Recessive resistance genes against potyviruses are localized in colinear genomic regions of the tomato ( Lycopersicon spp.) and pepper ( Capsicum spp.) genomes

Resistance against both Potato virus Y (PVY) and Tobacco etch virus (TEV) was identified in the wild tomato relative Lycopersicon hirsutum PI247087. Analysis of the segregation ratio in F(2)/F(3) and BC(1) interspecific progenies indicated that a single recessive gene, or two very tightly linked recessive loci, are involved in resistance to both potyviruses. This locus was named pot-1. Using amplified fragment length polymorphism markers and a set of L. hirsutum introgression lines, pot-1 was mapped to the short arm of tomato chromosome 3, in the vicinity of the recessive py-1 locus for resistance to corky root rot. Because of the occurrence of phenotypically similar genes in pepper ( Capsicum spp.), the comparative genetics of resistance to potyviruses between tomato and pepper was investigated. Unlike most of the comparative genetic studies on resistance genes, pot-1 was tightly flanked by the same restriction fragment length polymorphism (RFLP) markers than the pvr2/pvr5 locus for resistance to PVY and TEV from pepper. These results may indicate that recessive resistance genes against potyviruses evolve less rapidly than the majority of the dominant genes cloned so far, and consequently may belong to a different family of resistance genes.     

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.     

A single-base deletion mutation in <Emphasis Type="Italic">SlIAA9</Emphasis> gene causes tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) <Emphasis Type="Italic">entire</Emphasis> mutant

The entire (e) locus of tomato (Solanum lycopersicum L.) controls leaf morphology. Dominant E and recessive e allele of the locus produce pinnate compound and complex reduced leaves. Previous research had indicated that SlIAA9, an Aux/IAA gene, was involved in tomato leaf morphology. Down-regulation of SlIAA9 gene by antisense transgenic method decreased the leaf complex of tomato and converted tomato compound leaves to simple leaves. The leaf morphology of these transgenic lines was similar with leaf morphology of tomato entire mutant. In this paper, we report that a single-base deletion mutation in the coding region of SlIAA9 gene results in tomato entire mutant phenotypes.     

Ectopic expression of a recessive resistance gene generates dominant potyvirus resistance in plants

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.     

Engineering virus resistance using a modified potato gene

Natural mutations in translation initiation factor eIF4E confer resistance to potyviruses in many plant species. Potato is a staple food crop plagued by several potyviruses, yet to date no known eIF4E-mediated resistance genes have been identified. In this study, we demonstrate that transgenic expression of the pvr1(2) gene from pepper confers resistance to Potato virus Y (PVY) in potato. We then use this information to convert the susceptible potato ortholog of this allele into a de novo allele for resistance to PVY using site-directed mutagenesis. Potato plants overexpressing the mutated potato allele are resistant to virus infection. Resistant lines expressed high levels of eIF4E mRNA and protein. The resistant plants showed growth similar to untransformed controls and produced phenotypically similar tubers. This technique disrupts a key step in the viral infection process and may potentially be used to engineer virus resistance in a number of economically important plant-viral pathosystems. Furthermore, the general public may be more amenable to the 'intragenic' nature of this approach because the transferred coding region is modified from a gene in the target crop rather than from a distant species.     

A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E)

We show here that the pvr2 locus in pepper, conferring recessive resistance against strains of potato virus Y (PVY), corresponds to a eukaryotic initiation factor 4E (eIF4E) gene. RFLP analysis on the PVY-susceptible and resistant pepper cultivars, using an eIF4E cDNA from tobacco as probe, revealed perfect map co-segregation between a polymorphism in the eIF4E gene and the pvr2 alleles, pvr2(1) (resistant to PVY-0) and pvr2(2) (resistant to PVY-0 and 1). The cloned pepper eIF4E cDNA encoded a 228 amino acid polypeptide with 70-86% nucleotide sequence identity with other plant eIF4Es. The sequences of eIF4E protein from two PVY-susceptible cultivars were identical and differed from the eIF4E sequences of the two PVY-resistant cultivars Yolo Y (YY) (pvr2(1)) and FloridaVR2 (F) (pvr2(2)) at two amino acids, a mutation common to both resistant genotypes and a second mutation specific to each. Complementation experiments were used to show that the eIF4E gene corresponds to pvr2. Thus, potato virus X-mediated transient expression of eIF4E from susceptible cultivar Yolo Wonder (YW) in the resistant genotype YY resulted in loss of resistance to subsequent PVY-0 inoculation and transient expression of eIF4E from YY (resistant to PVY-0; susceptible to PVY-1) rendered genotype F susceptible to PVY-1. Several lines of evidence indicate that interaction between the potyvirus genome-linked protein (VPg) and eIF4E are important for virus infectivity, suggesting that the recessive resistance could be due to incompatibility between the VPg and eIF4E in the resistant genotype.     

High-resolution Mapping and Analysis of the Resistance Locus <Emphasis Type="Italic">Rpi-abpt</Emphasis> Against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Potato

Introduction of more durable resistance against Phytophthora infestans causing late blight into the cultivated potato is of importance for sustainable agriculture. We identified a new monogenically inherited resistance locus that is localized on chromosome 4. The resistance is derived from an ABPT clone, which is originally a complex quadruple hybrid in which Solanum acaule, S. bulbocastanum, S. phureja and S. tuberosum were involved. Resistance data of the original resistant accessions of the wild species and analysis of mobility of AFLP markers linked to the resistance locus suggest that the resistance locus is originating from S. bulbocastanum. A population of 1383 genotypes was screened with two AFLP markers flanking the Rpi-abpt locus and 98 recombinants were identified. An accurate high-resolution map was constructed and the Rpi-abpt locus was localized in a 0.5 cM interval. One AFLP marker was found to co-segregate with the Rpi-abpt locus. Its DNA sequence was highly similar with sequences found on a tomato BAC containing several resistance gene analogues on chromosome 4 and its translated protein sequence appeared to be homologous to several disease resistance related proteins. The results indicated that the Rpi-abpt gene is a member of an R gene cluster.     

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.     

<Emphasis Type="Italic">CaMi</Emphasis>, a root-knot nematode resistance gene from hot pepper (<Emphasis Type="Italic">Capsium annuum</Emphasis> L.) confers nematode resistance in tomato

Several root-knot nematode (Meloidogyne spp.) resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, none of them has yet been cloned. In this study, a candidate root-knot nematode resistance gene (designated as CaMi) was isolated from the resistant pepper line PR 205 by degenerate PCR amplification combined with the RACE technique. Expression profiling analysis revealed that this gene was highly expressed in roots, leaves, and flowers and expressed at a lower level in stems and was not detectable in fruits. To verify the function of CaMi, a sense vector containing the genomic DNA spanning the full coding region of CaMi was constructed and transferred into root-knot nematode susceptible tomato plants. Sixteen transgenic plants carrying one to five copies of T-DNA inserts were generated from two nematode susceptible tomato cultivars. RT-PCR analysis revealed that the expression levels of CaMi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some transgenic lines compared to untransformed susceptible plants, and that the resistance was inheritable. Ultrastructure analysis showed that nematodes led to the formation of galls or root knots in the susceptible lines while in the resistant transgenic plants, the CaMi gene triggered a hypersensitive response (HR) as well as many necrotic cells around nematodes. Rugang Chen and Hanxia Li are contributed equally to this work.     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Natural variation and functional analyses provide evidence for co-evolution between plant eIF4E and potyviral VPg

Amino acid substitutions in the eukaryotic translation initiation factor 4E (eIF4E) result in recessive resistance to potyviruses in a range of plant species, including Capsicum spp. Correspondingly, amino acid changes in the central part of the viral genome-linked protein (VPg) are responsible for the potyvirus's ability to overcome eIF4E-mediated resistance. A key observation was that physical interaction between eIF4E and the VPg is required for viral infection, and eIF4E mutations that cause resistance prevent VPg binding and inhibit the viral cycle. In this study, polymorphism analysis of the pvr2-eIF4E coding sequence in a worldwide sample of 25 C. annuum accessions identified 10 allelic variants with exclusively non-synonymous variations clustered in two surface loops of eIF4E. Resistance and genetic complementation assays demonstrated that pvr2 variants, each with signature amino acid changes, corresponded to potyvirus resistance alleles. Systematic analysis of the interactions between eIF4E proteins encoded by the 10 pvr2 alleles and VPgs of virulent and avirulent potato virus Y (PVY) and tobacco etch virus (TEV) strains demonstrated that resistance phenotypes arose from disruption of the interaction between eIF4E and VPg, and that viral adaptation to eIF4E-mediated resistance resulted from restored interaction with the resistance protein. Complementation of an eIF4E knockout yeast strain by C. annuum eIF4E proteins further shows that amino acid changes did not impede essential eIF4E functions. Altogether, these results argue in favour of a co-evolutionary 'arms race' between Capsicum eIF4E and potyviral VPg.     

Susceptibility to <Emphasis Type="Italic">Fusarium </Emphasis>head blight is associated with the <Emphasis Type="Italic">Rht-D1b</Emphasis> semi-dwarfing allele in wheat

Fusarium head blight (FHB) is an important disease of wheat worldwide. The cultivar Spark is more resistant than most other UK winter wheat varieties but the genetic basis for this is not known. A mapping population from a cross between Spark and the FHB susceptible variety Rialto was used to identify quantitative trait loci (QTL) associated with resistance. QTL analysis across environments revealed nine QTL for FHB resistance and four QTL for plant height (PH). One FHB QTL was coincident with the Rht-1D locus and accounted for up to 51% of the phenotypic variance. The enhanced FHB susceptibility associated with Rht-D1b is not an effect of PH per se as other QTL for height segregating in this population have no influence on susceptibility. Experiments with near-isogenic lines supported the association between susceptibility and the Rht-D1b allele conferring the semi-dwarf habit. Our results demonstrate that lines carrying the Rht-1Db semi-dwarfing allele are compromised in resistance to initial infection (type I resistance) while being unaffected in resistance to spread within the spike (type II resistance).     

Identification and fine mapping of <Emphasis Type="Italic">Pi39</Emphasis>(t), a major gene conferring the broad-spectrum resistance to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Blast, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. The Chinese native cultivar (cv.) Q15 expresses the broad-spectrum resistance to most of the isolates collected from China. To effectively utilize the resistance, three rounds of linkage analysis were performed in an F₂ population derived from a cross of Q15 and a susceptible cv. Tsuyuake, which segregated into 3:1 (resistant/susceptible) ratio. The first round of linkage analysis employing simple sequence repeat (SSR) markers was carried out in the F₂ population through bulked-segregant assay. A total of 180 SSR markers selected from each chromosome equally were surveyed. The results revealed that only two polymorphic markers, RM247 and RM463, located on chromosome 12, were linked to the resistance (R) gene. To further define the chromosomal location of the R gene locus, the second round of linkage analysis was performed using additional five SSR markers, which located in the region anchored by markers RM247 and RM463. The locus was further mapped to a 0.27 cM region bounded by markers RM27933 and RM27940 in the pericentromeric region towards the short arm. For fine mapping of the R locus, seven new markers were developed in the smaller region for the third round of linkage analysis, based on the reference sequences. The R locus was further mapped to a 0.18 cM region flanked by marker clusters 39M11 and 39M22, which is closest to, but away from the Pita/Pita ² locus by 0.09 cM. To physically map the locus, all the linked markers were landed on the respective bacterial artificial chromosome clones of the reference cv. Nipponbare. Sequence information of these clones was used to construct a physical map of the locus, in silico, by bioinformatics analysis. The locus was physically defined to an interval of ≈37 kb. To further characterize the R gene, five R genes mapped near the locus, as well as 10 main R genes those might be exploited in the resistance breeding programs, were selected for differential tests with 475 Chinese isolates. The R gene carrier Q15 conveys resistances distinct from those conditioned by the carriers of the 15 R genes. Together, this valuable R gene was, therefore, designated as Pi39(t). The sequence information of the R gene locus could be used for further marker-based selection and cloning. Xinqiong Liu and Qinzhong Yang contributed equally to this work.     

Fine mapping of the nematode resistance gene <Emphasis Type="Italic">Mi-3</Emphasis> in <Emphasis Type="Italic">Solanum peruvianum</Emphasis> and construction of a <Emphasis Type="Italic">S. lycopersicum</Emphasis> DNA contig spanning the locus

Currently, the only genetic resistance against root-knot nematodes in the cultivated tomato Solanum lycopersicum (Lycopersicon esculentum) is due to the gene Mi-1. Another resistance gene, Mi-3, identified in the related wild species Solanum peruvianum (Lycopersicon peruvianum) confers resistance to nematodes that are virulent on tomato lines that carry Mi-1, and is effective at temperatures at which Mi-1 is not effective (above 30°C). Two S. peruvianum populations segregating for Mi-3 were used to develop a high-resolution map of the Mi-3 region of chromosome 12. S. lycopersicum BACs carrying flanking markers were identified and used to construct a contig spanning the Mi-3 region. Markers generated from BAC-end sequences were mapped in S. peruvianum plants in which recombination events had occurred near Mi-3. Comparison of the S. peruvianum genetic map with the physical map of S. lycopersicum indicated that marker order is conserved between S. lycopersicum and S. peruvianum. The 600 kb contig between Mi-3-flanking markers TG180 and NR18 corresponds to a genetic distance of about 7.2 cM in S. peruvianum. We have identified a marker that completely cosegregates with Mi-3, as well as flanking markers within 0.25 cM of the gene. These markers can be used to introduce Mi-3 into cultivated tomato, either by conventional breeding or cloning strategies.