Overexpression of the wild potato eIF4E-1 variant Eva1 elicits Potato virus Y resistance in plants silenced for native eIF4E-1

Potato virus Y (PVY) is the most important viral pathogen of cultivated potato (Solanum tuberosum) from a commercial perspective, causing severe losses in both tuber quality and yield worldwide. Specific accessions of wild potato species exhibit resistance against PVY but efforts to transfer the trait to cultivated material have not yielded widely adopted varieties. Because amino acid substitutions at specific domains of host factor eIF4E-1 often confer resistance to various crops, we sequenced the associated genes expressed in wild potato plants. A novel eIF4E-1 variant, designated here as Eva1, was identified in S. chacoense, S. demissum, and S. etuberosum. The protein contains amino acid substitutions at ten different positions when compared to its cultivated potato (S. tuberosum) homolog. In the yeast two-hybrid system, Eva1 failed to bind VPg, a viral protein required for infectivity. Overexpression of the associated cDNA conferred PVY resistance to transgenic potato plants silenced for the native eIF4E-1 gene. Because the gene sources of Eva1 are sexually compatible with potato, the molecular strategies described can be employed to develop 'intragenic' potato cultivars.     

Effect of cDNA fragments in different length derived from Potato Virus Y coat protein gene on the induction of RNA-mediated virus resistance

Potato virus Y (PVY) is a main viral pathogen infecting economic crops such as potato and tobacco plants. Genetic engineering has been so far the most effective method to produce viral resistant plants. Be-cause of the shortage of viral resistant genes in plants, cDNAs derived from viral genes were often used for induction of resistance in transgenic plants (the so- called pathogen-derived resistance)[1]. Among the genes used in the pathogen-derived resistance strategy, the coat protein gen…     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

Transgenic Potato with PVY Coat Protein Gene and Its Small-Scale Field Test

Potato virus Y (PVY) infection may cause a severe yield depression up to 80%. To develop the potato (Solanum tuberosum L. ) cultivars that resist PVY infection is very crucial in potato production. The authors have been cloned the coat protein gene of PVY from its Chinese isolate. A chimaeric gene containing the cauliflower mosaic virus 35S promoter and PVY coat protein coding region was introduced into the potato cultivars “Favorita”, “Tiger head” and “K4” via Agrobacterium tumefaciens. Results from PCR and Southern blot analysis confirmed that the foreign gene has integrated into the potato chromosomes. These transgenic potato plants were mechanically inoculated with PVY virus (20 mg/L). The presence of the virus in the potato plants was determined by ELISA and method of back inoculation into tobacco. The authors observed a drastic reduction in the accumulation of virus in some transgenic potato lines. Furthermore, some transgenic potato lines produced more tubers per plant than the untransformed potato did, and the average weight of these transgenic plant tubers was also increased. In the field test, the morphology and development of these transgenic potato plants were normal, 3 transgenic lines of “Favorita” exhibited a higher yield than the untrasformed virus-free potato with an increase ranged from 20% to 30%. From these transgenic lines, it will be very hopeful to develop a potato cultivar which not only has a significant resistance to PVY infection, but also a good harvest in potato production.     

Strategies for antiviral resistance in transgenic plants

Genetic engineering offers a means of incorporating new virus resistance traits into existing desirable plant cultivars. The initial attempts to create transgenes conferring virus resistance were based on the pathogen-derived resistance concept. The expression of the viral coat protein gene in transgenic plants was shown to induce protective effects similar to classical cross protection, and was therefore distinguished as 'coat-protein-mediated' protection. Since then, a large variety of viral sequences encoding structural and non-structural proteins were shown to confer resistance. Subsequently, non-coding viral RNA was shown to be a potential trigger for virus resistance in transgenic plants, which led to the discovery of a novel innate resistance in plants, RNA silencing. Apart from the majority of pathogen-derived resistance strategies, alternative strategies involving virus-specific antibodies have been successfully applied. In a separate section, efforts to combat viroids in transgenic plants are highlighted. In a final summarizing section, the potential risks involved in the introduction of transgenic crops and the specifics of the approaches used will be discussed.     

Expression of the PVYO coat protein (CP) under the control of the PVX CP gene leader sequence: protection under greenhouse and field conditions against PVYO and PVYN infection in three potato cultivars

Coat protein-mediated resistance (CPMR), resistance conferred as a result of the expression of viral coat proteins in transgenic plants, has been illustrated to be an effective way of protecting plants against several plant viruses. Nonetheless, consistent protection has not been achieved for transgenic plants expressing the coat protein of potato virus Y (PVY), the type member of the potyvirus family. In this report, three different potato cultivars were transformed with a chimeric construct consisting of the capsid protein (CP) coding sequences of PVY flanked by the AUG codon and the translational enhancer from the coat protein gene of potato virus X (PVX). These cultivars were shown to express high levels of PVY CP and confer a high degree of protection against PVY^o and PVY^N under both greenhouse and field conditions. In addition, transgenic plants infected with potato virus A (PVA), a related potyvirus, exhibited a delay in virus accumulation, which could be easily overcome with increasing virus concentrations. Received: 26 October 1995 / Accepted: 14 June 1996     

Special origin of stem sequence influence the resistance of hairpin expressing plants against PVY

In this study, 16 hairpin RNA (hpRNA) vectors were constructed, each harboring 50 bp viral RNA sequence as the stem. They all targeted the coat protein (CP) gene of Potato virus Y (PVY). Virus resistance assay revealed that hairpin constructs targeting the anterior 200 bp regions of the CP gene were unable to induce virus resistance, while the 12 hpRNA constructs targeting posterior 600 bp regions induced high virus resistance up to 77.78 %. Northern blot analysis revealed that 50 bp-length hpRNA constructs could be transcribed efficiently and processed into siRNAs; however, no correlation between siRNA accumulation and degree of antiviral defense was observed. Results presented here indicated that the middle and 3′ end of the CP cDNA was important for hpRNA-mediated PVY resistance, improving the design of pathogen-derived hpRNA expression cassettes for transgenic plant against viruses.     

Mutations in potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum

The recessive resistance genes pot-1 and pvr2 in Lycopersicon hirsutum and Capsicum annuum, respectively, control Potato virus Y (PVY) accumulation in the inoculated leaves. Infectious cDNA molecules from two PVY isolates differing in their virulence toward these resistances were obtained using two different strategies. Chimeras constructed with these cDNA clones showed that a single nucleotide change corresponding to an amino acid substitution (Arg119His) in the central part of the viral protein genome-linked (VPg) was involved in virulence toward the pot-1 resistance. On the other hand, 15 nucleotide changes corresponding to five putative amino acid differences in the same region of the VPg affected virulence toward the pvr2(1) and pvr2(2) resistances. Substitution models identified six and five codons within the central and C terminal parts of the VPg for PVY and for the related potyvirus Potato virus A, respectively, which undergo positive selection. This suggests that the role of the VPg-encoding region is determined by the protein and not by the viral RNA apart from its protein-encoding capacity.     

Genetic variability of Potato virus Y (PVY) and Tobacco etch virus (TEV) from naturally infected pepper fields in the Hatay region of Turkey

The genetic structure of Potato virus Y (PVY) and Tobacco etch virus (TEV) (Potyvirus) populations was investigated in pepper fields in two regions in Turkey. The diversity of PVY and TEV populations according to coat protein (CP) and VPg coding regions showed some similarity. All the isolates built a monophyletic group due to a single introduction event or multiple introductions of genetically similar isolates. All the isolates of both viruses showed evidence to the diversification for a long time. Based on VPg and CP sequences, all PVY isolates corresponded to clade C1. Turkish potyvirus isolates were only able to break the pvr2¹ resistance allele and therefore belonged to pathotype (0,?1). The Pvr4 dominant gene was found to be efficient and durable against PVY but not at all efficient against TEV. Consequently, the pvr2² resistance allele, efficient resistance against PVY and TEV pathotype (0,?1) isolates, would be the most suitable strategy to control potyviruses.     

Genetic background matters: a plant-virus gene-for-gene interaction is strongly influenced by genetic contexts

Evolutionary processes responsible for parasite adaptation to their hosts determine our capacity to manage sustainably resistant plant crops. Most plant-parasite interactions studied so far correspond to gene-for-gene models in which the nature of the alleles present at a plant resistance locus and at a pathogen pathogenicity locus determine entirely the outcome of their confrontation. The interaction between the pepper pvr2 resistance locus and Potato virus Y (PVY) genome-linked protein VPg locus obeys this kind of model. Using synthetic chimeras between two parental PVY cDNA clones, we showed that the viral genetic background surrounding the VPg pathogenicity locus had a strong impact on the resistance breakdown capacity of the virus. Indeed, recombination of the cylindrical inclusion (CI) coding region between two PVY cDNA clones multiplied by six the virus capacity to break down the pvr2(3) -mediated resistance. High-throughput sequencing allowed the exploration of the diversity of PVY populations in response to the selection pressure of the pvr2(3) resistance. The CI chimera, which possessed an increased resistance breakdown capacity, did not show an increased mutation accumulation rate. Instead, selection of the most frequent resistance-breaking mutation seemed to be more efficient for the CI chimera than for the parental virus clone. These results echoed previous observations, which showed that the plant genetic background in which the pvr2(3) resistance gene was introduced modified strongly the efficiency of selection of resistance-breaking mutations by PVY. In a broader context, the PVY CI coding region is one of the first identified genetic factors to determine the evolvability of a plant virus.     

Virulence factor of potato virus Y, genome-attached terminal protein VPg, is a highly disordered protein

Potato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5' end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host.     

Complex formation between recombinant protein VPg of potato virus Y and heterologous eukaryotic translation initiation factors eIF4E

Genomes of some positive-strand RNA viruses do not contain cap-structure, but instead their 5'-end is covalently linked to a viral protein called VPg. Complex formation between VPg and cellular translation initiation factors (eIFs) has been extensively studied in the context of the model of this complex involvement in virus mRNA translation initiation and cellular protein translation shut down in infected cells. The potato virus (PVY) VPg was expressed in bacterial and baculovirus systems in order to investigate its binding capacity to wheat eIF4E and its isoform. Both purified recombinant eIF4E and eIF(iso)4E were identified in vitro as binding partners of the purified recombinant VPg by using affinity chromatography, as well in vivo by coexpressing of recombinant VPg and eIFs in insect cells with following complex purification using affinity chromatography. Besides it was shown that PVY VPg also formed a complex with endogenous insect eIF4E in vivo. PVY VPg interaction with eIF4E of wheat (non permissive plant for PVY), and also with so evolutionary distant partner as insect eIF4E suggests the conservation of general structural features of eIF4E implicated in the formation of the complex with VPg.     

Field resistance of transgenic burley tobacco lines and hybrids expressing the tobacco vein mottling virus coat protein gene

Fifty transgenic lines expressing the tobacco vein mottling virus (TVMV) coat protein (CP) gene in five genetic backgrounds were evaluated under field conditions for response to mechanic inoculation with TVMV, tobacco etch virus (TEV) and potato virus Y (PVY). TVMV CP transgenic lines conferred resistance to TVMV, TEV and PVY under field conditions. Combining two strategies, coat protein-mediated resistance (CPMR) coupled with an endogenous resistance gene (Virgin A Mutant, VAM) significantly extended the range and magnitude of virus resistance and provided a potential valuable new source of protection against potyviruses. CP transgenic lines lacking the VAM gene had high resistance to TEV, medium resistance to PVY, and a recovery phenotype to TVMV. A series of hybrids involving transgenic lines were generated and tested under field conditions for response to virus inoculation. One copy of TVMV-CP gene presented in lines homozygous for the VAM gene provided effective resistance to all three potyviruses. These studies also suggested that selection of a suitable recipient genotype was critical and that field evaluation was necessary in order to select elite resistant transgenic lines. Engineering viral CP genes into genotypes possessing some level of virus resistance could be critical to achieve an effective level of resistance.