Byoung-Cheorl Kang

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.

QTL analysis for capsaicinoid content in Capsicum

Pungency or “heat” found in Capsicum fruit results from the biosynthesis and accumulation of alkaloid compounds known as capsaicinoids in the dissepiment, placental tissue adjacent to the seeds. Pepper cultivars differ with respect to their level of pungency because of quantitative and qualitative variation in capsaicinoid content. We analyzed the segregation of three capsaicinoids: capsaicin, dihydrocapsaicin and nordihydrocapsaicin in an inter-specific cross between a mildly pungent Capsicum annuum ‘NuMex RNaky’ and the wild, highly pungent C. frutescens accession BG2814-6. F3 families were analyzed in three trials in California and in Israel and a dense molecular map was constructed comprised mostly of loci defined by simple sequence repeat (SSR) markers. Six QTL controlling capsaicinoid content were detected on three chromosomes. One gene from the capsaicinoid biosynthetic pathway, BCAT, and one random fruit EST, 3A2, co-localized with QTL detected in this study on chromosomes 3 and 4. Because one confounding factor in quantitative determination of capsaicinoid is fruit size, fruit weight measurements were taken in two trials. Two QTL controlling fruit weight were detected, however, they did not co-localize with QTL detected for capsaicinoid content. The major contribution to the phenotypic variation of capsaicinoid content (24–42% of the total variation) was attributed to a digenic interaction between a main-effect QTL, cap7.1, and a marker located on chromosome 2 that did not have a main effect on the trait. A second QTL, cap7.2 is likely to correspond to the QTL, cap, identified in a previous study as having pronounced influence on capsaicinoid content.

A GH3-like gene, CcGH3, isolated from Capsicum chinense L. fruit is regulated by auxin and ethylene*

Auxin, which has been implicated in multiple biochemical and physiological processes, elicits three classes of genes (Aux/IAAs, SAURs and GH3s) that have been characterized by their early or primary responses to the hormone. A new GH3-like gene was identified from a suppressive subtraction hybridization (SSH) library of pungent pepper (Capsicum chinense L.) cDNAs. This gene, CcGH3, possessed several auxin- and ethylene-inducible elements in the putative promoter region. Upon further investigation, CcGH3 was shown to be auxin-inducible in shoots, flower buds, sepals, petals and most notably ripening and mature pericarp and placenta. Paradoxically, this gene was expressed in fruit when auxin levels were decreasing, consistent with ethylene-inducibility. Further experiments demonstrated that CcGH3 was induced by endogenous ethylene, and that transcript accumulation was inhibited by 1-methylcyclopropene, an inhibitor of ethylene perception. When over-expressed in tomato, CcGH3 hastened ripening of ethylene-treated fruit. These results implicate CcGH3 as a factor in auxin and ethylene regulation of fruit ripening and suggest that it may be a point of intersection in the signaling by these two hormones.

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, pvr11, and pvr12. 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.

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.

Study on Inheritance of Potato virus X Resistance in Capsicum annuum

Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-921, Korea(Received on August 12, 2008; Accepted on November 6, 2008)Potato virus X (PVX) resistance in potato is one of thebest-characterized resistance models, however little isknown in pepper. To evaluate the resistance to PVX inCapsicum annuum, a total of eleven pepper accessionswere used for resistance screening against two PVXstrains, USA and UK3. None of them were resistantagainst strain UK3, whereas four resistant genotypeswere found against strain USA, three of which werefurther characterized. Two unlinked dominant geneswere identified for both genotypes Bukang andPerennial; resistance in the genotype CV3 seemed to beconferred by two complementary dominant genes.These results demonstrated that the resistance to PVXin C. annuum is different from that in potato. This is thefirst report on genetic analysis of PVX resistance in C.annuum. Keywords : Capsicum annuum, inheritance, Potato virus X,resistancePotato virus X (PVX), a flexuous rod-shaped virus con-taining a 6.4 kb plus-stranded RNA genome, is the typemember of the potexviruses (Park et al., 2006). Firstlyreported in potato (Solanum tuberosum) by Smith in theU.K in 1931 (Markham, 1977), PVX causes significantdamages in many economically important crops includingpotato, tobacco, and pepper. The virus can be easilytransmitted by mechanical inoculation and contact betweenplants in nature. Different PVX strains induce quitedifferent symptoms on various hosts. For instance, somePVX strains show symptomless while others inducenecrotic streaks in Solanum tuberosum; mild mosaicmottling, distortion of leaves and plant stunting symptomsare observed in Brassica rapa (http://image.fs.uidaho.edu/vide/refs.htm). PVX and its interaction with resistance genes have beenvery well characterized in potato (Solomon-Blackburn etal., 2001). Two types of resistance response have beenidentified in potato: hypersensitive resistance (HR) andextreme resistance (ER) (Cockerham, 1970). It has beenknown that HR is associated with prevention of virusmovement (Malcuit et al., 1999) whereas ER acts oninhibition of virus accumulation (Kohm et al., 1993). Bothtypes of resistance genes are inherited in a monogenicdominant fashion (Solomon-Blackburn et al., 2001). Nxand Nb, related to HR, are located on the long arm ofchromosome IX (Tommiska et al., 1998) and in the middleof chromosome V, respectively (Marano et al., 2002). TwoER type Rx genes, Rx1 and Rx2, have been cloned(Bendahmane et al., 1999; Moffett et al., 2002) and mappedto chromosome XII and V, respectively (Ritter et al., 1991;Bendahmane et al., 1997).Two kinds of methods have been used to classify PVXstrains primarily based on their ability to overcome resis-tance genes. Cockerham (1954) divided PVX strains intofour groups according to their compatibility with twodominant genes, Nx and Nb (Cockerham 1954; Kavanaghet al., 1992). The group 1 strain cannot infect plants havingeither Nx or Nb. The group 2 strains can only infects plantswith Nx, while the group 3 can infect plants with Nb. Andthe group 4 contains the widest host range that can inducesystemic infection in plants containing Nx and Nb genes.But Rx1 and Rx2 confer resistance to all four groups ofvirus. The second method classifies PVX strains into twogroup, designated as type X and B, by the coat protein (CP)amino acid identity and the avirulence on potato genotypescarrying the Nx resistance gene (Santa et al., 1995). Thereare 14 amino acid differences between type X and B. Thetype B coat proteins are capable of overcoming the Nxresistance. In fact, most strains of group 1 and 3 areincluded into type X and group 2 and 4 are into type B.Korean PVX strains, such as PVX-KR, PVX-KO1 andPVX-KO2, have been isolated previously. PVX-KO1 andPVX-KO2 have been assigned to the type X and noclassification analysis of PVX-KR has been performed, butthis strains has very high homolog with PVX-KO1 andPVX-KO2 (Jung et al., 2000). Although there have been many studies on PVX resis-tance in potato, little is known for PVX resistance in pepperwhich is one of the most importance fruit vegetable crops inKorea. Although several pepper accessions showing resis-

Evaluation of Resistance in Pepper Germplasm to Cucumber mosaic virus by High Resolution Melting Analysis

In this study, total number of 1941 Capsicum accessions conserved at RDA Genebank was evaluated for their response to Cucumber mosaic virus (CMV). These accessions were composed with 9 species originated from 89 countries, included 839 Capsicum annuum, 277 C. baccatum, 395 C. chinense, 343 C. frutescens, 49 C. pubescens, and other 38 wild pepper species (C. chacoense, C. galapagoense, etc.). Resistant to CMV was screened with the 240H02SP6 SNP marker related to the Cmr1 (Cucumber mosaic resistance 1). Eighty nine accessions of pepper germplasm were resistant to CMV based on the marker. One hundred sixty two accessions showed heterozygosity. One thousand two hundred seventy accessions were susceptible to CMV. Four hundred twenty accessions did not show distinction by 240H02SP6 marker. These 89 resistant pepper germplasm can be used in a pepper breeding program against CMV.

A New Nonsense Mutation in Capsanthin/Capsorubin Synthase Controlling Orange Pepper Fruit

Carotenoids are plant pigments that play a major role in conferring fruit color. Carotenoid content is often controlled by genetic variation in the biosynthetic genes. The color of mature pepper fruit is mainly classified as red, orange, and yellow. Orange and yellow fruit colors are determined by mutations in phytoene synthase (Psy) and capsanthin-capsorubin synthase (Ccs), respectively. In contrast to the current fruit color model, we hypothesized that genetic variation in Ccs also controls orange fruit color in pepper. Ripe fruit of Capsicum annuum ‘K146465’ is orange, and its carotenoid profile obtained by HPLC analysis showed a lack of the major pepper carotenoid capsanthin but an abundance of lutein, zeaxanthin, and β-carotene compared to red pepper. cDNA cloning and sequencing analysis detected a new nonsense mutation due to a T insertion in the coding region of Ccs, but no DNA sequence variation in Psy. We developed a derived cleaved amplified polymorphic sequence (dCAPS) marker to distinguish the nonsense mutation in Ccs. Genetic analysis of the F2 population derived from C. annuum ‘Sweet Banana’ (red fruit color) × C. annuum ‘K146465’ revealed that orange fruit color is determined by a single recessive gene. The nonsense mutation in Ccs distinguished by the dCAPS marker cosegregated with orange fruit color in the F2 population. This germplasm, coupled with the dCAPS marker and carotenoid profiling, will facilitate marker-assisted breeding to select orange fruit color and improve lutein, zeaxanthin, and β-carotene levels in pepper. OPEN ACCESS Received:

Genome-Wide Analysis and Evolution of the Pto-Like Protein Kinase (PLPK) Gene Family in Pepper.

The tomato Pto gene, which encodes a serine/threonine kinase (STK) domain-containing protein, confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato (Pst). In this study, in vivo recognition assays using PVX constructs showed that AvrPto was specifically recognized in the pepper genotypes. This AvrPto recognition caused a nonhost hypersensitive response (HR) and localization of the PVX::AvrPto fusion protein to inoculated pepper leaf tissues, which indicates the presence of a similar Pto recognition mechanism in pepper as in tomato. However, genome-wide analysis in pepper revealed no Pto clade corresponding to that in tomato, suggesting an alternative system for Pto recognition in pepper. Nevertheless, 25 Pto-like protein kinases (PLPKs) with a highly conserved STK domain have been identified in the pepper genome. For the majority of the amino acid sites in the STK domain of Ptos and PLPKs, nonsynonymous (dN) to synonymous (dS) nucleotide substitution ratios (ω) were less than one, suggesting that purifying selection played a predominant role in the evolutionary process. However, some amino acid sites were found to be subjected to episodic positive selection in the course of evolution of Pto homologs, and, thus, different evolutionary processes might have shaped the Pto gene family in plants. Based on RNA-seq data, PLPK genes and other Pto pathway genes, such as Prf, Pti1, Pti5, and Pti6 were expressed in all tested pepper genotypes. Therefore, the nonhost HR against Pst in pepper may be due to the recognition of the AvrPto effector by a PLPK homolog, and subsequent action of downstream components of the Pto signaling pathway. However, the possibility remains that the recognition of AvrPto in pepper plants may involve activities of other receptor like kinases (RLKs). The identification of the PLPKs in this study will serve as a foundation for further efforts to understand the roles of PLPKs in nonhost resistance.