Despite long-standing plant breeding investments and early successes in genetic engineering, plant viral pathogens still cause major losses in agriculture worldwide. Early transgenic approaches involved the expression of pathogen-derived sequences that provided limited protection against relatively narrow ranges of viral pathotypes. In contrast, this study demonstrates that the ectopic expression of pvr1, a recessive gene from Capsicum chinense, results in dominant broad-spectrum potyvirus resistance in transgenic tomato plants (Solanum lycopersicum). The pvr1 locus in pepper encodes the eukaryotic translation initiation factor eIF4E. Naturally occurring point mutations at this locus result in monogenic recessive broad-spectrum potyvirus resistance that has been globally deployed via plant breeding programmes for more than 50 years. Transgenic tomato progenies that over-expressed the Capsicum pvr1 allele showed dominant resistance to several tobacco etch virus strains and other potyviruses, including pepper mottle virus, a range of protection similar to that observed in pepper homozygous for the pvr1 allele.
For various reasons, including public perceptions of safety, regulatory and ecological considerations, transgenic strategies employing plant genes already used widely in agriculture present an attractive alternative to crop improvement strategies that depend on the expression of genes in crops that are derived from other classes of organisms (Baulcombe, 1996; Padgett et al., 1997). Using this approach, engineered gene transfer is employed to extend the boundaries of sexually defined gene pools within a family or kingdom. This paper demonstrates a powerful strategy by which recessively inherited genetic variation may be converted via transgenic expression to dominant expression for use in systems well beyond sexually defined gene pools.
With the advent of plant genetic transformation techniques, considerable effort has been focused on resistance to viral diseases in view of the limitations of conventional viral disease control measures. The expression of transgenes originating from a wide range of both plant and non-plant sources has resulted in varying degrees of resistance or tolerance to plant viruses in experimental systems, and, in some cases, has been successfully deployed in agriculture (Verberne et al., 2000; Roger, 2002; Goldbach et al., 2003; Rudolph et al., 2003; Boonrod et al., 2004). Resistance defines a condition in which viral infection is prevented, whereas tolerance may describe a spectrum of outcomes less damaging than full susceptibility. To date, pathogen-derived resistance (PDR) is by far the most commonly reported transgenic strategy for the control of plant viral disease (Lomonossoff, 1995; Baulcombe, 1996; Goldbach et al., 2003). PDR was first demonstrated in transgenic tobacco plants expressing the tobacco mosaic virus (TMV) coat protein (CP), resulting in resistance to TMV (Abel et al., 1986). Since that time, many reports have confirmed the utility of PDR (Baulcombe, 1996; Goldbach et al., 2003; Rudolph et al., 2003). The impact of the experimental success with PDR has been limited relative to its enormous potential as a result of public perceptions of risk and the high costs of de-regulation (Bradford et al., 2005). A few crop varieties expressing PDR have been de-regulated for commercial production in the USA, e.g. transgenic squash expressing CP genes from watermelon mosaic virus, zucchini mosaic virus and cucumber mosaic virus (CMV) (Tepfer, 2002; Nap et al., 2003), and transgenic papaya resistant to papaya ringspot virus (Jan et al., 1999).
To date, nine dominant and five recessive genes of plant origin involved in virus resistance have been isolated, cloned and characterized (Kang et al., 2005b). Heterologous expression of some dominant resistance genes has been shown to confer resistance to plant disease when expressed in closely related host taxa (Whitham et al., 1996; Bendahmane et al., 2000); however, field tests of transgenic plants expressing plant virus resistance (R) genes have been limited to the N gene from tobacco expressed in tobacco (Tepfer, 2002), and none have been deployed commercially.
Recently, we reported the isolation of a naturally occurring monogenic recessive gene, pvr1, a homologue of the eukaryotic initiation translation factor eIF4E (Kang et al., 2005a), implicated in resistance to at least three families of viruses in both monocots and dicots (Kang et al., 2005b). This allele, pvr1, originally identified in the 1950s (Greenleaf, 1956) in Capsicum chinense, is known to confer broad-spectrum recessive resistance to several potyviruses, such as potato virus Y (PVY) pathotypes 0, 1 and 2, pepper mottle virus (PepMoV) and most tobacco etch virus (TEV) strains (Kyle and Palloix, 1997). Because potyviruses, comprising 30% of all known plant viruses (Ward and Shukla, 1991), are very destructive in agriculture, pvr1 has been deployed globally through plant breeding over the past 50 years. Three previously described genes with narrower resistance spectra, formerly known as pvr21, pvr22 and pvr23, are now known to be alleles at the pvr1 locus (Ruffel et al., 2002, 2004; Kang et al., 2005a). Each allele at this locus is defined by a set of characteristic point mutations located near domains of the protein implicated in mRNA cap binding (Ruffel et al., 2002; Kang et al., 2005a). These mutations also interrupt the physical interaction of eIF4E with the viral protein covalently bound at the 5′ end of the single-stranded RNA genome, VPg, consistent with the hypothesis that physical interaction between wild-type eIF4E and VPg may be required for susceptibility (Kang et al., 2005a).
In both plants and animals, dominant negative approaches have been widely used to study gene function (Kruger et al., 2002; Sieczkarski and Whittaker, 2002; Chandler and Werr, 2003; Leiva-Neto et al., 2004; Wicker et al., 2004), but have been applied to the objective of crop virus resistance using only pathogen-derived sequences (Herskowitz, 1987; Rudolph et al., 2003). This paper describes a test of the hypothesis that transgenic expression of eIF4E alleles derived from a resistant Capsicum plant could perturb the interactions required for viral susceptibility in a heterologous system without an adverse effect on the host. Dwarf tomato (Solanum lycopersicum) variety ‘Micro-Tom’ was transformed with Capsicum Pvr1+ and pvr1 sequences in both sense and antisense orientations. Over-expression of the allele, pvr1, in the sense orientation in transgenic tomatoes resulted in a highly resistant phenotype showing dominant inheritance. This result illustrates, for the first time, a strategy that employs recessively inherited plant viral resistance genes to engineer extreme plant disease resistance, coupled with emerging strategies to engineer specificity of these alleles.
Generation and confirmation of transgenic tomatoes expressing Capsicum eIF4E
To test the hypothesis that the ectopic over-expression of the C. chinense eIF4E recessive resistance allele pvr1 could interrupt potyvirus infection in a susceptible tomato host, eIF4E levels in S. lycopersicum‘Micro-Tom’ were modulated by the ectopic expression of susceptible (Pvr1+) and resistant (pvr1) alleles. The full-length open reading frames (ORFs) of the resistant allele pvr1 and the susceptible allele Pvr1+ were cloned into the plant transformation vector pBI121. Plasmids containing the test constructs, 35S::Pvr1+ and 35S::pvr1, and control constructs, empty vector and 35S::GUS (GUS, β-glucuronidase), were transformed into the experimental dwarf tomato variety ‘Micro-Tom’ via the Agrobacterium-mediated leaf disc method (Meissner et al., 1997). Successful transgene insertion in the putative primary transformants was assessed via polymerase chain reaction (PCR) to detect the neomycin phosphotransferase II (NPTII) gene and 35S promoter/eIF4E sequence (Figure 1a,b). Furthermore, in order to confirm that the transgenes encoded the full-length pvr1 sequence, they were amplified via PCR and resequenced. Sequencing results showed that the 35S::pvr1 construct contained a 1-bp deletion at the adenine sequence of the start codon, which resulted in a five-amino-acid truncation of the eIF4E protein. To confirm that the five deleted amino acids did not affect virus resistance, transgenic plants were also developed containing the full-length pvr1 sequence.
Increased eIF4E transcript levels in primary transgenic tomatoes were shown via RNA blot analysis (Figure 1c). No detectable difference in eIF4E protein level, however, was observed between control plants and transgenic plants via immunoblot assays using an antibody for Capsicum eIF4E (data not shown). Control treatments in the immunoblot assays indicated that the rabbit antibody for the Capsicum eIF4E produced cross-reacts with endogenous tomato eIF4E, preventing the detection of the differential expression of Capsicum eIF4E relative to endogenous tomato eIF4E in transgenic plants. All primary transformants (T0) confirmed to contain pepper eIF4E transgenes appeared normal by visual observation. T1 transgenic progenies obtained by self-pollination from T0 transformants were again screened for the expression of the NPTII gene via foliar kanamycin application. Transgenic plants containing a single copy of the test transgene were selected for further assays.
T1 transgenic progenies were challenged with TEV-HAT (HAT, highly aphid transmissible) to examine how the ectopic expression of Capsicum eIF4E would affect the outcome of viral challenge. Ten T1 plants representing each of the following T0 treatments were screened via mechanical inoculation with TEV-HAT: empty vector, 35S-GUS, 35S-pvr1 and 35S-Pvr1+. Symptom development was monitored, and the accumulation of viral antigen was assayed via indirect enzyme-linked immunosorbent assay (ELISA) of non-inoculated and inoculated tissues (Figure 2). Visible symptoms did not develop on any T1 plants carrying the 35S:pvr1 transgene after inoculation with TEV-HAT. At 21 days post-inoculation (dpi), all inoculated T1 plants carrying the 35S:pvr1 construct were indistinguishable from non-inoculated and mock-inoculated treatments (Figure 2a), and were comparable with non-inoculated control plants with respect to ELISA values (Figure 2b). When the resistance was compared between transgenic plants with the truncated and full-length pvr1 sequences, no differences in resistance were detected to TEV-HAT (data not shown), indicating that the resistance was not caused by the point mutations found in the resistance allele. 35S:pvr1 transgenic plants with the truncated sequence were used for further analysis. By contrast, typical systemic TEV-HAT symptoms (terminal leaflet cupping, downward bent petioles and stunting) developed at 7–9 dpi in all positive control treatments: untransformed (UT), vector-transformed (VT) and plants transformed with 35S::GUS (GT) and 35S::Pvr1+ constructs (Figure 2a). Three sets of T1 progeny per T0 transformant were evaluated independently with similar results.
To characterize the transgenic resistance phenotype in more detail, two primary transformants, 35S::pvr1 plants 9 and 10, were selected on the basis of DNA blot hybridization analysis indicating the presence of a single copy of the transgene (Figure 1d). Progenies from each of these T0 transformants, consisting of 16 T1 individuals, were tested for resistance to kanamycin and TEV-HAT. In both T1 progenies, kanamycin resistance and disease resistance perfectly co-segregated, consistent with a 3 : 1 segregation ratio (data not shown). Homozygous resistant T2 lines were selected on the basis of the absence of kanamycin sensitivity in their progeny, and used for further study.
In Capsicum, resistance in plants homozygous for the pvr1 allele (pvr1/pvr1) eliminates or limits potyviral replication to undetectable levels when assessed at the single-cell level by blocking replication (Murphy et al., 1998). To test whether the resistant transgene, 35S::pvr1, would confer the same extreme potyvirus resistance in tomato as found in Capsicum, TEV-HAT accumulation in the inoculated leaves of transgenic tomatoes was examined by both indirect ELISA at 2, 5, 8 and 15 dpi and green fluorescent protein (GFP) accumulation (Figure 3a). ELISA results showed that virus accumulation was clearly evident in the leaves of positive control treatments by 5 dpi; however, transgenic tomatoes containing 35S::pvr1 showed no detectable virus accumulation throughout the duration of the experiment (Figure 3a). To confirm this result at the cellular level, engineered TEV-HAT expressing GFP (Schaad et al., 1997a) was used to monitor viral infection in infected leaf tissue. TEV-GFP contains the GFP coding sequence between the P1- and Hc-Pro region and retains the same disease specificity as wild-type TEV-HAT (data not shown). Pepper plants naturally homozygous for either the pvr1 or Pvr1+ allele were compared with homozygous T2 tomato transgenic lines containing 35S::GUS and 35S::pvr1. Infection proceeded without a detectable interference in all inoculated leaves of susceptible peppers and 35S::GUS-transformed tomatoes (Figure 3b). Visible infection foci were detected by 3 dpi and continued to expand until the entire inoculated leaf showed GFP expression at 15 dpi. By contrast, no infection foci were detected in the resistant pepper plants or 35S::pvr1 transgenic tomatoes. These results indicate that TEV-HAT does not replicate to a detectable level via this assay in 35S::pvr1 transgenic plants or in Capsicum plants homozygous for the pvr1 allele.
The resistance spectrum of pvr1 was maintained in transgenic tomatoes expressing 35S::pvr1
Resistance conferred by the pvr1 allele in Capsicum controls most strains of TEV, including TEV-HAT, the necrotic strain of TEV (TEV-N) and TEV-NW (non-wilting), although at least one strain is known, TEV-Mex21, that overcomes pvr1-mediated resistance (Murphy et al., 1998). To determine whether a similar range of protection was afforded by the transgenic expression of the allele in tomato, groups of six plants from homozygous T2 35S::pvr1 tomato lines and homozygous 35S::GUS lines were mechanically inoculated at the four- to six-leaf stage with three TEV strains (TEV-HAT, TEV-N and TEV-Mex21). By 7–9 dpi, the positive control, 35S::GUS, developed clear symptoms after inoculation with TEV-HAT, TEV-N and TEV-Mex21, including leaf deformation and dwarfing that were very apparent by 21 dpi (data not shown). Infection was confirmed by positive ELISA absorbance values. By contrast, neither visible symptom development nor viral accumulation was detected in 35S::pvr1 transgenic tomato plants after inoculation with TEV-HAT and TEV-N (Figure 4a). In the case of TEV-Mex21, 35S::pvr1 transgenic tomatoes eventually developed very mild symptoms; however, virus accumulation was lower than the levels observed in the positive control plants, as expected on the basis of infectivity data from the Capsicum pvr1 genotype (Figure 4a,b).
Resistance to PepMoV is also controlled by the pvr1 gene in pepper (Murphy et al., 1998; Yeam et al., 2005). In a similar test with PepMoV, positive controls developed clear symptoms, including stunting and leaf distortion, at 8–10 dpi. Transgenic tomato plants expressing 35S::pvr1 remained free of symptoms and free of detectable accumulation of virus (Figure 4a). Because pvr1 is also known to control most PVY pathotypes, including PVY-0, PVY-1 and PVY-2, in pepper (Kyle and Palloix, 1997), a similar comparison was performed using PVY0 In this case, both controls and 35S::pvr1 transgenic plants showed no symptoms (data not shown), and no PVY CP accumulation was detected in inoculated and upper non-inoculated leaves via ELISA (Figure 4a). Preliminary data also showed that 35S::pvr1 conferred PVYNTN resistance (data not shown), a strain which has been shown to infect control ‘Micro-Tom’ plants. To further define the spectrum of the resistance conferred by the ectopic expression of 35S::pvr1 in ‘Micro-Tom’ plants, transgenic plants were also challenged with type members of two important viral genera, TMV and CMV. Transgenic plants inoculated with either CMV or TMV showed clear symptom development and viral accumulation, implying that the 35S::pvr1 construct does not confer resistance against TMV and CMV (Figure 4a).
In summary, virus infectivity studies demonstrated that transgenic tomatoes expressing the construct 35S::pvr1 were extremely resistant to TEV-HAT, TEV-N and PepMoV. In addition, 35S::pvr1 transgenic plants showed delayed onset and reduced symptom intensity with TEV-Mex21. Because ‘Micro-Tom’ is naturally resistant to most PVY strains, a thorough study of the response to this virus was not possible. This analysis clearly demonstrates that the ectopic expression of the pvr1 transgene in tomato confers a nearly identical resistance spectrum to that observed in pepper.
All Capsicum eIF4E encoded at the pvr1 locus can interact with eIF4G in yeast
We have recently shown that TEV requires functional eIF4E to bind TEV VPg for full susceptibility (Kang et al., 2005a), and that the point mutations in pvr1 abolish this interaction between its protein product and TEV VPg in vitro. To account for the shift to dominant inheritance observed in transgenic tomatoes expressing Capsicum pvr1 ectopically, it is possible that the mutant eIF4E functions to inhibit or interfere with an interaction that is essential for viral infectivity, but not required for host viability. On the basis of a large body of evidence in other systems (Morley et al., 1997; Gallie, 2001; Michon et al., 2006), it is probable that eIF4E complexes with a second translation factor, eIF4G, to support viral infectivity in this system. It is also probable that the cellular pool of eIF4E becomes enriched relative to endogenous eIF4E for the particular eIF4E protein encoded by an ectopically expressed allele driven by a very strong promoter. If the over-expressed version of eIF4E fails to bind viral VPg (Kang et al., 2005a), but still retains the capacity to interact with eIF4G, it is probable that the eIF4E-binding sites on endogenous eIF4G will become saturated with a form of eIF4E that cannot support viral infectivity.
When eIF4E levels were assayed in this system, elevated levels of eIF4E were not observed in transgenic tomatoes (data not shown), as might be expected in the light of evidence from other systems suggesting that eIF4E levels are regulated post-translationally (Dinkova et al., 2000). It is still probable, however, that over-expression of the transcript encoding the pvr1-eIF4E protein (Figure 1c) will result in a predominance of mutant eIF4E in the cellular pool, out-competing endogenous eIF4E for interaction with eIF4G, and saturating the eIF4E-binding sites on eIF4G with a version of the protein that cannot bind viral VPg, and hence failing to support viral infection. Using this model, the mutant eIF4E will effectively sequester eIF4G from the virus, resulting in a strongly reduced or absent viral infectivity and dominant inheritance of resistance.
This model is based on the assumption that the eIF4G-binding domain on pvr1-encoded eIF4E is not affected by the mutations that result in resistance. If this model is correct, the resistance will show dominant inheritance, as already demonstrated above, and the eIF4G-binding function of eIF4E proteins encoded by resistance alleles will be retained, despite defects in VPg binding. To test this hypothesis, a systematic analysis of the physical interactions between proteins encoded by resistance alleles at pvr1 and eIF4G was undertaken via yeast two-hybrid assay.
To evaluate the eIF4G-binding activity of each of the Capsicum eIF4E alleles, fragments of the eIF4G gene were cloned from Arabidopsis ecotype Columbia for yeast two-hybrid analysis that contained the putative eIF4E-binding domain based on results from mammalian systems (Morino et al., 2000) (Figure 5a). Arabidopsis eIF4E served as a positive control for the interaction (Figure 5b). All Capsicum eIF4E proteins used in this study interacted as strongly with Arabidopsis eIF4G fragments as the Arabidopsis eIF4E control. This result suggests that the mutations in pvr1 alleles that result in virus resistance do not interfere with the capacity of eIF4E to bind eIF4G in planta (Figure 5b). Ternary complexes between VPg, eIF4E and eIF4G have been detected in planta in the lettuce mosaic virus–lettuce pathosystem (Michon et al., 2006). Depletion of the eIF4G pool complexed with a version of the eIF4E protein that is capable of interacting with viral VPg provides a plausible explanation for the dominant inheritance of resistance observed when Capsicum eIF4E is over-expressed in tomato.
In this study, it has been demonstrated that the ectopic expression of an eIF4E homologue containing point mutations responsible for virus resistance in pepper results in a very high level of resistance to viral infection that extends to multiple viral species in the tomato system. This study also provides evidence for dominant inheritance of disease resistance conferred by transgenes derived from plant sources, previously observed in only one case (Deslandes et al., 2002). This work establishes, for the first time, that recessively inherited disease resistance genes in plants can function across intergeneric boundaries, well outside sexually defined gene pools. Clearly, our results establish that recessively inherited disease resistance genes in plants represent an untapped and valuable source of genetic variation for deployment for crop protection via transgenesis. Although prevalent in nature and considered to be highly durable relative to dominant R genes (Fraser, 1992; Kang et al., 2005a,b), much less is understood in general about the identity and function of these genes. In fact, only five recessively inherited plant viral resistance genes have been cloned to date, all of which encode the translation factor eIF4E, the focus of this study, or its isoform, eIF(iso)4E (Kang et al., 2005b). This remarkable identity of recessive viral resistance genes isolated from plants to date from across the kingdom strongly suggests that the results of this study will be broadly applicable. Clearly, the gene that is the focus of the present study, eIF4E, has been widely conserved across eukaryotes with respect to its role in eukaryotic translation and its critical importance in both animal and plant viral infection (Kleijn et al., 1996; Whitham and Wang, 2004).
The shift observed in this study from recessive inheritance of a gene in its native location to dominant inheritance via ectopic expression has important mechanistic and practical implications. Mechanistically, the observation of a shift to dominance suggests that the mutant protein may function to interfere with a process or sequester a factor required for viral infection, possibly eIF4G, based on evidence drawn from many systems (Morley et al., 1997; Gallie, 2001; Michon et al., 2006). It is probable that one important component of the mechanism of resistance in this system, and in naturally occurring recessive resistance, is the failure of pvr1-encoded eIF4E to bind the viral VPg, a step that appears to be necessary, although not sufficient, to establish viral infection (Gao et al., 2004; Kang et al., 2005a,b). In the case of transgenic expression of this protein, it is probable that this same feature of the protein is important. This alone, however, cannot account for the shift to dominance observed in this study. In order to account for this shift, it is predicted that there is a physical interaction in the cell between pvr1-eIF4E and another protein, most probably eIF4G (Morley et al., 1997; Gallie, 2001; Michon et al., 2006). If pvr1-eIF4E binds eIF4G, but is unable to function in the viral infection cycle as a result of the failure of transgene-encoded eIF4E to bind viral VPg, eIF4G can be effectively sequestered from the virus. Even if endogenous wild-type eIF4E is present in the cell, the depletion of eIF4G available for the virus may result in the failure of infection. Consistent with this hypothesis, it has been shown that the capacity to bind eIF4G is retained in pvr1-eIF4E (Figure 5). A survey of naturally occurring resistance alleles at eIF4E-encoding loci across the plant kingdom indicates that the domain predicted to be required for eIF4G binding is highly conserved (Marcotrigiano et al., 1997), consistent with the possibility that the host cannot tolerate interruption of this function.
The use of dominant negative approaches to control viral infection in plants is not unique. Success to date, however, has been reported only for sequences of viral origin (Padgett et al., 1997). On the basis of the observed shift in inheritance, the specific resistance mechanism by which functional eIF4E is sequestered from the virus probably differs between the dominant transgenic system reported here and the naturally existing recessively inherited eIF4E-based resistance systems. It may be possible to make valid inferences about the stability and durability of transgenic resistance using pvr1 on the basis of decades of experience employing this allele in breeding programmes with no shift towards full susceptibility (Kyle and Palloix, 1997). On the basis of decades of global deployment of eIF4E-based resistance in both monocots and dicots, it appears that plant viruses do not readily mutate to compensate for the interruption in the VPg–eIF4E interaction (Kang et al., 2005b). Even when viral isolates are identified that overcome these resistance genes, it is common that symptom intensity and viral accumulation are reduced to levels below agricultural significance (Kang et al., 2005b). Although the mechanism by which the VPg–eIF4E interaction is disrupted probably differs in the transgenic system relative to naturally occurring recessive resistance, it is likely that transgenic plants expressing appropriate eIF4E alleles will also show stability and durability through large-scale deployment in agriculture. Taken together, these results open up a new area of crop genetics with important implications for both conventional and transgenic crop improvement strategies.
There are clear advantages to the strategy illustrated by these results when compared with existing strategies to engineer disease resistance in crops. Strategies based on RNA silencing or RNA interference (Baulcombe, 2004; Tenllado et al., 2004) can be disabled by strong silencing suppressors of non-related viruses in mixed infections (Mitter et al., 2001). In this study, high levels of eIF4E transcript rule out the possibility that the extreme resistance observed is a result of post-transcriptional gene silencing (Chicas and Macino, 2001). In fact, very high levels of 35S::pvr1 transcript were observed in transgenic lines, but levels of eIF4E protein were equivalent to those of UT tomato controls when assayed using an eIF4E antibody. Similar levels of eIF4E protein in control and test transgenic plants suggest that the expression of eIF4E is regulated by translational or post-translational control, consistent with what is already known regarding the regulation of eIF4E and eIF(iso)4E in plants (Dinkova et al., 2000). This observation suggests that it is not necessary to accumulate significantly elevated levels of protein to achieve extreme resistance.
Ultimately, our goal is the identification of genetic strategies that would preclude the susceptibility of crops to a wide array of plant pathogens. Current investments in large-scale genomics of crop plant species and the availability of a wide array of tools to link proteins to their functions will provide new targets for engineered resistance (Noueiry and Ahlquist, 2003; Diaz-Pendon et al., 2004). In Arabidopsis, several host genes have been identified recently in which mutations impair the infection cycle of plant viruses (Whitham and Wang, 2004; Kang et al., 2005a,b). In theory, each of these genes defines a locus at which recessive resistance alleles may occur. However, to date, all recessive viral resistance genes isolated from plants as diverse as barley and Arabidopsis are homologues of eIF4E or eIF(iso)4E and viruses with RNA genomes that include members of the Potyviridae, Cucumoviridae and Carmoviridae (Kang et al., 2005b). Although it remains unknown which shifts in the protein account for the range of viruses controlled by variation in this gene, the involvement of this protein in a wide array of plant virus–host interactions is now indisputable. Additional tests are underway to further define the breadth of host taxa in which such constructs can function to confer resistance. Current work is also focusing on the dissection of point mutations in naturally occurring resistance alleles to identify the specific amino acid substitutions that account for the spectrum of viral isolates controlled, and the shift to dominance after over-expression. Once the rules that govern the physical interactions of these proteins are understood, an endogenous host allele could be cloned from a susceptible target crop species, mutagenized via a site-directed process and subsequently returned to the source species. If the results from this study can be generalized, the engineered allele driven by a strong promoter would probably then function in a dominant negative mode to interfere with the endogenous wild-type proteins and to establish successful viral infection. Using this scenario, point mutations found in naturally occurring resistance alleles in eIF4E homologues from various hosts would guide site-directed strategies in which naturally occurring variations have not been identified to date. The subsequent reintroduction of mutated eIF4E alleles driven by a strong promoter may allow new strategies of engineering new resistance alleles within taxa in which naturally occurring resistance at this locus has not yet been reported. The wide involvement of these translation factors across the plant kingdom in viral infection cycles of several large and important plant viral genera suggests significant potential for the broad application of our results in agriculture.
Plant materials and viral cultures
Capsicum annuum‘Early Cal Wonder’ was obtained from Asgrow Seed Co. (San Juan Bautista, CA, USA). C. chinense PI 159234 (234) was obtained from the United States Department of Agriculture (USDA) Southern Regional Plant Introduction Station (Experiment, GA, USA). S. lycopersicum cultivar ‘Micro-Tom’ (Ball Seed, West Chicago, IL, USA) was used to generate transgenic plants (Meissner et al., 1997). TEV-HAT was obtained from T. Pirone (University of Kentucky, Lexington, KY, USA). TEV-N, TEV-Mex and TEV-GFP were obtained from J. Carrington (Washington State University, Pullman, WA, USA). PepMoV-FL (Florida) was obtained from T. Zitter (Cornell University, Ithaca, NY, USA). All potyviruses were maintained on TMV-resistant Nicotiana tabacum‘Kentucky 14’ and were transferred every 4–8 weeks. PVY0, CMV and TMV strains were obtained from K. Perry (Cornell University, Ithaca, NY, USA). PVYNTN was obtained from Stewart Gray (Cornell University, Ithaca, NY, USA).
TEV-GFP RNA transcripts were prepared as described previously (Schaad et al., 1997b; Kang et al., 2005a), and were employed to inoculate N. tabacum‘Samsun’ using a microprojectile bombardment gun (Biorad, Hercules, CA, USA). At 5 dpi, infected leaves were harvested and used to inoculate pepper and tomato plants. GFP fluorescence was monitored to check infection of TEV-GFP using a Leica TCS SP2 scanning confocal microscope (Leica, Wetzlar, Germany).
Yeast two-hybrid analysis
Yeast two-hybrid analysis was performed as described previously (Kang et al., 2005a). Yeast strains and plasmid vectors were provided by G.B. Martin (Boyce Thompson Institute, Ithaca, NY, USA). A bait plasmid, pEG202, was used for the fusion of VPg and eIF4G to the DNA-binding domain of LexA; a prey plasmid, pJG4-5, was used to express Capsicum eIF4E alleles and Arabidopsis thaliana eIF4E. Cloning of Capsicum eIF4E alleles and VPg has been described previously (Kang et al., 2005a). The DNA sequences encoding Arabidopsis eIF4E and eIF4G were amplified via PCR from DNA samples extracted from A. thaliana Columbia (ABRC) and cDNA reverse-transcribed from leaf mRNA. The full-length cDNA sequence of Arabidopsis eIF4E and a 1200-bp cDNA fragment containing the putative eIF4E-binding domain of Arabidopsis eIF4G were used for yeast two-hybrid analysis. The position of the eIF4E-binding domain was predicted on the basis of results from humans (Morino et al., 2000).
Production of eIF4E antibody
Primers 5′-CATATGGCTTTTGCATTACCATCA-3′ and 5′-CTCGAGGTTGACCGTAAACTTCCGTTG-3′ were used to amplify the eIF4E ORF. Fragments were cloned into the NdeI and XhoI sites of pET16b (Novagen, Madison, WI, USA). The N-terminus His-tagged eIF4E recombinant protein was over-expressed in Escherichia coli (DE3) and affinity purified using nickel-nitrilotriacetic agarose (Qiagen, Valencia, CA, USA). The purified eIF4E protein was 26 kDa as expected. After gel purification, the recombinant protein was used to immunize rabbits for antibody production (Spring Valley Laboratories, Woodbine, MD, USA).
Evaluation of resistance to potyviruses
Seeds of test tomato genotypes to be evaluated for response to viral inoculation were sown in Styrofoam trays in Cornell Mix and held in a glasshouse at Cornell University (28 °C during the day and 21 °C at night; 16 h daylight). Plants were inoculated at the four- to six-leaf stage. A light application of carborundum was applied to the two oldest leaves (leaves 1 and 2), followed by rub-inoculation with inoculum produced by grinding systemically infected tobacco tissue in 50 mm potassium phosphate buffer, pH 7.5 (1 g tissue : 20 mL buffer). Mock-inoculated and non-inoculated controls were routinely included. Transgenic progenies at the T1 or T2 generation, as indicated, were inoculated with TEV-HAT, TEV-N, TEV-Mex21, PVY0, PVYNTN, PepMoV, CMV or TMV. Viral cultures were maintained in screened cages and routinely monitored for purity. Treatments typically consisted of at least 8–10 plants. After inoculation, the plants were monitored daily for the appearance of symptoms. Leaf tissue was tested for the presence of virus using antigen plate-coating indirect ELISA, as described previously (Kang et al., 2005a). Anti-viral immunoglobulins were obtained from Agdia, Inc. (Elkhart, IN, USA) and used according to the manufacturer's instructions. Virus accumulation was tested at 10 dpi for inoculated leaves or at 21 dpi for upper non-inoculated leaves.
We thank J.D. Frantz, M. Deom, G.B. Martin, K.L. Perry and S.M. Gray for experimental materials and G. Moriarty, H. Griffiths and M. Kreitinger for technical assistance. We thank J.F. Murphy, S.M. Gray and G.B. Martin for critical review of the manuscript. This work was supported in part by USDA National Research Initiative Competitive Grants Program (NRICGP) Plant Genome Award no. 94-37300-0333, USDA Initiative for Future Agricultural and Food Systems (IFAFS) Award no. 2001-52100-113347 and National Science Foundation (NSF) Plant Genome Award no. 0218166. IY was supported in part by a fellowship from the Kwanjeong Educational Foundation. KP was supported by a supplemental award from the NSF Plant Genome Program. We gratefully acknowledge Rosario Provvidenti whose work on inheritance of resistance to plant viruses remains a treasure for the future.