-t4xBY21 1167 1117 1124 1116 1075 1463 1554 1385 1555 1321 18KY-al2xKY8959 1142 - - - 904 1537 - - - 1735 19TN-a46xTN86 1041 - - - 822 1435 - - - 1735 20BY-a5xBY21 1041 - - - 934 1201 - - - 1207 21 KY-al2xKY-t41 1082 903 1191 960 996 1533 1583 1701 1645 1673 22KY-al7xKY-t41 1096 903 1171 1035 846 1585 1370 1826 1617 1471 23TN-a3xTN-tl9 1186 912 1093 1005 957 1543 1375 1591 1687 1540 24 TN-a3 x TN-t46 1023 975 1103 1005 974 1402 1624 1589 1633 1431 25 BY-a5 x BY-t2 1153 937 1125 1087 918 1295 1171 1291 1332 1377 26 BY-a5 x BY-t4 1063 911 1124 951 882 1266 1082 1292 1129 1321 LSD (005) 126 130 116 121 254 251 264 238 252 293 Contrast 1 KY-t-a vs KY-t x KY NS NS NS NS NS NS NS * NS ** 2 KY-t-a vs KY-t NS NS NS NS NS NS NS * NS * * 3 KY-t-a vs KY-a x KY NS - - - NS NS - - - NS 4 KY-t-a vs KY-a NS NS NS NS NS NS NS * NS * 5 KY-t-a vs KY NS NS * NS NS NS NS NS NS * 6 TN-t-a vs TN-t x TN NS NS NS NS NS NS NS NS NS ** 7 TN-t-a vs TN-t * NS NS * ** NS * NS NS ** 8 TN-t-a vs TN-axTN NS - - - NS NS - - - NS 9 TN-t-a vs TN-a NS NS NS NS NS NS NS NS NS NS 10 TN-t-a vs TN NS NS NS NS NS NS NS NS NS ** 11 BY-t-a vs BY-t x BY NS NS NS NS NS ** NS ** NS 12 BY-t-a vs BY-t NS ** NS NS NS * ** NS ** NS 13 BY-t-a vs BY-axBY NS - - - NS NS - - - NS 14 BY-t-a vs BY-a NS * NS NS NS NS NS NS NS NS 15 BY-t-a vs BY NS NS NS NS NS ** NS * ** NS 16 VAM-pos-a vs VAM-pos NS NS NS NS NS NS NS NS NS ** 17VAM-pos-t-avs VAM-pos-t" 2 NS NS NS NS NS NS NS NS NS ** 18 VAM-pos-a vs VAM-pos-a" 2 NS - - - NS NS - - - * *, ** Difference of contrasts at the level of P=005 and P= =001, respectively Agronomic performance of transgenic lines and hybrids that were not inoculated was similar to their appropriate controls (Table 13). Plant height and yield were reduced in control genotypes and in some transgenic lines following inoculation with TEV, PVY, and AMV (Table 13). Most contrasts showed no significant differences in plant height following virus inoculation. The KY 8959 hybrids possessing both CP genes were taller than the KY 8959 nontransformed control following inoculation with TEV. The TN 86 hybrid with both CP genes was taller than TVMV CP transgenic lines under noninoculated conditions and following PVY and AMV infections. Hybrid of BY 21 transgenics were taller than the corresponding TVMV CP parents and their TVMV CP hemizygous hybrids following TEV infection. Table 14 Correlation coefficients between yield and infection scores, ELISA value and plant height for hybrids combining TVMV and AMV CP and their parents following TEV, PVY, TVMV, and AMV infection Virus Score 14 DPI Score 46 DPI ELISA 46 DPI Plant Height TEV Yield -0.722" -0.661" -0.619" 0.460" PVY Yield -0.532" -0.380" -0.329 ** 0.06 TVMV Yield - -0.471" - 0.244 * AMV Yield -0.807" -0.867" -0.526 ** 0.508" Yield was also affected by virus infection and the resistance level of the lines. BY 21 hybrids expressing two CP genes had lower yield than their TVMV CP transgenic parent lines following TEV and PVY infection. However, KY 8959 hybrids containing both CP genes had higher yield than their parental transgenic lines when inoculated with TVMV. AMV infection scores were highly correlated with yield (Table 14, r=0.8). Most AMV resistant lines and hybrids had significantly higher yield than susceptible lines and hybrids following AMV inoculation (Table 13). Contrasts revealed that hybrids containing the AMV and TVMV CP genes had significantly higher yield than the TVMV CP parent lines, their hemizygotes, and their nontransformed host genotypes for KY 8959 and TN 86 backgrounds, but not for BY 21. All hybrids containing both CP genes had the same yield level as lines hemizygous for the AMV CP gene. Only the homozygous KY 8959 AMV CP lines had higher yields than hybrids with both CP genes. There was a high correlation between infection scores and yield in virus inoculated treatments, especially for the AMV infection (Table 14). Stepwise regression revealed that plant height and infection scores of 67 DPI accounted for 60% of the variation in yield increase following inoculation with AMV (Y(Yield) = 149.8 - 8.23X, (Scores) + 0.27X 2 (Plant height), R 2 =0.6074). Table 15 Correlation coefficients for yield among the four virus inoculation treatments TEV PVY TVMV AMV NONE 0.229 0.610" 0.507 * 0.097 TEV 0.558" 0.391 -0.084 PVY 0.758 * 0.214 TVMV 0.124 Yield of transgenic lines and hybrids under different potyvirus inoculation treatments were correlated, except yield under TEV and TVMV treatments (Table 15). Yield performance following AMV inoculation was not correlated with yield production under any of the other treatments. The above-described experiments evaluated the extent of resistance of the hybrids from two parental transgenic lines, each of which expresses a single copy of the AMV or TVMV CP genes. Hybrids with both CP genes maintained resistance to AMV but not to the potyviruses. Indeed, it appeared that the presence of the AMY CP gene in the hybrids enhanced the potyvirus, especially TEV, infection (Table 12). The presence of the TVMV CP gene did not affect protection against AMV infection conferred by the AMV CP gene. TVMV CP gene did enhance AMV infection when the AMV CP was not present. This influence is more evident in the VAM-pos background transgenic lines than for the VAM-neg TVMV CP transgenics. The VAM-pos TVMV CP homozygotes and hemizygotes had an average yield that was 24.6% and 5.3%, respectively, lower than the susceptible controls when infected by AMV. To our knowledge, this is the first report of a viral CP transgene interacting with infections caused by an unrelated virus. In summary, these studies demonstrated that the VAM-pos hybrids hemizygous for the TVMV CP gene confer effective protection against infection with three potyviruses. AMV hemizygotes significantly reduced the yield loss due to AMV infection and had the same level of resistance as the AMV homozygous transgenic lines. Combining two CP genes together effectively protected against AMV, but was less effective against potyviruses. The above-described results were obtained using the following materials and methods. Gene cloning and plant transformation The coat protein gene of alfalfa mosaic virus strain 425 (Barker et al. , Nucleic Acids Res. 11 : 2881-2891, 1983) was recovered from pSP65 by preparative agarose gel electrophoresis. The resulting fragment was cloned as an EcoR I - Xba I fragment into a plasmid (pBSK) and further digested with Hind III and Xba I. This Hind III - Xba I fragment was cloned into the plasmid pKYLX71 :35S 2 (Maiti et al, Proc. Natl. Acad. Sci. 90: 6110-6114, 1993; Fig. 17). The AMV CP gene was introduced into the recipient genotypes using an Agrobacterium tumefaciens mediated leaf disk transformation system (Schardl et al, Gene 61 : 1-11, 1987). Three cultivars of burley tobacco Nicotiana tabacum, 'BY 21', 'TN 86', and 'KY 8959' were used as the recipient genotypes. Leaf tissue from 4- week-old, aseptically-grown plants was cut into pieces and incubated in liquid medium containing A. tumefaciens (strain GV3850) with the desired construct. Tissues were allowed to grow on basic media lacking any antibiotics for two days to enhance tissue infection. On the third day, tissues were plated on basic media containing kanamycin (300mg/L), and Mefoxin (500mg/L) for transformant selection and to kill the remaining A. tumafaciens, respectively. Media were refreshed once per week until good calli developed. Plant shoots derived from the calli were transferred to media containing mefoxin for rooting, and subsequently, rooted plantlets were transplanted into 4" pots containing commercial growth media. Plants were grown to maturity in a greenhouse and self-pollinated. Screening and regenerating transgenic lines R, seeds derived from selfing the primary transformants were germinated and grown on media containing 300 mg/L kanamycin. The number of kanamycin resistant and sensitive seedlings were determined 2-3 weeks after germination when the sensitive seedlings were chlorotic and unable to produce true leaves. These data were used to compute χ 2 values for segregation ratios, and only those lines segregating for 3:1 or 15:1, respectively, single or double gene insertions were kept. Plants resistant to kanamycin were grown to maturity in a greenhouse and self- pollinated. Seeds were collected from each plant and were then sown on media containing kanamycin to determine homozygosity or hemizygosity of R, plants. Analysis of expression of coat protein genes in transformed plants Immunoblot analyses were used to assess the level of expression of the coat protein gene in selected transgenic lines. Leaves from 3-week-old seedlings were homogenized in extraction buffer (0.0625 M Tris-HCl, pH 6.8/10% glycerol/2%SDS/10% 2-mercaptoethanol), boiled for 10 min, and centrifuged at 12,000 x g for 10 min. Proteins (50 μg of total soluble leaf protein) were separated on 12.5% polyacrylamide gels/SDS, transferred to nitrocellulose, and probed with an AMV antiserum. Inoculation and evaluation of transgenic lines in the greenhouse R, and R 2 generation plants from all independent transgenic lines were inoculated with AMV 425 and one AMV Kentucky isolate. Selected lines were challenged with an AMV isolate from North Carolina. The source of inocula was fresh sap, obtained by grinding AMV infected leaves of the susceptible cultivar TN 86. Inoculum concentration was 1 :50 (w:v) with 0.05 M potassium phosphate buffer (pH 7.0). Five-week-old tobacco seedlings growing in soil were mechanically inoculated on the second leaf from the bottom after lightly dusting the leaf with carborundum. Inoculated plants were examined visually, and symptoms of disease development were recorded daily. Virus accumulation in plants was detected by direct ELISA (Clark and Adams, J. Gen. Virol. 34: 475-483, 1977; Ghabrial, Virology 31 : 154-162, 1967). Five leaf discs (3 cm 2 ) from the third systemic infected leaf of each infected plant were sampled for ELISA analysis. Virus in the sample was extracted by homogenization with 5ml of PBS-Tween (phosphate-buffered saline, 0.05 (v/v) Tween, pH 7.3) per gram of tissue. Duplicates of each sample were applied to microtiter plates. Polyclonal antibodies were used at concentrations of 1:1000 and 1 :3000. Reactions were measured spectrophotometrically at 405 nm using a BIO- RAD Microplate Reader Model 450. Samples were rated as positive for the presence of the virus when the sample ELISA absorbance value was three times the mean absorbance value for non-inoculated plants. A reinoculation assay was also performed by back inoculating sap from leaf samples on an indicator species, cowpea (Vigna unguiculata). 1994 field experiment Nine BY 21 AMV CP transgenic lines and two non-transformed genotypes, BY 21 and TN 86, were planted in the field as subplots in a split plot design with four replications. Main plots were three treatments: no AMV inoculation, inoculation with AMV one day before transplanting, and inoculation with AMV 14 days after transplanting. The experiment was conducted in an area where there was no other tobacco plants in the immediate vicinity. A plot row of the AMV susceptible TN 86 was planted after every 10th test plot row as an indicator of the level of natural infection. Each transgenic line evaluated was in the R 2 generation and homozygous for the CP gene. Seeds of the transgenic lines and control genotypes were germinated in 4 inch pots containing a commercial potting mix on April 22, 1994. Four weeks after seeding, plants were transferred to trays designed for greenhouse transplant production. Plants were fertilized once a week for five weeks with a water soluble fertilizer until plants reached 15-20 cm in height and were suitable for transplanting to the field. Plants were transplanted to the field described as a Maury silt loam soil (fine, mixed, mesic Typic Paleudalfs), using a mechanical transplanter on June 3. Each plot consisted of 20 plants spaced 46 cm apart. Row width was 107 cm. All production practices used were those recommended for commercial burley tobacco production. 1995 field experiment This experiment was a split-plot design with three replications. The three background or recipient genotypes, BY 21, TN 86, and KY 8859, comprised the main plots. Sub-plots consisted of the original nontransformed genotype and 10 transgenic lines that were randomly selected from all the transgenic lines developed for each genotype. Therefore, each replication included 33 entries. Three weeks prior to tobacco transplanting, maize was planted between the experiments to provide a partial barrier to the movement of aphids among the experiments. All transplanting and production practices used were the same as those used in 1994. The plants were inoculated with AMV one day before transplanting. Field inoculation and evaluation An AMV local isolate (AMV-KY) was used as the field inoculum. Virus stocks were maintained in susceptible TN 86 plants grown in a greenhouse. When virus infections reached a peak stage (2 weeks post inoculation), infected leaf tissue was triturated into 0.05 M Na 2 HPO 4 KH 2 PO 4 buffer (pH 7.0) at the rate of 1 g of tissue per 10 ml (1994) or lg per 5 ml (1995) of buffer and used as inoculum. Relative infectivity of each inoculum was determined by local lesion assay on cowpea (Vigna unguiculata). One gram of 600-mesh carborundum was added to each 100 ml of inoculum. Inoculum was placed in flasks and kept on ice for transport to the field for inoculation on the same day it was prepared. Plants were inoculated mechanically by applying inoculum to one expanding leaf per plant. Beginning one week after inoculation, each plant was inspected visually weekly. In the 1994 trial, disease incidence (percent infected plants) was recorded throughout the season. For 1995, disease incidence was recorded at the beginning of the season, and individual plants were rated for symptom expression at weekly intervals throughout the season. Symptom expression was rated using a scale from 0 (no symptoms) to 7 (severe symptoms as indicated by stunting and leaf distortion and stunting). The average symptom score was calculated for each plot and was used in the statistical analysis. Virus detection in inoculated plants was accomplished by direct ELISA (Clark and Adams, J. Gen. Virol. 34: 475-483, 1977). Tissue samples were taken twice, at approximately 14 and 46 days post inoculation (DPI). Each sample consisted of a bulk of 12 cm 2 discs from five plants per plot. Details of IgG purification and ELISA procedure were described above. Plant material, transformation, and crosses Five transgenic lines expressing the TVMV CP gene and four transgenic lines expressing the AMV CP gene were selected as parent lines based on results from greenhouse tests for virus resistance. These lines had Nicotiana tabaccum L. cvs. KY 8959, TN 86 or BY 21 as background genotypes. Details of the transformation and transgenic line development have been described above. These transgenic lines were crossed to generate hybrids that should express the AMV and TVMV CP genes. In addition, the lines were also crossed with their nontransformed recipient parents to generate lines hemizygous for the TVMV CP or AMV CP gene. Two transgenic lines expressing only vector, BY-kylx and BRv-pos-kylx were also included in this field experiment. Field experiment design With the same basic design as described above, 26 hybrids and homozygous transgenic lines and their corresponding controls comprised all the genetic material for this experiment. The basic randomized complete block design with three replications was repeated five times: one repeat was for the non-inoculated test and the other four repeats allowed for separate inoculations with TEV, PVY, TVMV, and AMV. Plot size was 20 plants for noninoculated plots and 12 plants for inoculated plots. The non-inoculated and AMV inoculated experiments included all 26 genotypes, while other three inoculation experiments had 23 lines which did not include three AMV CP hemizygous lines. Cultural practices Seed germination, transplant production, and transplanting were previously described herein. Production recommended practices for burley tobacco were followed throughout the growing season. Cured tobacco was weighed separately as top, middle, and bottom portions for noninoculated studies, and all leaves were weighed together for virus inoculated studies. The date at which 50% of the plants flowered was recorded. Plant height, leaf number, leaf length and width, and stalk diameter were ascertained after topping for 10 plants in each plot for noninoculated plots. Plant height was measured for virus inoculated plots. Field inoculation and evaluation Locally collected isolates of TVMV, TEV, PVY, and AMV were used as inocula in these studies. Virus stock maintenance and inoculation methods were previously described. Disease symptoms were monitored visually and recorded, and virus accumulation in systemic infected leaves was detected by the direct-ELISA. Details of sampling, infection rating, and ELISA methodology were as described above. Data analysis All data from each of the experiments were analyzed using the Generalized Linear Modeling procedures of SAS (SAS/STAT, User's Guide, SAS Institute Inc., 1993). Line means for observed traits were separated using the least square difference test. Various contrast statements were used to compare factors contributing to virus resistance and field performance. Pearson correlation coefficients for yield among transgenic lines inoculated with TEV, PVY, and TVMV were determined in order to describe the relationship of reactions to the different virus infections. Correlation coefficients between yield and infection intensity were also determined. Stepwise regression was used to build a regression model that estimated the contribution of virus resistance to yield. V. Agronomic Performance of Transgenic Burley Tobaccos Expressing TVMV or AMV Coat Protein Genes With and Without Vims Challenge Agronomic performance of transgenic lines expressing the TVMV CP gene under non-challenged conditions The incidence of viral disease in transgenic plants expressing the TVMV CP gene determined by visual inspection, was very low (< 5%) in the non-challenged experiment, and, thus, these plants were considered virus-free and not affected by virus infection. Analysis of variance for agronomic traits revealed that the main genotype effects for plant height, number of leaves per plant, leaf length and width, stalk diameter, and cured leaf yield were significant. Most notably, plants of the BY 21, BRv-neg, and BRv-pos transgenic lines were taller than plants of the KY 8959 and TN 86 lines. The average yield of TN 86 transgenic lines was also greater than those of the other lines (Table 16). The average performance of the transgenic lines was measured by comparing the mean of the 10 transgenic lines with the corresponding nontransgenic parent genotype (Table 16). There were only a few instances in which the transgenic lines differed significantly from their respective nontransformed parental genotype. The majority of the transgenic lines exhibited a similar agronomic performance to the controls. The only notable difference was that the means for leaf size and stalk diameter of the BY 21 TVMV CP lines were significantly smaller than BY 21. Table 16 Agronomic performance of 10 TVMV CP transgenic lines and their respective host genotypes Plant Leaves Leaf Leaf Stalk Yield height Plant ' length width diameter plant ' cm No. g KY 8959 107 3 16.9 66.6 30.1 38.6 168.2 Line mean" 106.8 16.4 * 64.0 * 28.6 38.1 149.2 * TN 86 115 0 19.2 65.5 28.2 38.7 171.9 Line mean 110.5 17.9" 63.7 27.2 38.8 158.9 BY 21 122.3 18.0 68.8 28.0 41.4 149.8 Line mean 119.1 17.3 65.3" 25.8" 38.7" 147.5 BRv-neg 126.7 18.7 63.6 29.3 37.9 159.4 Line mean 122.0 18.0 63.2 28.7 37.3 141.3 * BRv-pos 133.3 19.2 64.3 29.4 39.7 148.8 Line mean 124.5 18.2 63.2 28.7 37 3" 142.1 * Means of 10 TVMV CP transgenic lines derived from same genotype For KY 8959 genotype, line mean is from 9 transgenic lines. * The difference between line mean and the host genotype was significant at the P=0.05 level. " The difference between line mean and the host genotype was significant at the P=0.01 level. Analysis of variance revealed that the variation among transgenic lines within a genetic background was significant for all traits. However, most of the transgenic lines exhibited similar agronomic performances as the nontransformed controls (Table 17). Forty of the 49 transgenic lines (82%) did not show significant differences for yield when compared with the corresponding controls under the virus- free conditions. In each genetic background, there were one or two lines with reduced plant height and low yield, except for KY 8959 which had 4 low-yielding transgenic lines (Table 17). Table 17 Plant height and yield of transgenic lines expressing TVMV CP no virus (NONE) and following inoculation with TEV, PVY, and TVMV Inoculation treatment* Inoculation treatment NONE TEV PVY TVMV NONE TEV PVY TVMV Plant height (ι zva) Yield Plant'(g) KY 8959 $ 1073 958 988 1032 1682 1677 1180 1709 1 1038 907 888 1009 1578 1592 1500 * 1652 2 1017 987 774" 1054 1364" 1500 1316 1506 3 1000 987 990 1072 1629 1487 1629" 1613 5 1117 996 808 * 1182 1465 1275" 1515 * 1624 6 1127 968 998 1034 1581 1459 1299 1549 7 1047 996 1028 1053 1695 1650 1787" 1787 8 1103 913 1033 1020 1342" 1332" 1362 1543 9 1107 1050 988 1058 1342" 1253" 1116 1409 * 10 1053 1005 949 996 1428" 1418 * 1231 1424 * Line mean 1068 C& 95 T 940 C 1025° 1492 b 144 l b 1416 ab 156 T TN86 1150 1015 948 1112 1719 1629 1562 1834 1 1357" 1068 1030 1164 1411" 1359 * 1378 1459" 2 1077 1032 858 1067 1802 1811 1608 1610 3 1110 927 874 1100 1720 1747 1553 1788 4 1210 999 839 1162 1725 1704 1576 1545 * 5 1003 1022 979 1072 1554 1625 1555 1709 6 873" 793" 685" 903 * 1003" 918" 796" 945" 7 1067 1018 847 1010 1636 1590 1502 1532 8 1170 921 990 1074 1730 1587 1608 1796 9 1147 973 1015 1095 1684 1701 1749 1695 10 1037 87 r 885 1043 1634 1413 * 1561 1535 Line mean 110 5 C 96 2 C 90 0 C 106 9 b 158 9 a 154 5 1 148 9 a 156 l a BY 21 1223 1038 1080 1166 1498 1409 1352 1508 1 1110 1158 * 1055 1141 1448 1550 1443 1606 2 1217 1217" 1084 1204 1562 1566 1459 1533 3 1240 1144 1066 1137 1489 1457 1477 1396 4 1090 * 1057 1071 1085 1401 1319 1345 1444 5 1263 1002 1074 1181 1540 1318 1310 1616 6 1123 1144 1002 1102 1557 1517 1451 1368 7 1287 1057 1142 1184 1628 1292 1455 1654 8 1253 1084 1043 1161 1337 1195 1143 1341 9 1090 * 956 * 857" 955 * 1322 1348 1167 1353 10 1240 1141 1071 1103 1468 1352 1387 1428 Line mean 119 l ab 1096 b 1046 b 1125 b 1475 b 1392 b 1364 b 1474 ab Table l7 Con Inoculation treatment Inoculation treatment NONE TEV PVY TVMV NONE TEV PVY TVMV Plant height ( cm) Yield P lant '(g) BRv-neg 126 7 110 2 108 1 117 1 159 4 159 5 139 3 145 5 1 117 3 113 9 113 7 159 1 135 7 * 119 0" 123 5 131 1 2 126 7 118 0 120 8 118 5 140 8 151 0 147 9 152 1 3 119 0 113 8 1104 116 5 140 1 135 0 * 135 0 136 3 4 117 3 104 8 113 0 115 5 144 9 139 2 126 0 157 6 5 115 3 110 3 107 8 121 9 145 5 140 9 136 2 150 2 6 125 0 108 6 110 8 122 0 148 1 149 2 138 0 149 3 7 122 0 124 7 ** 121 6 124 4 144 2 137 5 137 0 150 4 8 122 3 116 2 111 1 127 0 146 7 159 8 128 3 162 5 9 128 0 114 1 103 9 120 4 121 1" 137 8 127 7 150 5 10 127 3 107 7 105 6 115 0 145 8 142 2 129 9 144 5 Line mean 122 0 a 113 2 a 111 9 d 124 0 1 141 3 141 2 b 133 0 b 148 5 a BRv-pos 133 3 114 911 106 4 122 2 148 8 155 4 137 3 156 9 1 130 7 24 1 113 7 127 3 152 3 151 0 163 2 148 0 2 131 7 124 8 105 0 125 2 148 8 148 9 153 9 150 1 3 134 0 121 5 100 6 130 7 156 6 171 3 154 0 162 7 4 127 0 118 2 101 8 127 5 147 0 149 3 142 5 154 4 5 106 7" 92 1" 87 2 * 103 4 * 93 7" 111 5" 94 4 104 5" 6 123 7 124 0 114 0 123 2 156 4 149 6 164 1 149 4 7 121 7 109 9 96 6 115 3 141 2 129 0 133 0 139 7 8 115 0" 106 1 96 1 127 1 141 2 144 1 142 0 145 8 9 129 3 122 3 107 7 126 5 138 2 151 6 141 0 144 2 10 125 3 129 9" 111 8 130 5 146 0 158 7 150 4 144 2 Line mean 124 5 1 117 3 a 103 5 b 123 7 a 142 l 1 146 5 ab 144 3 q 144 3 b # Inoculation treatment includes nonmoculation (NONE) and inoculation with TEV, PVY and TVMV $ Transgenic lines (1-10) derive from corresponding genotypes * and ** Difference between transgenic lines and their respective genotypes was significant at P=0 05 and P=0 01 levels, respectively & Means of 10 transgenic lines followed by the same letter were not significantly different at P=0 05 level Significant differences between the mean of 10 transgenic lines and the nontransformed control were caused pπmarily by a few of the transgenic lines having inferior agronomic performance For instance, two TN 86 lines had a low leaf number, and one KY 8959 line and two BY 21 lines had narrower leaves than their controls (Fig 18) One KY 8959 transgenic line, KY-4, grew so poorly that it was not harvestable, and was eliminated from the data analysis Performance of TVMV CP transgenic lines following virus challenge The performance of the 50 TVMV CP transgenic lines was also evaluated following separate challenge with TEV, PVY, and TVMV. Plant height and yield were the traits chosen as performance indicators. Data from the three experiments, one for each challenged virus, were subjected separately to analyses of variance. Results indicated that the main effect of genotype and the effect of the transgenic lines within a genotype were significant for both plant height and yield. By comparing the mean leaf yield and plant height of the nontransgenic control genotypes (Table 17), the relative disease severity of the three viruses could be estimated as: PVY>TEV>TVMV. The TVMV CP transgenic lines did not have any significant performance advantage over the nontransgenic control genotypes following TVMV and TEV challenge. It should be noted that three of the five genotypes used in this experiment possess the VAM gene that confers high resistance to TVMV and medium resistance to TEV. The transgenic lines from VAM-neg backgrounds had little or no yield reduction, indicating CPMR was also effective against TVMV and TEV infection. A few of the transgenic lines had significantly lower yield and plant height than their nontransgenic counterparts, but in virtually all cases, these were the same lines that also had poor performance under the non-challenged conditions. Under challenge by PVY-KY, plant height and yield means for the 10 transgenic lines of each genotype were not significantly different from the mean of the nontransgenic lines. This was despite the fact that PVY infection resulted in varying levels of adverse effects on yield and plant height for all five nontransformed controls. However, a number of individual transgenic lines had superior performance, compared to their respective nontransgenic controls, following challenge with PVY. Eight lines of KY 8959, eight lines of BRv-pos, and four lines of TN 86 genotype background had higher yield relative to their respective controls. Transgenic lines KY-3, KY-5, and KY-7 had much greater yield than KY 8959 following PVY challenge. Indeed, it should be noted that KY-7 had a very consistent high yield level in all four experiments. Several transgenic lines in each of the other four backgrounds had acceptable yield under PVY infection. Agronomic performance of AMV CP transgenic lines under non-challenged conditions Analyses of variance for the 1995 test of the AMV CP lines under non- challenged conditions showed that some traits had significant mean squares for the main genotype effect and for the effect of lines within a genotype. Lines in a BY 21 background were taller and had narrower leaves than TN 86 and KY 8959 lines. Contrasts between the means of the transgenic lines and their respective nontransformed control were not significant (Table 18). Some transgenic lines had reduced leaf size and stalk diameter compared to their control, but similar comparisons for all other traits were not significant, except one KY 8959 AMV CP line which had significantly less cured leaf yield than KY 8959. Table 18 Agronomic performance of 10 AMV CP transgenic lines and their respective host genotypes Plant Leaves Leaf Leaf Stalk Yield Height Plant '1 Length Width Diameter Plant "1 No. g KY 8959 108.3 16.7 64.9 31.5 38.5 180.4 Line mean* 96.4 15.4 66.3 31.2 39.1 161.9 TN 86 111.0 17.0 68.7 32.1 38.4 178.8 Line mean 110.7 17.6 66.7 31.1 38.2 170.0 BY 21 113.3 17.7 66.0 28.7 39.3 156.1 Line mean 112.2 16.5 65.9 28.9 38.7 150.6 Contrast* NS NS NS NS NS NS " Means of 10 AMV CP transgenic lines. $ Contrast between mean of 10 transgenic lines and their respective host controls. NS Not significant at P=0.05 level. Performance of AMV CP following AMV inoculation A high level of resistance responses following AMV infection was evident in the AMV CP transgenic lines. AMV infection caused dramatic reductions in the agronomic performance of the parental lines following AMV challenge (Figs. 19 and 20). Yield reductions for KY 8959, TN 86, and BY 21 background genotypes were 30.0%), 25.5%, and 22.5%, respectively. In contrast, yield of transgenic lines showed no effect of AMV infection (Fig. 20). Eight transgenic lines from KY 8959, 9 lines from TN 86 and 4 lines from BY 21, had significantly greater yield than their nontransformed parent genotypes following AMY inoculation. The percentage yield gains ranged from 4.2-52.2% (Fig. 21). It should be noted that genotype effects on AMV protection was evident. The TN 86 and KY 8959 AMV CP lines had relatively higher levels of protection than the BY 21 AMV CP lines (Fig. 21). Agronomic performance evaluations of AMV CP transgenic lines were also conducted in 1994. The principal objectives of the 1994 field trials were to evaluate resistance of the BY 21 transgenic lines expressing AMV CP and to evaluate the effects inoculation time on AMV resistance. Results revealed no significant difference between the two inoculation treatments: inoculation one day before transplanting and inoculation 14 days after transplanting. Thus, data from these two treatments were combined (Table 19). Table 19 Agronomic performance of 9 AMV CP BY 21 transgenic lines and untransformed controls: no AMV and following AMV inoculation in 1994 Plant Leaves Leaf Leaf Stalk Yield Height Plant ' Length Width Diameter Plant ' No. g No Inoculation TN 86 115.8 19.9 65.0 32.9 33.0 185.0 BY 21 118.4 19.1 64.0 33.0 34.3 172.0 Mean of 9 lines* 119.8 19.4 61.1 * 30.4" 33.1 157.5 * AMV Inoculation TN 86 81.1" 15.7" 58.9 29.0 29.6 ** 121.2 BY 21 111.8 18.8 61.2 29.9 32.5 153.9 Mean of 9 lines 111.4 19.0 60.0 29.9 33.1 159.6" Means of nine BY 21 transgenic lines expressing AMV CP * and " Difference between mean of 9 transgenic lines or TN 86 and BY 21 host genotype was significant at the P=0.05 and P=0 01 levels, respectively. The performance of BY 21 transgenic lines in non-challenged plots was similar to that observed in 1995 trial: no significant difference between transgenic lines and the controls for most traits (Table 19). When inoculated with AMV, the controls exhibited reduced plant size (plant height, leaf size, and stalk diameter) and resulted in a yield loss of 10.5% for BY 21 and 34.4% for TN 86 compared to the noninoculated treatment. In contrast, seven BY 21 AMV CP lines (78%) did not have any yield reduction compared with the noninoculated treatment (Fig. 22). One line, BY-3, had higher yield than the BY 21 control. Most of the AMV CP transgenic lines had higher yield than TN 86 following AMV inoculation but lower yield than TN 86 under non-challenged conditions (Fig. 22). Correlation between yield production and resistance traits, and yield performance with different virus challenge Data obtained from disease resistance tests of the same lines under virus challenge were analyzed with data on yield production under the same challenge condition using a Pearson correlation analysis (Table 20). The data revealed low correlation between infection scores, ELISA readings and yield for TEV, PVY, and TVMV challenge. However, a considerable negative correlation between infection rating and yield performance was observed in AMV CP transgenic lines following AMV challenge (Table 20). This resulted from the heavy challenge and dramatic yield reduction of severely infected plants. Stepwise regression analysis suggested that the level of resistance can be used to predict potentially high yielding lines (Y=24.3- 0.36X,+1.39X 2 , where Y=yield; x,=infection score, X 2 =plant height), but the R 2 value was low (0.4). Table 20 Correlation coefficients between infection scores, ELISA values and yield for transgenic lines infected with TEV, PVY, and TVMV Score 14 DPI Score 46 DPI ELISA 46 DPI Plant Height TEV Yield -0.1620 * -0.1565 * -0.0948 * 0.2081" PVY Yield -0.0184 -0.1391 -0.1100 0.2896" TVMV Yield - -0.1968 * -0.0268 0.0170 AMV Yield -0.3756" -0.4708" -0.4510" 0.5588" * , " Correlation coefficients were significant at P=0.05 and P=0.01 levels, respectively. Yield of the TVMV CP lines in the non-challenged, TEV challenged, and TVMV challenged treatments were highly correlated. Correlation between transgenic line yield following PVY challenge and the other treatments (i.e., TVMV challenged, TEV challenged, and non-challenged) were significant but rather low (Table 21). In contrast, a low correlation between yield of AMV CP transgenic lines under non- challenged conditions, and that under AMV challenge conditions, suggested that virus resistance evaluation is the most important step. Table 21 Correlation coefficients between yield for transgenic lines uninfected and infected with TEV, PVY, and TVMV TEV PVY TVMV NONE 0.7858" 0.4634" 0.8126" TEV 0.3326 * 0.7377 ** PVY 0.3572 * * , " Correlation coefficients were significant at P=0.05 and P=0.01 levels, respectively. The experiments above-described have documented the effectiveness of the TVMV CP and AMV CP genes in reducing the symptoms of virus disease under virus challenge conditions. The present experiments focused on the agronomic performance of these transgenic lines. In other words, the field performance of transgenic lines offers a direct and reliable way to assess the value of this genetic engineering approach on crop improvement. The principle aims of the following studies were to determine whether transgenic lines exhibit any undesirable agronomic properties and whether virus resistance of transgenic lines is manifested as improved performance under disease conditions. The effectiveness of combining two strategies, CPMR and endogenous resistance, on potyvirus infection was evident, as shown above. This combination also positively affected yield gains. This was characterized by small yield reductions for most transgenic lines after virus infection. Furthermore, measured by field performance, CPMR enhanced the effect of the VAM gene against TEV. This synergistic effect was even more evident following inoculation with PVY-KY, against which the VAM gene is less effective. The average yield of nontransformed genotypes possessing the VAM gene was 15.6% less in the PVY inoculated plots than in the noninoculated plots (Table 17). In contrast, 13 of 29 TVMV CP transgenic lines with VAM-pos backgrounds did not have any yield reduction when compared with the same lines in noninoculated plots (Table 17). Thirteen TVMV CP lines had increased yield from 10-52% compared with their respective controls. The yield advantage provided by CPMR measured by the pure yield gain relative to nontransformed controls following PVY inoculation were 10.3% and 5.1% for VAM-pos and VAM-neg transgenic lines. The yield gain difference between two isogenic lines, BRv-pos and BRv-neg, which permits a direct comparison of disease reaction to virus infection solely caused by the VAM gene, strongly supported this synergistic effect. This proves the feasibility of using TVMV CPMR as a promising source of resistance in potyvirus control in tobacco. The effectiveness of CPMR in AMV transgenic lines was even more evident. The average yield gains relative to nontransformed controls following AMV inoculation were 36.7%, 36.3%, and 19.0% for KY 8959, TN 86, and BY 21 AMV CP transgenic lines, respectively. Indeed, all AMV transgenic lines had slight yield reduction after infection (Fig. 21). The severely infected control plants not only had significantly reduced yield (22.5%>-30.0%), but also had poor leaf texture, which was essentially not harvestable. Therefore, CPMR to AMV would appear to be the most promising source of resistance for a breeding program. This is especially useful in KY 8959 and TN 86 genotypes, as genotypes possessing the VAM gene appeared to be more sensitive to AMV (Tedford and Nielson, Plant Dis. 74: 956-958, 1990). The above-described results were obtained using the following materials and methods. Plant materials and transformation The methods used for transformation and development of transgenic lines were provided in studies described above. The same 50 TVMV CP transgenic lines that were provided for potyvirus resistance in the field were also used in this study. Similarly, the 30 AMV CP transgenic lines evaluated in the field for response to AMV infection were also chosen for the field performance study. Experimental design TVMV CP Transgenic Lines: A split-plot experimental design with three replications was used. The five recipient genotypes were designated as main plots. Sub-plots in each main plot consisted of the original nontransformed genotype and 10 TVMV CP transgenic lines that were randomly selected from among all transgenic lines developed for each genotype. This basic design was repeated four times using a different randomization each time, for the four different inoculation treatments. No plants were inoculated in one experiment and the remaining three repeats allowed for separate inoculations with TEV, PVY, and TVMV. There were 20 plants per plot for the noninoculated experiment, and 12 plants per plot for the experiments involving virus inoculation. The three inoculated experiments were established in close proximity in the same field. The no inoculation experiment was about 15 M from the closest inoculated plots. Three weeks prior to tobacco transplanting, maize was planted between all experiments to provide a partial barrier against the movement of aphids among the experiments. 1995 AMV experiment Thirty AMV CP transgenic lines and three nontransformed parental genotypes were planted in a split plot design as described above. The basic experiment was repeated twice. Plants in one experiment were not inoculated and plants in the remaining experiment were inoculated with AMV. Both experiments were planted in the same field approximately 190 M apart and surrounded by maize. 1994 AMV experiment A split plot design was used with three virus inoculation treatments, inoculation with AMV-KY one day before transplanting, inoculation 2 weeks after transplanting and no inoculation, were considered as the main plots. Subplots included 9 BY 21 transgenic lines and 2 controls, TN 86 and BY 21. Each plot consisted of 20 plants of each line with the end plants not being harvested. The first inoculation occurred on June 2, one day before transplanting, and the second AMV inoculation was on June 17, 14 days after transplanting. The plants in the other block were not inoculated and served as controls. Cultural practices Seed germination, plant growth, and transplanting were described above. Cultural, fertilization, harvesting, and curing practices recommended for burley tobacco production were followed. The 1994 experiment was irrigated three times during the season, and all 1995 experiments were irrigated once. The date when 50% of the plants flowering in each plot was used as estimate of maturity. Plant height, leaf number, leaf length and width, and stalk diameter for 10 plants in each plot were recorded before topping for noninoculated plots in 1995 and for all plots in 1994. Only plant height and cured leaf weight were determined for the virus inoculated experiments in 1995. Cured tobacco was weighed for top, middle and bottom stalk position for noninoculated studies, and leaves from all stalk positions were bulked for yield determination in the inoculated treatments. Field inoculation and evaluation Isolates of TVMV, TEV, PVY, and AMV that were collected in Kentucky were used as inocula in these studies. The inocula stock maintenance and inoculation methods were described above. Details of sampling, infection rating, and ELISA procedure were described above. Data analysis Data from each of the experiments were analyzed using standard analysis of variance procedures of SAS (SAS/STAT, User's Guide, SAS Institute Inc., 1993). Contrasts between the means of the 10 transgenic lines and their corresponding non- transformed controls were performed. Further contrasts included CP lines versus VAM-pos lines, and CP transgenic lines with the VAM gene versus CP transgenic lines without the VAM gene. Yield gain associated with CPMR was calculated as the percentage increase in yield over the nontransformed controls following virus challenge. Pearson correlation coefficients among yield means of transgenic lines inoculated with TEV, PVY, and TVMV were determined. Correlation coefficients between yield and infection intensity were also determined. Stepwise regression was used to determine the components contributing to yield gain. VI. Further Applications The above-described methods may be used to confer disease resistance to other crop plants. The following examples are intended to illustrate, but not limit, the invention. Soybean Soybean mosaic virus (SMV) is a common pathogen in soybean. SMV is a potyvirus and is closely related to the tobacco potyviruses TVMV, TEV, and PVY. Soybeans have at least two endogenous genes, RSvl and RSv2, which confer resistance to some but not all of the seven SMV strains found in soybean. Using the techniques described herein, the coat protein gene of an SMV strain (SMV-CP) (Eggenberger et al, J. Gen. Virol. 70: 1853-1860, 1989) is introduced into soybean genotypes expressing Rsvl, RSv2, and/or other endogenous SMV resistance genes. Transgenic soybean plants are evaluated for viral disease resistance according to standard methods. Transgenic plants that express a coat protein having resistance to at least one more strain of SMV, or another potyvirus, relative to control, untransformed soybean plants, are taken as being useful in the invention. Common Bean Bean common mosaic virus (BCMV) is a pathogen of the common bean (Phaseolus vulgaris). There are several endogenous resistance genes, bc-1, bc-2, and bc-3, that confer resistance to BCMV strains (Spence and Walkey, Plant Pathol. 44: 537-546, 1995; Drijfhout, E., Agric. Res. Rept. Cent. Agr. Publ. Doc. Wageningen, pp. 1-90, 1978). Using the methods described herein, a transgenic bean plant may be generated by introducing a BCMV CP gene (Saiz et al, Virus Res. 31: 39-48, 1994) into a plant carrying one or more of the endogenous resistance genes. Transgenic common bean plants are evaluated for viral disease resistance according to standard methods. Transgenic plants expressing a coat protein having resistance to other strains or isolates of BCMV or another potyvirus relative to control, untransformed common bean plants, are taken as being useful in the invention. The recessive endogenous resistance gene, by-3, also controls resistance of the common bean to bean yellow mosaic virus (BYMV) (Provvidenti and Schroeder, Phytopathology 63: 196-197, 1973). The coat protein (CP) gene of BYMV (Hammond and Hammond, J. Gen. Virol. 70: 1961-1974, 1989), may be introduced into a by-3 expressing bean plant according to standard techniques. Transgenic common bean plants are evaluated for viral disease resistance according to standard methods. Transgenic plants expressing a coat protein having resistance to at least one more strain of potyvirus relative to control, untransformed common bean plants, are taken as being useful in the invention. Maize and Sorghum Maize dwarf mosaic virus (MDMV) is a major virus disease that affects both maize (corn) and sorghum (Louie et al, Plant Dis. Rep. 59: 518-522, 1976). MDMV is a potyvirus and exists as many different strains. Genotypes of these plants exist which show resistance to certain strains of MDMV (Louie et al., Plant Dis. Rep. 60: 838-842, 1976). These tolerant genotypes are transformed with the coat protein gene of one or more strains of MDMV (see, e.g., Salomon and Bernardi, Virology 213: 676-679, 1995), to generate transgenic maize and sorghum plants. Transgenic plants are evaluated for viral disease resistance according to standard methods. Transgenic maize and sorghum plants expressing a coat protein having resistance to at least one additional strain or isolate of MDMV or another potyvirus relative to control, untransformed plants, are taken as being useful in the invention. Papaya Certain papaya plant genotypes are endogenously resistant to papaya ringspot virus (PRV); for example, one such genotype called Cariflora, has recently been described (Conover et al, HortScience 21: 1072, 1986). Furthermore, CPMR with PRV coat protein (PRV-CP) gene (Quemada et al, J. Gen. Virol. 71 (Pt 1): 203-210, 1990) has been developed experimentally (Fitch et al, Bio/Technology 10: 1466-1472, 1992). By applying the concept of this invention to papaya plants, a Cariflora genotype endogenously resistant to a PRV strain is transformed with a PVR- CP gene to generate a transgenic papaya plant. Transgenic papaya plants are evaluated for viral disease resistance according to standard methods. Transgenic plants expressing a coat protein having resistance to at least one additional, or isolate strain of PRV or another potyvirus relative to control, untransformed papaya plants, are taken as being useful in the invention. Sugar Beet The recessive endogenous resistance gene, Bm, confers resistance in sugar beet to beet mosaic virus (BMV) (Lewellen, R. J., Phytopathology 63: 877-881, 1973). The coat protein gene of BMV may be isolated by standard molecular biology techniques (see, e.g. , Ausubel et al. , Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, 1994) and used to transform a sugar beet genotype carrying the Bm gene to generate transgenic sugar beet plants. Transgenic plants are evaluated for viral disease resistance according to standard methods. Transgenic sugar beet plants expressing a coat protein having resistance to at least one additional strain or isolate of BMV or another potyvirus relative to control, untransformed plants, are taken as being useful in the invention. Potato Certain potato cultivars contain PVY resistance genes, for example, the "Pentland Crown" cultivar (Scottish Research Institute, 1980). Yet another transgenic plant of the invention is a potato cultivar containing an endogenous PVY resistance gene transformed with a PVY coat protein (PVY-CP) gene (Chu et al, Acta Bot. Sin. 34: 191-196, 1992), or the coat protein gene of some other potyvirus. Transgenic potato plants are evaluated for viral disease resistance according to standard methods. Transgenic plants expressing a coat protein having resistance to at least one additional strain or isolate of PVY or another potyvirus relative to control, untransformed potato plants, are taken as being useful in the invention. Alfalfa The staple grain crop, alfalfa, is highly susceptible to disease caused by the alfalfa mosaic virus (AMV). As described above, AMV also infects tobacco. Certain alfalfa genotypes possess the recessive endogenous gene Am-1 , which confers resistance to AMV (Crill et al, Phytopathology 61: 369-371, 1971). These resistant genotypes could be transformed with the coat protein gene of AMV, according to the methods described above, or with the coat protein gene of an AMV-related virus (e.g., a virus that also infects alfalfa). Transgenic plants are evaluated for viral disease resistance according to standard methods. Transgenic alfalfa plants expressing a coat protein having resistance to at least one additional strain or isolate of AMV or AMV- related virus relative to control, untransformed plants, are taken as being useful in the invention. Melon The cucumber mosaic virus (CMV) is virus related to AMV whose coat protein gene (CMV-CP) (Namba et αl, Gene 107: 181-188, 1991) has been transformed into cucumber, tomato, and cantaloupe to generate CMV-resistant strains of these crops. In addition, certain genotypes of melon show an increased resistance to CMV infection, which is controlled by an incompletely dominant or recessive gene (Karchi et αl., Phytopathology 65: 479-481, 1975). Hence, these resistance genotypes of melon may be transformed with the CMV-CP gene to generate transgenic melons. Transgenic melon plants are evaluated for viral disease resistance according to standard methods. Transgenic plants expressing a coat protein having resistance to at least one additional strain or isolate of CMV or a CMV-virus relative to control, untransformed melon plants, are taken as being useful in the invention. Tomato Certain genotypes of tomato plants express the recessive endogenous resistance gene, Tm-2, which renders the plants more resistant to infection with Tomato Mosaic Virus (ToMV) than non-TM-2-expressing tomato plants. Furthermore, tomato plants are infected by the following viruses which have been utilized in coat protein mediated resistance (CPMR): Alfalfa Mosaic Virus (AMV; sometimes written as AIMV), Cucumber Mosaic Virus (CMV), Tomato Mosaic Virus (ToMV), and Tobacco Mosaic Virus (TMV) (Kaniewski and Thomas, Sem. Virol. 4: 389-396, 1993). Tomato viruses expressing Tm-2 can be transformed with one or more coat protein genes from the above list of viruses to generate transgenic tomato plants. Transgenic plants are evaluated for viral disease resistance according to standard methods. Transgenic tomato plants expressing a coat protein having resistance to at least one additional strain or isolate of AMV, CMV, ToMV, TMV, or related viruses relative to control, untransformed tomato plants, are taken as being useful in the invention. What is claimed is:

Claims: Claims 1. A method for providing resistance to a first viral pathogen in a plant, said method comprising the steps of: (a) selecting a plant having an endogenous level of resistance to a second viral pathogen that is at least two times that of a non-resistant plant; (b) transforming cells of said plant with a nucleic acid molecule encoding a viral coat protein gene; (c) regenerating said plant cells to provide a differentiated plant; and (d) selecting a transgenic plant which expresses the viral coat protein gene, wherein expression of the gene in said transformed plant provides resistance to said first viral pathogen. 2. The method of claim 1, wherein said transgenic plant is a dicotyledonous plant. 3. The method of claim 2, wherein said dicotyledonous plant is a member of the Solanaceae. 4. The method of claim 3, wherein said solanaceous plant is a member of the genus Nicotiana. 5. The method of claim 1, wherein said transgenic plant is a monocotyledonous plant. 6. The method of claim 1, wherein said transgenic plant has increased resistance to said second viral pathogen. 7. The method of claim 1, wherein said plant of step (a) comprises a gene derived from a Virgin A mutant. 8. The method of claim 1, wherein said viral coat protein gene is from alfalfa mosaic virus. 9. The method of claim 1, wherein said viral coat protein gene is from a potyvirus. 10. The method of claim 9, wherein said viral coat protein gene is from tobacco vein mottling virus. 11. The method of claim 9, wherein said viral coat protein gene is from potato virus Y. 12. The method of claim 1, wherein said transgenic plant has greater resistance and a broader spectrum of resistance than a transgenic plant without an endogenous level of resistance to said second viral pathogen. 13. The method of claim 1, wherein said transgenic plant is resistant to more than two different potyviruses. 14. The method of claim 1, wherein said first and said second viruses are related viruses. 15. A transgenic plant having resistance to a first viral pathogen, comprising a plant having an endogenous level of resistance to a second viral pathogen that is at least two times that of a non-resistant plant transformed with a nucleic acid molecule encoding a viral coat protein, wherein expression of the gene in said transformed plant provides resistance to said first viral pathogen. 16. A seed from a transgenic plant of claim 15. 17. A cell from a transgenic plant of claim 15. 18. The transgenic plant of claim 15, wherein said transgenic plant is a dicotyledonous plant. 19. The transgenic plant of claim 18, wherein said dicotyledonous plant is a member of the Solanaceae. 20. The transgenic plant of claim 19, wherein said solanaceous plant is a member of the genus Nicotiana. 21. The transgenic plant of claim 15, wherein said transgenic plant is a monocotyledonous plant. 22. The transgenic plant of claim 15, wherein said transgenic plant has increased resistance to said second viral pathogen. 23. The transgenic plant of claim 15, wherein said transgenic plant comprises a gene derived from a Virgin A mutant. 24. The transgenic plant of claim 15, wherein said viral coat protein gene is from alfalfa mosaic virus. 25. The transgenic plant of claim 15, wherein said viral coat protein gene is from a potyvirus. 26. The transgenic plant of claim 25, wherein said viral coat protein gene is from tobacco vein mottling virus. 27. The transgenic plant of claim 25, wherein said viral coat protein gene is from potato virus Y. 28. The transgenic plant of claim 15, wherein said transgenic plant has greater resistance and a broader spectrum of resistance than a transgenic plant without an endogenous level of resistance to said second viral pathogen. 29. The transgenic plant of claim 15, wherein said transgenic plant is resistant to more than two different potyviruses. 30. The transgenic plant of claim 15, wherein said first and said second viruses are related viruses.

Inventors: COLLINS GLENN B, NIELSEN MARK T, XU DONGMEI, COLLINS, GLENN, B., NIELSEN, MARK, T., XU, DONGMEI
Applicants: UNIV KENTUCKY, THE BOARD OF TRUSTEES OF THE UNIVERSITY OF KENTUCK
Application year: 1997
Main classification: A01H1
IPC codes: C07K14, C12N15