Erratum: Development of diagnostic molecular markers for marker-assisted breeding against bacterial wilt in tomato.

[This corrects the article DOI: 10.1270/jsbbs.20027.].

Salicylic Acid, a Plant Hormone, Suppresses Phytophagous Insect Immune Response by Interrupting HMG-Like DSP1.

Salicylic acid is a plant hormone that can mediate various plant physiological processes. Salicylic acid can bind to human high mobility group box 1 (HMGB1) and interrupt its role in mediating immune responses. Dorsal switch protein 1 (DSP1) is an insect homolog of HMGB1. In this study, a DSP1 (Se-DSP1) encoded in Spodoptera exigua, a phytophagous insect, was characterized, and its potential role in immune response was explored. Upon bacterial challenge, Se-DSP1 was localized in the nucleus and released into the hemolymph. The released Se-DSP1 could mediate both cellular and humoral immune responses by activating eicosanoid biosynthesis. Salicylic acid could bind to Se-DSP1 with a high affinity. The immune responses of S. exigua were significantly interrupted by SA feeding. Larvae reared on tomatoes with high endogenous SA levels became more susceptible to entomopathogens. Taken together, these results suggest a tritrophic defensive role of plant SA against phytophagous insects.

QTL-Seq Analysis for Identification of Resistance Loci to Bacterial Canker in Tomato.

Bacterial canker caused by Clavibacter michiganensis (Cm) is one of the most economically important vascular diseases causing unilateral leaf wilting, stem canker, a bird's-eye lesion on fruit, and whole plant wilting in tomato. There is no commercially available cultivar with bacterial canker resistance, and genomics-assisted breeding can accelerate the development of cultivars with enhanced resistance. Solanum lycopersicum "Hawaii 7998" was found to show bacterial canker resistance. A Quantitative trait loci (QTL)-seq was performed to identify the resistance loci using 909 F2 individuals derived from a cross between S. lycopersicum "E6203" (susceptible) and "Hawaii 7998," and a genomic region (37.24-41.15 Mb) associated with bacterial canker resistance on chromosome 6 (Rcm6) was found. To dissect the Rcm6 region, 12 markers were developed and several markers were associated with the resistance phenotypes. Among the markers, the Rcm6-9 genotype completely matched with the phenotype in the 47 cultivars. To further validate the Rcm6 as a resistance locus and the Rcm6-9 efficiency, subsequent analysis using F2 and F3 progenies was conducted. The progeny individuals with homozygous resistance allele at the Rcm6-9 showed significantly lower disease severity than those possessing homozygous susceptibility alleles. Genomes of five susceptible and two resistant cultivars were analyzed and previously known R-genes were selected to find candidate genes for Rcm6. Nucleotide-binding leucine-rich repeat, receptor-like kinase, and receptor-like protein were identified to have putative functional mutations and show differential expression upon the Cm infection. The DNA markers and candidate genes will facilitate marker-assisted breeding and provide genetic insight of bacterial canker resistance in tomato.

Development and Application of Gene-Specific Markers for Tomato Yellow Leaf Curl Virus Resistance in Both Field and Artificial Infections.

Tomato yellow leaf curl virus (TYLCV) is a disease that is damaging to tomato production worldwide. Resistance to TYLCV has been intensively investigated, and single resistance genes such as Ty-1 have been widely deployed in breeding programs. However, resistance-breaking incidences are frequently reported, and achieving durable resistance against TYLCV in the field is important. In this study, gene-specific markers for Ty-2 and ty-5, and closely-linked markers for Ty-4 were developed and applied to distinguish TYLCV resistance in various tomato genotypes. Quantitative infectivity assays using both natural infection in the field and artificial inoculation utilizing infectious TYLCV clones in a growth chamber were optimized and performed to investigate the individual and cumulative levels of resistance. We confirmed that Ty-2 could also be an effective source of resistance for TYLCV control, together with Ty-1. Improvement of resistance as a result of gene-pyramiding was speculated, and breeding lines including both Ty-1 and Ty-2 showed the strongest resistance in both field and artificial infections.

Development of diagnostic molecular markers for marker-assisted breeding against bacterial wilt in tomato.

Bacterial wilt, caused by the Ralstonia pseudosolanacearum species complex, is an important vascular disease that limits tomato production in tropical and subtropical regions. Two major quantitative trait loci (QTL) of bacterial wilt resistance on chromosome 6 (Bwr-6) and 12 (Bwr-12) were previously identified in Solanum lycopersicum 'Hawaii 7996'; however, marker-assisted breeding for bacterial wilt resistance is not well established. To dissect the QTL, six cleaved amplified polymorphic sites (CAPS) and derived CAPS (dCAPS) markers within the Bwr-6 region and one dCAPS marker near Bwr-12 were developed, and resistance levels in 117 tomato cultivars were evaluated. Two markers, RsR6-5 on chromosome 6 and RsR12-1 on chromosome 12, were selected based on the genotypic and phenotypic analysis. The combination of RsR6-5 and RsR12-1 effectively distinguishes resistant and susceptible cultivars. Furthermore, the efficiency of the two markers was validated in the F3 generation derived from the F2 population between E6203 (susceptible) and Hawaii 7998 (resistant). Resistant alleles at both loci led to the resistance to bacterial wilt. These markers will facilitate marker-assisted breeding of tomato resistant to bacterial wilt.

Inferring the Genetic Determinants of Fruit Colors in Tomato by Carotenoid Profiling.

Carotenoids are essential for plant and animal nutrition, and are important factors in the variation of pigmentation in fruits, leaves, and flowers. Tomato is a model crop for studying the biology and biotechnology of fleshy fruits, particularly for understanding carotenoid biosynthesis. In commercial tomato cultivars and germplasms, visual phenotyping of the colors of ripe fruits can be done easily. However, subsequent analysis of metabolic profiling is necessary for hypothesizing genetic factors prior to performing time-consuming genetic analysis. We used high performance liquid chromatography (HPLC), employing a C30 reverse-phase column, to efficiently resolve nine carotenoids and isomers of several carotenoids in yellow, orange, and red colored ripe tomatoes. High content of lycopene was detected in red tomatoes. The orange tomatoes contained three dominant carotenoids, namely δ-carotene, ß-carotene, and prolycopene. The yellow tomatoes showed low levels of carotenoids compared to red or orange tomatoes. Based on the HPLC profiles, genes responsible for overproducing δ-carotene and prolycopene were described as lycopene ε-cyclase and carotenoid isomerase, respectively. Subsequent genetic analysis using DNA markers for segregating population and germplasms were conducted to confirm the hypothesis. This study establishes the usefulness of metabolic profiling for inferring the genetic determinants of fruit color.

Molecular marker development and genetic diversity exploration by RNA-seq in Platycodon grandiflorum.

Platycodon grandiflorum, generally known as the bellflower or balloon flower, is the only species in the genus Platycodon of the family Campanulaceae. Platycodon plants have been traditionally used as a medicinal crop in East Asia for their antiphlogistic, antitussive, and expectorant properties. Despite these practical uses, marker-assisted selection and molecular breeding in platycodons have lagged due to the lack of genetic information on this genus. In this study, we performed RNA-seq analysis of three platycodon accessions to develop molecular markers and explore genetic diversity. First, genic simple sequence repeats (SSRs) were retrieved and compared; dinucleotide motifs were the most abundant repeats (39%-40%) followed by trinucleotide (25%-31%), tetranucleotide (1.5%-1.9%), and pentanucleotide (0.3%-1.0%) repeats. The result of in silico SSR analysis, three SSR markers were detected and showed possibility to distinguish three platycodon accessions. After several filtering procedures, 180 single nucleotide polymorphisms (SNPs) were used to design 40 cleaved amplified polymorphic sequence (CAPS) markers. Twelve of these PCR-based markers were validated as highly polymorphic and utilized to investigate genetic diversity in 21 platycodon accessions collected from various regions of South Korea. Collectively, the 12 markers yielded 35 alleles, with an average of 3 alleles per locus. Polymorphism information content (PIC) values ranged from 0.087 to 0.693, averaging 0.373 per locus. Since platycodon genetics have not been actively studied, the sequence information and the DNA markers generated from our research have the potential to contribute to further genetic improvements, genomic studies, and gene discovery in this genus.

Infection processes of xylem-colonizing pathogenic bacteria: possible explanations for the scarcity of qualitative disease resistance genes against them in crops.

KEY MESSAGE: Disease resistance against xylem-colonizing pathogenic bacteria in crops. Plant pathogenic bacteria cause destructive diseases in many commercially important crops. Among these bacteria, eight pathogens, Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, Erwinia amylovora, Pantoea stewartii subsp. stewartii, Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. actinidiae, and Xylella fastidiosa, infect their host plants through different infection sites and paths and eventually colonize the xylem tissues of their host plants, resulting in wilting symptoms by blocking water flow or necrosis of xylem tissues. Noticeably, only a relatively small number of resistant cultivars in major crops against these vascular bacterial pathogens except X. oryzae pv. oryzae have been found or generated so far, although these pathogens threaten productivity of major crops. In this review, we summarize the lifestyles of major xylem-colonizing bacterial pathogens and then discuss the progress of current research on disease resistance controlled by qualitative disease resistance genes or quantitative trait loci against them. Finally, we propose infection processes of xylem-colonizing bacterial pathogens as one of possible reasons for why so few qualitative disease resistance genes against these pathogens have been developed or identified so far in crops.

Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST).

Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.

Tobacco etch virus infectivity in Capsicum spp. is determined by a maximum of three amino acids in the viral virulence determinant VPg.

Potyvirus resistance in Capsicum spp. has been attributed to amino acid substitutions at the pvr1 locus that cause conformational shifts in eukaryotic translation initiation factor eIF4E. The viral genome-linked protein (VPg) sequence was isolated and compared from three Tobacco etch virus (TEV) strains, highly aphid-transmissible (HAT), Mex21, and N, which differentially infect Capsicum genotypes encoding Pvr1(+), pvr1, and pvr1(2). Viral chimeras were synthesized using the TEV-HAT genome, replacing HAT VPg with Mex21 or N VPg. TEV HAT did not infect pepper plants homozygous for either the pvr1 or pvr1(2) allele. However, the novel chimeric TEV strains, TEVHAT(Mex21-VPg) and TEV-HAT(N-VPg), infected pvr1 and pvr1(2) pepper plants, respectively, demonstrating that VPg is the virulence determinant in this pathosystem. Three dimensional structural models predicted interaction between VPg and the susceptible eIF4E genotype in every case, while resistant genotypes were never predicted to interact. To determine whether there is a correlation between physical interaction of VPg with eIF4E and infectivity, the effects of amino acid variation within VPg were assessed. Interaction between pvr1(2) eIF4E and N VPg was detected in planta, implying that the six amino acid differences in N VPg relative to HAT VPg are responsible for restoring the physical interaction and infectivity.

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.

Two virulence determinants of type III effector AvrPto are functionally conserved in diverse Pseudomonas syringae pathovars.

*The Pseudomonas syringae pv. tomato type III effector protein AvrPto has two functional domains that contribute additively to its ability to promote pathogen virulence in susceptible tomato plants and also defense responses in resistant tomato and tobacco genotypes. Here, we test the hypothesis that key amino acid residues in these two domains will be conserved even in sequence-divergent AvrPto proteins expressed by diverse P. syringae pathovars. *We cloned avrPto homologs from diverse P. syringae pathovars and characterized the four most diverse homologs from P. syringae pathovars mori, lachrymans, myricae and oryzae for their virulence activity and ability to elicit resistance in tomato and tobacco. *Key residues within the two AvrPto domains are conserved in three of the four homologs and are required for virulence activity and defense elicitation. AvrPto(oryzae), lacks conserved residues in each domain, but was found to be recognized by a previously unknown resistance gene in both tomato and tobacco. *Our results indicate that the two virulence domains of AvrPto are conserved in diverse pathovars despite the fact these domains are recognized by certain plant species. AvrPto may therefore function in pathovars infecting diverse plant species by targeting conserved host processes.

Phosphorylation of the Pseudomonas syringae effector AvrPto is required for FLS2/BAK1-independent virulence activity and recognition by tobacco.

The type III effector protein AvrPto from Pseudomonas syringae pv. tomato is secreted into plant cells where it promotes bacterial growth and enhances symptoms of speck disease on susceptible tomato plants. The virulence activity of AvrPto is due, in part, to its interaction with components of host pattern recognition receptor complexes, which disrupts pathogen-associated molecular pattern-triggered immunity. This disruption mechanism requires a structural element of the AvrPto protein, the CD loop, which is also required for triggering Pto/Prf-mediated resistance in tomato. We have shown previously that the carboxyl-terminal domain (CTD) of AvrPto is phosphorylated and also contributes to bacterial virulence. Here we report that phosphorylation of the CTD on S147 and S149 promotes bacterial virulence in an FLS2/BAK1-independent manner, which is mechanistically distinct from the CD loop. In a striking corollary with Pto recognition of the CD loop in tomato, the tobacco species Nicotiana sylvestris and Nicotiana tabacum have a recognition mechanism that specifically detects the phosphorylation status of the CTD. Thus different species in the Solanaceae family have evolved distinct recognition mechanisms to monitor the same type III effector.

Double mutations in eIF4E and eIFiso4E confer recessive resistance to Chilli veinal mottle virus 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 F(1) plants showed resistance, and F(2) individuals segregated in a resistant-susceptible ratio of 166:21, indicating that many resistance loci were involved. Seventy-five F(2) and 329 F(3) 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.

Functional dissection of naturally occurring amino acid substitutions in eIF4E that confers recessive potyvirus resistance in plants.

Naturally existing variation in the eukaryotic translation initiation factor 4E (eIF4E) homolog encoded at the pvr1 locus in Capsicum results in recessively inherited resistance against several potyviruses. Previously reported data indicate that the physical interaction between Capsicum-eIF4E and the viral genome-linked protein (VPg) is required for the viral infection in the Capsicum-Tobacco etch virus (TEV) pathosystem. In this study, the potential structural role(s) of natural variation in the eIF4E protein encoded by recessive resistance alleles and their biological consequences have been assessed. Using high-resolution three-dimensional structural models based on the available crystallographic structures of eIF4E, we show that the amino acid substitution G107R, found in many recessive plant virus resistance genes encoding eIF4E, is predicted to result in a substantial modification in the protein binding pocket. The G107R change was shown to not only be responsible for the interruption of VPg binding in planta but also for the loss of cap binding ability in vitro, the principal function of eIF4E in the host. Overexpression of the Capsicum-eIF4E protein containing the G107R amino acid substitution in Solanum lycopersicum indicated that this polymorphism alone is sufficient for the acquisition of resistance against several TEV strains.

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.

Megaprimer-mediated domain swapping for construction of chimeric viruses.

Clones that encode viral genomes constructed from two viruses with contrasting biological properties have been widely used in studies of viral-host interactions, particularly when the objective is to determine the identity of the viral component recognized by the host in a resistant response, known as the avirulence factor. This paper presents an efficient method based on megaprimer-mediated domain swapping for the construction of clones encoding chimeric viral genomes as a versatile and widely applicable alternative to conventional restriction enzyme digestion and ligation methods. Potato virus X (PVX)-derived vectors expressing genes encoding fluorescent proteins were used to demonstrate this concept. The cyan fluorescent protein (CFP) gene was cloned into a binary PVX vector and subsequently replaced with the yellow fluorescent protein (YFP) gene using the megaprimer amplification reaction. DNA fragments up to 1480 bp could be replaced efficiently and quickly. Most viral clones showed the expected change in phenotype without altered infectivity. Sequence analysis revealed mutations were not introduced into the four domain-swapped plasmids. This approach will provide a valuable tool for determining which domains of a viral genome are essential for infectivity, avirulence, or otherwise determine biologically significant properties of plant viruses.

Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum.

Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 (1), and pvr1 (2). These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.

Genetics of plant virus resistance.

Genetic resistance to plant viruses has been used for at least 80 years to control agricultural losses to viral diseases. To date, hundreds of naturally occurring genes for resistance to plant viruses have been reported from studies of both monocot and dicot crops, their wild relatives, and the plant model, Arabidopsis. The isolation and characterization of a few of these genes in the past decade have resulted in detailed knowledge of some of the molecules that are critical in determining the outcome of plant viral infection. In this chapter, we have catalogued genes for resistance to plant viruses and have summarized current knowledge regarding their identity and inheritance. Insofar as information is available, the genetic context, genomic organization, mechanisms of resistance and agricultural deployment of plant virus resistance genes are also discussed.

The pvr1 locus in Capsicum encodes a translation initiation factor eIF4E that interacts with Tobacco etch virus VPg.

Mutations in the eIF4E homolog encoded at the pvr1 locus in Capsicum result in broad-spectrum potyvirus resistance attributed to the pvr1 resistance allele, a gene widely deployed in agriculture for more than 50 years. We show that two other resistance genes, previously known to be eIF4E with narrower resistance spectra, pvr2(1) and pvr2(2), are alleles at the pvr1 locus. Based on these data and current nomenclature guidelines, we have re-designated these alleles, pvr1(1) and pvr1(2), respectively. Point mutations in pvr1, pvr1(1), and pvr1(2) grouped to similar regions of eIF4E and were predicted by protein homology models to cause conformational shifts in the encoded proteins. The avirulence determinant in this potyvirus system has previously been identified as VPg, therefore yeast two-hybrid and GST pull-down assays were carried out with proteins encoded by the pvr1 alleles and VPg from two different strains of Tobacco etch virus (TEV) that differentially infected Capsicum lines carrying these genes. While the protein encoded by the susceptible allele pvr1+ interacted strongly, proteins translated from all three resistance alleles (pvr1, pvr1(1), and pvr1(2)) failed to bind VPg from either strain of TEV. This failure to bind correlated with resistance or reduced susceptibility, suggesting that interruption of the interaction between VPg and this eIF4E paralog may be necessary, but is not sufficient for potyvirus resistance in vivo. Among the three resistance alleles, only the pvr1 gene product failed to bind m7-GTP cap-analog columns, suggesting that disrupted cap binding is not required for potyvirus resistance.