Deletion of a large genomic segment in tobacco varieties that are resistant to potato virus Y (PVY)

Recessive alleles (va, va ¹ , va ² , etc) of the tobacco Va locus confer resistance to potato virus Y (PVY). To elucidate the mechanism underlying this resistance, we attempted to identify randomly amplified polymorphic (RAPD) markers that reveal polymorphism between two nearly isogenic lines (NILs) that differ in their susceptibility to PVY. Using each of 500 primers and 800 pairs of primers, we identified over 100 RAPD fragments that differed between the NILs. We applied these RAPD primers or primer combinations to an F₂ population obtained from a cross between the susceptible line BY4 and the resistant va ² -bearing NIL, F55. It was found that only 10 RAPD markers were polymorphic between resistant and susceptible plants. Unexpectedly, these markers were all linked to Va. All 10 RAPD markers were present in all 8 susceptible varieties tested. At least one RAPD marker was not detected in 8 out of 10 resistant varieties. Southern analysis revealed that the sequences of markers were not present in the genomes of resistant varieties, and the markers were found in individually distinct positions on the chromosomes of susceptible tobacco varieties. These results strongly suggest that the resistance conferred by va is due to deletions at the Va locus governing susceptibility to PVY. Received: 20 May 1999 / Accepted: 17 August 1999     

NtTPN1: a RPP8‐like R gene required for Potato virus Y‐induced veinal necrosis in tobacco

Potato virus Y (PVY) is one of the most damaging viruses of tobacco. In particular, aggressive necrotic strains (PVY^N) lead to considerable losses in yield. The main source of resistance against PVY is linked to the va locus. However, va‐overcoming PVY isolates inducing necrotic symptoms were observed in several countries. In this context, it is important to find va‐independent protection strategies. In a previous study, the phenotyping of 162 tobacco varieties revealed 10 accessions that do not carry the va allele and do not exhibit typical PVY^N‐induced veinal necrosis. Despite the absence of necrotic symptoms, normal viral accumulation in these plants suggests a va‐independent mechanism of tolerance to PVY^N‐induced systemic veinal necrosis. Fine mapping of the genetic determinant(s) was performed in a segregating F2 population. The tolerance trait is inherited as a single recessive gene, and allelism tests demonstrated that eight of the 10 tolerant varieties carry the same determinant. Anchoring the linkage map to the tobacco genome physical map allowed the identification of a RPP8‐like R gene, called NtTPN1 (for t abacum P VY‐induced 相似文献   

Quantitative trait loci from the host genetic background modulate the durability of a resistance gene: a rational basis for sustainable resistance breeding in plants

The combination of major resistance genes with quantitative resistance factors is hypothesized as a promising breeding strategy to preserve the durability of resistant cultivar, as recently observed in different pathosystems. Using the pepper (Capsicum annuum)/Potato virus Y (PVY, genus Potyvirus) pathosystem, we aimed at identifying plant genetic factors directly affecting the frequency of virus adaptation to the major resistance gene pvr2³ and at comparing them with genetic factors affecting quantitative resistance. The resistance breakdown frequency was a highly heritable trait (h²=0.87). Four loci including additive quantitative trait loci (QTLs) and epistatic interactions explained together 70% of the variance of pvr2³ breakdown frequency. Three of the four QTLs controlling pvr2³ breakdown frequency were also involved in quantitative resistance, strongly suggesting that QTLs controlling quantitative resistance have a pleiotropic effect on the durability of the major resistance gene. With the first mapping of QTLs directly affecting resistance durability, this study provides a rationale for sustainable resistance breeding. Surprisingly, a genetic trade-off was observed between the durability of PVY resistance controlled by pvr2³ and the spectrum of the resistance against different potyviruses. This trade-off seemed to have been resolved by the combination of minor-effect durability QTLs under long-term farmer selection.     

The recessive potyvirus resistance gene <Emphasis Type="Italic">pot-1</Emphasis> is the tomato orthologue of the pepper <Emphasis Type="Italic">pvr2-eIF4E</Emphasis> gene

The translation initiation factor 4E (eIF4E) has been implicated in naturally occurring resistance to Potato virus Y (PVY) determined by the pvr2 locus in pepper (Capsicum annuum). Here, the molecular basis of the recessive resistance to PVY and Tobacco etch virus (TEV) controlled by the pot-1 locus in tomato (Lycopersicon esculentum; now Solanum lycopersicum) was investigated. On the basis of genetic mapping data that indicated that pot-1 and pvr2 are located in syntenic regions of the tomato and pepper genomes, the possible involvement of eIF4E in pot-1-mediated resistance was assessed. Genetic mapping of members of the eIF4E multigenic family in tomato introgression lines revealed that an eIF4E locus indeed maps in the same genomic region as pot-1. By comparing eIF4E coding sequences between resistant and susceptible Lycopersicon genotypes, a small number of polymorphisms that co-segregate with the pot-1 locus were identified, suggesting that this gene could be involved in resistance to potyviruses. Functional complementation experiments using Potato virus X-mediated transient expression of eIF4E from a susceptible genotype in a resistant pepper genotype confirmed that a small number of amino acid substitutions in the eIF4E protein indeed account for resistance/susceptibility to both the PVY and TEV, and consequently that pot-1 and pvr2 are orthologues. Taken together, these results support the role of this eIF4E gene as a key component of recessive resistance to potyviruses, and validate the comparative genomic approach for the molecular characterization of recessive resistance genes.     

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.     

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.     

A TILLING approach to generate broad‐spectrum resistance to potyviruses in tomato is hampered by eIF4E gene redundancy

Genetic resistance to pathogens is important for sustainable maintenance of crop yields. Recent biotechnologies offer alternative approaches to generate resistant plants by compensating for the lack of natural resistance. Tomato (Solanum lycopersicum) and related species offer a model in which natural and TILLING‐induced potyvirus resistance alleles may be compared. For resistance based on translation initiation factor eIF4E1, we confirm that the natural allele Sh–eIF4E1^PI24–pot1, isolated from the wild tomato species Solanum habrochaites, is associated with a wide spectrum of resistance to both potato virus Y and tobacco etch virus isolates. In contrast, a null allele of the same gene, isolated through a TILLING strategy in cultivated tomato S. lycopersicum, is associated with a much narrower resistance spectrum. Introgressing the null allele into S. habrochaites did not extend its resistance spectrum, indicating that the genetic background is not responsible for the broad resistance. Instead, the different types of eIF4E1 mutations affect the levels of eIF4E2 differently, suggesting that eIF4E2 is also involved in potyvirus resistance. Indeed, combining two null mutations affecting eIF4E1 and eIF4E2 re‐establishes a wide resistance spectrum in cultivated tomato, but to the detriment of plant development. These results highlight redundancy effects within the eIF4E gene family, where regulation of expression alters susceptibility or resistance to potyviruses. For crop improvement, using loss‐of‐function alleles to generate resistance may be counter‐productive if they narrow the resistance spectrum and limit growth. It may be more effective to use alleles encoding functional variants similar to those found in natural diversity.     

Drosophila Me31B is a Dual eIF4E-Interacting Protein

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. We show that Me31B interacts with eIF4E-1 and eIF4E-3 by means of yeast two-hybrid system, FRET in D. melanogaster S2 cells and coimmunoprecipitation in testis. Truncation and point mutations of Me31B proteins show two eIF4E-binding sites located in different protein domains. Residues Y401-L407 (at the carboxy-terminus) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminus) are critical for interaction with eIF4E-3. The residue W117 in eIF4E-1 and the homolog position F103 in eIF4E-3 are necessary for Me31B-eIF4E interaction suggesting that the change of tryptophan to phenylalanine provides specificity. Me31B represents a novel type of eIF4E-interacting protein with dual and specific interaction domains that might be recognized by different eIF4E isoforms in different tissues, adding complexity to the control of gene expression in eukaryotes.     

Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-22 from Lycopersicon esculentum

In tomato, infections by tomato mosaic virus are controlled by durable Tm-2² resistance. In order to gain insight into the processes underlying disease resistance and its durability, we cloned and analysed the Tm-2² resistance gene and the susceptible allele, tm-2. The Tm-2² gene was isolated by transposon tagging using a screen in which plants with a destroyed Tm-2² gene survive. The Tm-2² locus consists of a single gene that encodes an 861 amino acid polypeptide, which belongs to the CC-NBS-LRR class of resistance proteins. The putative tm-2 allele was cloned from susceptible tomato lines via PCR with primers based on the Tm-2² sequence. Interestingly, the tm-2 gene has an open reading frame that is comparable to the Tm-2² allele. Between the tm-2 and the Tm-2² polypeptide 38 amino acid differences are present of which 26 are located in the second half of the LRR-domain. Susceptible tomato plants, which were transformed with the Tm-2² gene, displayed resistance against ToMV infection. In addition, virus specificity, displayed by the Tm-2² resistance was conserved in these transgenic lines. To explain the durability of this resistance, it is proposed that the Tm-2²-encoded resistance is aimed at the Achilles' heel of the virus.     

Trans‐species synthetic gene design allows resistance pyramiding and broad‐spectrum engineering of virus resistance in plants

To infect plants, viruses rely heavily on their host's machinery. Plant genetic resistances based on host factor modifications can be found among existing natural variability and are widely used for some but not all crops. While biotechnology can supply for the lack of natural resistance alleles, new strategies need to be developed to increase resistance spectra and durability without impairing plant development. Here, we assess how the targeted allele modification of the Arabidopsis thaliana translation initiation factor eIF4E1 can lead to broad and efficient resistance to the major group of potyviruses. A synthetic Arabidopsis thaliana eIF4E1 allele was designed by introducing multiple amino acid changes associated with resistance to potyvirus in naturally occurring Pisum sativum alleles. This new allele encodes a functional protein while maintaining plant resistance to a potyvirus isolate that usually hijacks eIF4E1. Due to its biological functionality, this synthetic allele allows, at no developmental cost, the pyramiding of resistances to potyviruses that selectively use the two major translation initiation factors, eIF4E1 or its isoform eIFiso4E. Moreover, this combination extends the resistance spectrum to potyvirus isolates for which no efficient resistance has so far been found, including resistance‐breaking isolates and an unrelated virus belonging to the Luteoviridae family. This study is a proof‐of‐concept for the efficiency of gene engineering combined with knowledge of natural variation to generate trans‐species virus resistance at no developmental cost to the plant. This has implications for breeding of crops with broad‐spectrum and high durability resistance using recent genome editing techniques.     

Diversity of genetic backgrounds modulating the durability of a major resistance gene. Analysis of a core collection of pepper landraces resistant to Potato virus Y

The evolution of resistance‐breaking capacity in pathogen populations has been shown to depend on the plant genetic background surrounding the resistance genes. We evaluated a core collection of pepper (Capsicum annuum) landraces, representing the worldwide genetic diversity, for its ability to modulate the breakdown frequency by Potato virus Y of major resistance alleles at the pvr2 locus encoding the eukaryotic initiation factor 4E (eIF4E). Depending on the pepper landrace, the breakdown frequency of a given resistance allele varied from 0% to 52.5%, attesting to their diversity and the availability of genetic backgrounds favourable to resistance durability in the plant germplasm. The mutations in the virus genome involved in resistance breakdown also differed between plant genotypes, indicating differential selection effects exerted on the virus population by the different genetic backgrounds. The breakdown frequency was positively correlated with the level of virus accumulation, confirming the impact of quantitative resistance loci on resistance durability. Among these loci, pvr6, encoding an isoform of eIF4E, was associated with a major effect on virus accumulation and on the breakdown frequency of the pvr2‐mediated resistance. This exploration of plant genetic diversity delivered new resources for the control of pathogen evolution and the increase in resistance durability.     

Characterization of mammalian eIF4E-family members.

The translational factor eukaryotic initiation factor 4E (eIF4E) is a central component in the initiation and regulation of translation in eukaryotic cells. Through its interaction with the 5' cap structure of mRNA, eIF4E functions to recruit mRNAs to the ribosome. The accumulation of expressed sequence tag sequences has allowed the identification of three different eIF4E-family members in mammals termed eIF4E-1, eIF4E-2 (4EHP, 4E-LP) and eIF4E-3, which differ in their structural signatures, functional characteristics and expression patterns. Unlike eIF4E-1, which is found in all eukaryotes, orthologues for eIF4E-2 appear to be restricted to metazoans, while those for eIF4E-3 have been found only in chordates. Like prototypical eIF4E-1, eIF4E-2 was found to be ubiquitously expressed, with the highest levels in the testis. Expression of eIF4E-3 was detected only in heart, skeletal muscle, lung and spleen. Similarly to eIF4E-1, both eIF4E-2 and eIF4E-3 can bind to the mRNA cap-structure. However, in contrast to eIF4E-1 which interacts with both the scaffold protein, eIF4G and the translational repressor proteins, the eIF4E-binding proteins (4E-BPs), eIF4E-2 and eIF4E-3 each possesses a range of partial activities. eIF4E-2 does not interact with eIF4G, but does interact with 4E-BPs. Conversely, eIF4E-3 interacts with eIF4G, but not with 4E-BPs. Neither eIF4E-2 nor eIF4E-3 is able to rescue the lethality of eIF4E gene deletion in yeast. It is hypothesized that each eIF4E-family member fills a specialized niche in the recruitment of mRNAs by the ribosome through differences in their abilities to bind cap and/or to interact with eIF4G and the 4E-BPs.     

The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance

Hypersensitive resistance (HR) is an efficient defense strategy in plants that restricts pathogen growth and can be activated during host as well as non-host interactions. HR involves programmed cell death and manifests itself in tissue collapse at the site of pathogen attack. A novel hypersensitivity gene, Ny-1, for resistance to Potato virus Y (PVY) was revealed in potato cultivar Rywal. This is the first gene that confers HR in potato plants both to common and necrotic strains of PVY. The locus Ny-1 mapped on the short arm of potato chromosome IX, where various resistance genes are clustered in Solanaceous genomes. Expression of HR was temperature-dependent in cv. Rywal. Strains PVY^O and PVY^N, including subgroups PVY^NW and PVY^NTN, were effectively localized when plants were grown at 20°C. At 28°C, plants were systemically infected but no symptoms were observed. In field trials, PVY was restricted to the inoculated leaves and PVY-free tubers were produced. Therefore, the gene Ny-1 can be useful for potato breeding as an alternative donor of PVY resistance, because it is efficacious in practice-like resistance conferred by Ry genes.     

Suppression of Nonsense and Frameshift Mutations Obtained by Different Methods for Inactivating the Translation Termination Factor eRF3 in Yeast Saccharomyces cerevisiae

Mutations in genes of omnipotent nonsense suppressors SUP35 and SUP45 in yeast Saccharomyces cerevisiae encoding translation termination factors eRF3 and eRF1, respectively, and prionization of the eRF3 protein may lead to the suppression of some frameshift mutations (CPC mutations). Partial inactivation of the translation termination factor eRF3 was studied in strains with unstable genetically modified prions and also in transgenic yeast S. cerevisiae strains with the substitution of the indigenous SUP35 gene for its homolog from Pichia methanolica or for a recombinant S. cerevisiae SUP35gene. It was shown that this partial inactivation leads not only to nonsense suppression, but also to suppression of the frameshift lys2-90 mutation. Possible reasons for the correlation between nonsense suppression and suppression of the CPC lys2-90 mutation and mechanisms responsible for the suppression of CPC mutations during inactivation of translation termination factors are discussed.     

Multiple copies of eukaryotic translation initiation factors in Brassica rapa facilitate redundancy,enabling diversification through variation in splicing and broad‐spectrum virus resistance

Recessive strain‐specific resistance to a number of plant viruses in the Potyvirus genus has been found to be based on mutations in the eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E. We identified three copies of eIF(iso)4E in a number of Brassica rapa lines. Here we report broad‐spectrum resistance to the potyvirus Turnip mosaic virus (TuMV) due to a natural mechanism based on the mis‐splicing of the eIF(iso)4E allele in some TuMV‐resistant B. rapa var. pekinensis lines. Of the splice variants, the most common results in a stop codon in intron 1 and a much truncated, non‐functional protein. The existence of multiple copies has enabled redundancy in the host plant's translational machinery, resulting in diversification and emergence of the resistance. Deployment of the resistance is complicated by the presence of multiple copies of the gene. Our data suggest that in the B. rapa subspecies trilocularis, TuMV appears to be able to use copies of eIF(iso)4E at two loci. Transformation of different copies of eIF(iso)4E from a resistant B. rapa line into an eIF(iso)4E knockout line of Arabidopsis thaliana proved misleading because it showed that, when expressed ectopically, TuMV could use multiple copies which was not the case in the resistant B. rapa line. The inability of TuMV to access multiple copies of eIF(iso)4E in B. rapa and the broad spectrum of the resistance suggest it may be durable.     

Double mutations in eIF4E and eIFiso4E confer recessive resistance to <Emphasis Type="Italic">Chilli veinal mottle virus</Emphasis> in pepper

To evaluate the involvement of translation initiation factors eIF4E and eIFiso4E in Chilli veinai mottle virus (ChiVMV) infection in pepper, we conducted a genetic analysis using a segregating population derived from a cross between Capsicum annuum ‘Dempsey’ containing an eIF4E mutation (pvr1 ² ) and C. annuum ‘Perennial’ containing an eIFiso4E mutation (pvr6). C. annuum ‘Dempsey’ was susceptible and C. annuum ‘Perennial’ was resistant to ChiVMV. All F₁ plants showed resistance, and F₂ individuals segregated in a resistant-susceptible ratio of 166:21, indicating that many resistance loci were involved. Seventy-five F₂ and 329 F₃ plants of 17 families were genotyped with pvr1 ² and pvr6 allele-specific markers, and the genotype data were compared with observed resistance to viral infection. All plants containing homozygous genotypes of both pvr1 ² and pvr6 were resistant to ChiVMV, demonstrating that simultaneous mutations in eIF4E and eIFiso4E confer resistance to ChiVMV in pepper. Genotype analysis of F2 plants revealed that all plants containing homozygous genotypes of both pvr1 ² and pvr6 showed resistance to ChiVMV. In protein-protein interaction experiments, ChiVMV viral genome-linked protein (VPg) interacted with both eIF4E and eIFiso4E. Silencing of eIF4E and eIFiso4E in the VIGS experiment showed reduction in ChiVMV accumulation. These results demonstrated that ChiVMV can use both eIF4E and eIFiso4E for replication, making simultaneous mutations in eIF4E and eIFiso4E necessary to prevent ChiVMV infection in pepper. These authors contributed equally to this work.     

Eukaryotic initiation factor 4E-3 is essential for meiotic chromosome segregation, cytokinesis and male fertility in Drosophila

Gene expression is translationally regulated during many cellular and developmental processes. Translation can be modulated by affecting the recruitment of mRNAs to the ribosome, which involves recognition of the 5' cap structure by the cap-binding protein eIF4E. Drosophila has several genes encoding eIF4E-related proteins, but the biological role of most of them remains unknown. Here, we report that Drosophila eIF4E-3 is required specifically during spermatogenesis. Males lacking eIF4E-3 are sterile, showing defects in meiotic chromosome segregation, cytokinesis, nuclear shaping and individualization. We show that eIF4E-3 physically interacts with both eIF4G and eIF4G-2, the latter being a factor crucial for spermatocyte meiosis. In eIF4E-3 mutant testes, many proteins are present at different levels than in wild type, suggesting widespread effects on translation. Our results imply that eIF4E-3 forms specific eIF4F complexes that are essential for spermatogenesis.