[Show abstract][Hide abstract] ABSTRACT: Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.
[Show abstract][Hide abstract] ABSTRACT: Tomato spotted wilt virus (TSWV) causes one of the most destructive viral diseases that threatens global tomato production. Sw-5b was reported, as the resistance gene effective against TSWV. The objective of this research was to develop a single-nucleotide polymorphism (SNP) marker to distinguish tomato cultivars resistant to TSWV from susceptible cultivars for marker-assisted breeding. First, we determined genotypes for TSWV resistance in 32 commercial tomato cultivars using the previously reported Sw-5b gene-based marker. Then, DNA sequences of Sw-5b alleles in tomato cultivars showing resistant or susceptible genotypes were analyzed; a single SNP was found to distinguish tomato cultivars resistant to TSWV from susceptible cultivars. Based on the confirmed SNP, a SNP primer pair was designed. Using this new SNP sequence and high-resolution melting analysis, the same 32 tomato cultivars were screened. The results were perfectly correlated with those from screening with the Sw-5b gene-based marker. These results indicate that the SNP maker developed in this study will be useful for better tracking of resistance to TSWV in tomato breeding.
No preview · Article · Oct 2015 · Wonye kwahak kisulchi = Korean journal of horticultural science and technology /
[Show abstract][Hide abstract] ABSTRACT: The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bβ). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bβ- or eEF1Bɣ-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bβ interacted with eEF1A and that eEF1A and eEF1Bβ interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bβ play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bβ deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bβ are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bβ is involved in the interaction with eEF1A. These results suggest that eEF1Bβ could be a potential target for engineering virus-resistant plants.
[Show abstract][Hide abstract] ABSTRACT: Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species.
[Show abstract][Hide abstract] ABSTRACT: The protein-protein interaction between VPg proteins of potyviruses and eIF4E or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected based on other previous results and comparison of protein models of cap-binding pockets and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine if these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of TuMV resistance of T1 and T2 transformants demonstrated that over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for engineering broad-spectrum resistance in Chinese cabbage.
No preview · Article · Jan 2014 · Molecular Plant Pathology
[Show abstract][Hide abstract] ABSTRACT: Two Chinese cabbage (Brassica rapa L. ssp. pekinensis) lines resistant to Turnip mosaic virus (TuMV) CHN5 were identified and found to have broad-spectrum resistance against three other TuMV strains (CHN2, 3, and 4). Genetic analysis indicated that this TuMV resistance is recessive, and a candidate gene approach was used to identify the resistance gene, which we named trs (TuMV resistance discovered at Seoul National University). Based on previous research in Arabidopsis showing that mutations in eIF(iso)4E determine TuMV resistance, the eIF(iso)4E gene was selected as a candidate for the trs gene in Brassica rapa. Three copies of eIF(iso)4E, Braiso4Ea, Braiso4Eb, and Braiso4Ec, were amplified, and polymorphisms between resistant and susceptible lines were analyzed. Sequence polymorphisms were found in Braiso4Ea and Braiso4Eb; in contrast, no sequence differences were found in Braiso4Ec between resistant and susceptible lines. A CAPS marker developed to test the linkage between Braiso4Eb and TuMV resistance displayed no linkage. A SCAR marker, trsSCAR, developed using allele-specific deletions and SNPs in Braiso4Ea, did co-segregate perfectly with trs in three F
2 populations. However, the presence or absence of the Braiso4Ea sequence deletion was not consistent between resistant lines and susceptible lines, indicating that Braiso4Ea is not the actual resistance gene. Results from mapping analysis indicated that the trs is located at chromosome A04, between scaffold 000104 and scaffold 040552. This location demonstrated that trs may be another recessive resistance gene tightly linked to retr02 or another allele. The molecular markers developed in this study will be useful for breeding durable resistance.
Full-text · Article · Aug 2013 · Molecular Breeding
[Show abstract][Hide abstract] ABSTRACT: A pepper genetic map was constructed from F2 mapping population of 93 individuals from a cross between Capsicum annuum ‘F68’ and C. chinense ‘Habanero’. Surrey was made for the map distribution and polymorphism level of these marker groups; reverse random amplification microsatellite polymorphism (rRAMP), WRKY and amplified fragment length polymorphisms (AFLP). A total of 912 molecular markers [356 rRAMP, 190 WRKY, 305 AFLP, and 61 simple sequence repeats (SSR)] were developed in this study. The rRAMP and WRKY markers were more evenly scattered in the pepper genome than the AFLP and SSR markers, and filled the gaps not populated by the other markers. The interspecific pepper map contained 28 linkage groups with 625 linked markers and covered 3377.2 cM with an average interval of 5.9 cM. On the basis of the map, the fruit length quantitative trait locus (QTL) was analyzed and these QTL regions were detected near rRAMP and WRKY markers on the chromosome 3, 5, 11, and LG3. These marker system, map information, and detected QTLs could be one of basic information for pepper research.
Full-text · Article · Dec 2012 · Horticulture, Environment and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: The Cmr1 gene in peppers confers resistance to Cucumber mosaic virus isolate-P0 (CMV-P0). Cmr1 restricts the systemic spread of CMV strain-Fny (CMV-Fny), whereas this gene cannot block the spread of CMV isolate-P1 (CMV-P1) to the upper leaves, resulting in systemic infection. To identify the virulence determinant of CMV-P1, six reassortant viruses and six chimeric viruses derived from CMV-Fny and CMV-P1 cDNA clones were used. Our results demonstrate that the C-terminus of the helicase domain encoded by CMV-P1 RNA1 determines susceptibility to systemic infection, and that the helicase domain contains six different amino acid substitutions between CMV-Fny and CMV-P1(.) To identify the key amino acids of the helicase domain determining systemic infection with CMV-P1, we then constructed amino acid substitution mutants. Of the mutants tested, amino acid residues at positions 865, 896, 957, and 980 in the 1a protein sequence of CMV-P1 affected the systemic infection. Virus localization studies with GFP-tagged CMV clones and in situ localization of virus RNA revealed that these four amino acid residues together form the movement determinant for CMV-P1 movement from the epidermal cell layer to mesophyll cell layers. Quantitative real-time PCR revealed that CMV-P1 and a chimeric virus with four amino acid residues of CMV-P1 accumulated more genomic RNA in inoculated leaves than did CMV-Fny, indicating that those four amino acids are also involved in virus replication. These results demonstrate that the C-terminal region of the helicase domain is responsible for systemic infection by controlling virus replication and cell-to-cell movement. Whereas four amino acids are responsible for acquiring virulence in CMV-Fny, six amino acid (positions at 865, 896, 901, 957, 980 and 993) substitutions in CMV-P1 were required for complete loss of virulence in 'Bukang'.
[Show abstract][Hide abstract] ABSTRACT: The well-conserved NBS domain of resistance (R) genes cloned from many plants allows the use of a PCR-based approach to isolate resistance gene analogs (RGAs). In this
study, we isolated an RGA (CapRGC) from Capsicum annuum “CM334” using a PCR-based approach. This sequence encodes a protein with very high similarity to Rx genes, the Potato Virus X (PVX) R genes from potato. An evolutionary analysis of the CapRGC gene and its homologs retrieved by an extensive search of a Solanaceae database provided evidence that Rx-like genes (eight ESTs or genes that show very high similarity to Rx) appear to have diverged from R1 [an NBS-LRR R gene against late blight (Phytophthora infestans) from potato]-like genes. Structural comparison of the NBS domains of all the homologs in Solanaceae revealed that one novel
motif, 14, is specific to the Rx-like genes, and also indicated that several other novel motifs are characteristic of the R1-like genes. Our results suggest that Rx-like genes are ancient but conserved. Furthermore, the novel conserved motifs can provide a basis for biochemical structural–function
analysis and be used for degenerate primer design for the isolation of Rx-like sequences in other plant species. Comparative mapping study revealed that the position of CapRGC is syntenic to the locations of Rx and its homolog genes in the potato and tomato, but cosegregation analysis showed that CapRGC may not be the R gene against PVX in pepper. Our results confirm previous observations that the specificity of R genes is not conserved, while the structure and function of R genes are conserved. It appears that CapRGC may function as a resistance gene to another pathogen, such as the nematode to which the structure of CapRGC is most similar.
–Solanaceae–Resistance gene analogs (RGAs)–Evolution
No preview · Article · Oct 2011 · Plant Biotechnology Reports
[Show abstract][Hide abstract] ABSTRACT: Tobamovirus is one of most destructive viruses in Capsicum. Accordingly, the L locus, a resistance gene against tobamoviruses, has been used for pepper breeding programs. Previously, the L
gene, one of the L alleles, was isolated through map-based cloning, and a L
gene candidate was isolated by homology-based PCR methods. Here, the L4segF&R marker was developed based on the leucine-rich repeat (LRR) region of the L
candidate, and co-segregation analysis was performed using two L
-segregating F2 populations derived from the commercial cultivars Special and Myoung-sung. The L4segF&R marker was located within 0.3 cM of the L
gene but did not completely co-segregate with the L
gene, indicating that the candidate is not actually L
. To confirm the mapping result, L4segF&R genotypes of L
-containing breeding lines from three different seed companies were analyzed, resulting in the identification of several recombinants in the breeding lines. Based on these results, we postulate several genetic models that show different introgression histories and genetic structures for the L
-containing segment in different breeding lines. All of the models demonstrate that resistance conferred by the L
segment could not be explained by the L
gene candidate alone. Although the presence of the L
gene candidate could not fully explain the L
resistance, we were able to develop allele-specific markers for the L locus using the candidate sequence. To develop allele-specific markers for the L locus, HRM analysis was performed using primer pairs based on the LRR sequence of the L
gene candidate. When commercial breeding lines homozygous for L
were analyzed, L4RP-3F/L4RP-3R correctly detected the L allele in 90 out of 91 lines. We believe that the L allele-specific marker developed in the study provides a solution for pepper breeders developing improved resistance lines against tobamoviruses.
No preview · Article · Aug 2011 · Molecular Breeding
[Show abstract][Hide abstract] ABSTRACT: In plants, the primary defense against pathogens is mostly inducible and associated with cell wall modification and defense-related gene expression, including many secreted proteins. To study the role of secreted proteins, a yeast-based signal-sequence trap screening was conducted with the RNA from Phytophthora capsici-inoculated root of Capsicum annuum 'Criollo de Morelos 334' (CM334). In total, 101 Capsicum annuum secretome (CaS) clones were isolated and identified, of which 92 were predicted to have a secretory signal sequence at their N-terminus. To identify differences in expressed CaS genes between resistant and susceptible cultivars of pepper, reverse Northern blots and real-time reverse-transcription polymerase chain reaction were performed with RNA samples isolated at different time points following P. capsici inoculation. In an attempt to assign biological functions to CaS genes, we performed in planta knock-down assays using the Tobacco rattle virus-based gene-silencing method. Silencing of eight CaS genes in pepper resulted in suppression of the cell death induced by the non-host bacterial pathogen (Pseudomonas syringae pv. tomato T1). Three CaS genes induced phenotypic abnormalities in silenced plants and one, CaS259 (PR4-l), caused both cell death suppression and perturbed phenotypes. These results provide evidence that the CaS genes may play important roles in pathogen defense as well as developmental processes.
Full-text · Article · Jun 2011 · Molecular Plant-Microbe Interactions
[Show abstract][Hide abstract] ABSTRACT: Cucumber mosaic virus (CMV) is one of the most destructive viruses in the Solanaceae family. Simple inheritance of CMV resistance in peppers has not previously been documented; all previous studies have reported that resistance to this virus is mediated by several partially dominant and recessive genes. In this study, we showed that the Capsicum annuum cultivar 'Bukang' contains a single dominant resistance gene against CMV(Korean) and CMV(FNY) strains. We named this resistance gene Cmr1 (Cucumber mosaic resistance 1). Analysis of the cellular localization of CMV using a CMV green fluorescent protein construct showed that in 'Bukang,' systemic movement of the virus from the epidermal cell layer to mesophyll cells is inhibited. Genetic mapping and FISH analysis revealed that the Cmr1 gene is located at the centromeric region of LG2, a position syntenic to the ToMV resistance locus (Tm-1) in tomatoes. Three SNP markers were developed by comparative genetic mapping: one intron-based marker using a pepper homolog of Tm-1, and two SNP markers using tomato and pepper BAC sequences mapped near Cmr1. We expect that the SNP markers developed in this study will be useful for developing CMV-resistant cultivars and for fine mapping the Cmr1 gene.
No preview · Article · Feb 2010 · Theoretical and Applied Genetics
[Show abstract][Hide abstract] ABSTRACT: In pepper, the TMV resistance locus L is syntenic to the tomato I2 and the potato R3 loci on chromosome 11. In this report, we identified pepper bacterial artificial chromosome (BAC) clones corresponding to
the I2 and R3 loci and developed L-linked markers using the BAC sequence information. A BAC library was screened using the tomato I2C-1 gene as a probe. The resulting clones were sorted further by PCR screening, sequencing, and genetic mapping. A linkage analysis
revealed that BAC clone 082F03 could be anchored to the target region near TG36 on chromosome 11. Using the 082F03 sequence,
more BAC clones were identified and a BAC contig spanning 224kb was constructed. Gene prediction analysis showed that there
were at least three I2/R3 R gene analogs (RGAs) in the BAC contig. Three DNA markers closely linked (about 1.2cM) to the L
gene were developed by using the BAC contig sequence. The single nucleotide polymorphism marker 087H3T7 developed in this
study was subjected to linkage analysis in L
- and L
-segregating populations together with previously developed markers. The 189D23M marker, which is known to co-segregate with
, was located on the opposite side of 087H3T7, about 0.7cM away from L
. This supports the idea that L
may be different genes closely linked within the region instead of different alleles at the same locus. Finally, use of flanking
markers in molecular breeding program for introgression of L
to elite germplasm against most aggressive tobamoviruses pathotype P1,2,3 is discussed.
No preview · Article · Nov 2009 · Molecular Breeding
[Show abstract][Hide abstract] ABSTRACT: Microsatellites or simple sequence repeats (SSR) are widely distributed in eukaryotic genomes and are informative genetic markers. Despite many advantages of SSR markers such as a high degree of allelic polymorphisms, co-dominant inheritance, multi-allelism, and genome-wide coverage in various plant species, they also have shortcomings such as low polymorphic rates between genetically close lines, especially in Capsicum annuum. We developed an alternative technique to SSR by normalizing and alternating anchored primers in random amplified microsatellite polymorphisms (RAMP). This technique, designated reverse random amplified microsatellite polymorphism (rRAMP), allows the detection of nucleotide variation in the 3' region flanking an SSR using normalized anchored and random primer combinations. The reproducibility and frequency of polymorphic loci in rRAMP was vigorously enhanced by translocation of the 5' anchor of repeat sequences to the 3' end position and selective use of moderate arbitrary primers. In our study, the PCR banding pattern of rRAMP was highly dependent on the frequency of repeat motifs and primer combinations with random primers. Linkage analysis showed that rRAMP markers were well scattered on an intra-specific pepper map. Based on these results, we suggest that this technique is useful for studying genetic diversity, molecular fingerprinting, and rapidly constructing molecular maps for diverse plant species.
Full-text · Article · May 2008 · Molecules and Cells
[Show abstract][Hide abstract] ABSTRACT: Tomato spotted wilt virus (TSWV) is an important viral disease affecting pepper production worldwide. A single dominant resistance gene to TSWV, Tsw, has been known to originate from Capsicum chinense. Tsw has been mapped on chromosome 10 in Capsicum. To perform genome-based fine mapping of the Tsw gene, approaches of mapping comparison, pooled transcriptome analysis, and genome walking were applied. Eleven SNPs tightly linked to the Tsw gene were developed using tomato and pepper whole genome sequencing databases. Among them, four SNP markers, SNP7715-1, SNP68-1, SNP17918-1, and SNP1072-1, showed no recombination in two segregating populations of F2 ‘Telmo’ (210 individuals) and ‘SP’ (843 individuals). Three scaffold sequences from the pepper genome database and two BAC clones from the BAC library of C. annuum ‘CM334’ covering the Tsw gene were identified by transcriptome analysis and genome walking. The region of Tsw was delimited within 259 kb by alignment analysis of two BAC clone sequences and the pepper scaffold sequence. A total of 22 predicted genes were resided in the target. Among them, eight predicted genes showing annotations of NBS-LRR resistance proteins were identified in genome of C.annuum ‘CM334’. To confirm accurate sequence of candidate genes in target, eight genes were blasted to WGS of C. chinense. The homolog sequences of seven candidate genes out of eight genes were identified in C. chinense.