A hot pepper cDNA encoding a pathogenesis-related protein 4 is induced during the resistance response to tobacco mosaic virus

Hot pepper (Capsicum annuum) plants exhibit a hypersensitive response (HR) against infection by many tobamoviruses. A clone (CaPR-4) encoding a putative pathogenesis-related protein 4 was isolated by differential screening of a cDNA library prepared from resistant pepper plant leaves inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaPR-4 is very similar to those of other plant PR-4s. Southern blot analysis showed that small gene families of PR-4-related sequences were present in the pepper genome. Hot pepper cultivar Bugang, resistant to TMV-P0 and susceptible to TMV-P1.2, induced CaPR-4 expression by pathotype P0 inoculation in inoculated and systemic leaves, but not by pathotype P1.2. Effects of exogenously applied abiotic elicitors upon the CaPR-4 expression were also examined. The expression of the CaPR-4 gene was stimulated by methyl jasmonate (MeJA), ethephon and wounding treatment. However, application of salicylic acid (SA) did not trigger the expression. Evidence is emerging that jasmonic acid and ethylene play key roles in the SA-independent pathways of plant-pathogen interaction. Taken together, these results suggest that the CaPR-4 gene is one of the defense-related genes conferring resistance on pepper plants by the SA-independent pathway and the cross-talk between signaling compounds, jasmonic acid and ethylene could have a great regulatory potential in a plant's defense against TMV.     

Functional study of hot pepper 26S proteasome subunit RPN7 induced by Tobacco mosaic virus from nuclear proteome analysis

Two-dimensional gel electrophoresis (2-DE) was applied for the screening of Tobacco mosaic virus (TMV)-induced hot pepper (Capsicum annuum cv. Bugang) nuclear proteins. From differentially expressed protein spots, we acquired the matched peptide mass fingerprint (PMF) data, analyzed by MALDI-TOF MS, from the non-redundant hot pepper EST protein FASTA database using the VEMS 2.0 software. Among six identified nuclear proteins, the hot pepper 26S proteasome subunit RPN7 (CaRPN7) was subjected to further study. The level of CaRPN7 mRNA was specifically increased during incompatible TMV-P(0) interaction, but not during compatible TMV-P(1.2) interaction. When CaRPN7::GFP fusion protein was targeted in onion cells, the nuclei had been broken into pieces. In the hot pepper leaves, cell death was exacerbated and genomic DNA laddering was induced by Agrobacterium-mediated transient overexpression of CaPRN7. Thus, this report presents that the TMV-induced CaRPN7 may be involved in programmed cell death (PCD) in the hot pepper plant.     

Functional study of Capsicum annuum fatty acid desaturase 1 cDNA clone induced by Tobacco mosaic virus via microarray and virus-induced gene silencing

A series of microarray analyses employing the expressed sequence tags (ESTs) of hot pepper was conducted in an effort to elucidate the molecular mechanisms inherent to hypersensitive response (HR) by viral or bacterial pathogens. There were 2535 ESTs exhibiting differential expression (over 2-fold changes) among about 5000 ESTs during viral or bacterial response. Further, via virus-induced gene silencing (VIGS) and TMV-infection studies, we were able to isolate several ESTs, which may be relevant to defense response against TMV. Of these ESTs, Capsicum annuum fatty acid desaturase 1 (CaFAD1) showed the distinct phenotype against TMV infection and thus was subjected to further study. CaFAD1-silenced plants showed weaker resistance against TMV-P0 infection compared to TRV2 control plants. Also the suppression of FAD1 expression caused blocking of cell death induced by Bcl2-associated X (Bax) protein in tobacco plants. Therefore, this report presents that both microarray and VIGS approaches are feasible in hot pepper plants and the TMV-induced CaFAD1 plays a role in HR response.     

Pathogen-induced expression of cyclo-oxygenase homologue in hot pepper (Capsicum annuum cv. Pukang).

The hypersensitive reaction (HR) in plants is typified by a rapid and localized cell death at the site of pathogen infection. To understand better the molecular and cellular defence mechanism controlling HR, hot pepper leaves (Capsicum annuum cv. Pukang) were inoculated with the soybean pustule pathogen Xanthomonas campestris pv. glycine 8ra. By using the DD-PCR technique, a cDNA fragment was identified that exhibited a sequence similarity to the recently identified tobacco pathogen-induced oxygenase (PIOX) with homology to animal cyclo-oxygenase (COX). Subsequently, the full-length cDNA clone, pCa-COX1, encoding the COX homologue from the pathogen-inoculated hot pepper leaf cDNA library was isolated. The deduced amino acid sequence of Ca-COX1 shares 85.8% identity with tobacco PIOX and displays a significant degree of sequence identity (21.7-23.7%) with mammalian COXs. The expression of Ca-COX1 was markedly induced at 4-12 h after pathogen infection, while HR cell death on pepper leaves appeared at approximately 15 h post-inoculation. These results are consistent with the notion that the lipid-derived signalling pathway is involved in the initial response of hot pepper plants to pathogen infection.     

The CaTin1 (Capsicum annuum TMV-induced clone 1) and CaTin1-2 genes are linked head-to-head and share a bidirectional promoter

CaTin1 was expressed relatively early in the TMV-inoculated leaves of hot pepper which is resistant to TMV-P(0) infection. Interestingly, there was another homologous gene (CaTin1-2) located in front of CaTin1 in a head-to-head fashion and they shared a single promoter. The expression profile of the CaTin1-2 was very similar to CaTin1 in all the treatments except the slower induction time compared to CaTin1 upon TMV-P(0) inoculation. The promoter analysis of CaTin1 and CaTin1-2 revealed bidirectionality both in cis-elements and activity. The CaTin1-2 promoter had two TATA-boxes, four GCC-boxes, the root responsive element, and a W1-box. The ethylene-inducible promoter activity depended on GCC-boxes and TMV-inducible activity of the CaTin1-2 promoter reached its highest activity when this promoter had a W1-box.     

Capsicum annuum tobacco mosaic virus-induced clone 1 expression perturbation alters the plant's response to ethylene and interferes with the redox homeostasis

Capsicum annuum tobacco mosaic virus (TMV)-induced clone 1 (CaTin1) gene was expressed early during incompatible interaction of hot pepper (Caspsicum annuum) plants with TMV and Xanthomonas campestris. RNA-blot analysis showed that CaTin1 gene was expressed only in roots in untreated plants and induced mainly in leaf in response to ethylene, NaCl, and methyl viologen but not by salicylic acid and methyl jasmonate. The ethylene dependence of CaTin1 induction upon TMV inoculation was demonstrated by the decrease of CaTin1 expression in response to several inhibitors of ethylene biosynthesis or its action. Transgenic tobacco (Nicotiana tabacum) plants expressing CaTin1 gene in sense- or antisense-orientation showed interesting characteristics such as the accelerated growth and the enhanced resistance to biotic as well as abiotic stresses. Such characteristics appear to be caused by the elevated level of ethylene and H2O2. Moreover, in transgenic plants expressing antisense CaTin1 gene, the expression of some pathogenesis-related genes was enhanced constitutively, which may be mainly due to the increased ethylene level. The promoter of CaTin1 has four GCC-boxes, two AT-rich regions, and an elicitor-inducible W-box. The induction of the promoter activity by ethylene depends on GCC-boxes and by TMV on W-box. Taken together, we propose that the CaTin1 up-regulation or down-regulation interferes with the redox balance of plants leading to the altered response to ethylene and biotic as well as abiotic stresses.     

Virulence of Meloidogyne incognita to expression of N gene in pepper

Four pepper genotypes classified as resistant and four pepper genotypes classified as susceptible to several avirulent populations of M. incognita were compared for their reactions against a population of Meloidogyne incognita (Chitwood) Kofoid and White which had been shown to be virulent to resistant bell pepper (Capsicum annuum) in preliminary tests. The virulent population of M. incognita originated from a commercial bell pepper field in California. The resistant pepper genotypes used in all experiments were the Capsicum annuum cultivars Charleston Belle, Carolina Wonder, and Carolina Cayenne, and the C. chinense cultigen PA-426. The susceptible pepper genotypes used in the experiments were the C. annuum cultivars Keystone Resistant Giant, Yolo Wonder B, California Wonder, and the C. chinense cultigen PA-350. Root gall indices (GI) were ≥ 3.0 for all genotypes in both tests except for PA-426 (GI=2.57) in test 1 and 'Carolina Cayenne' (GI=2.83) in test 2. Numbers of eggs per gram fresh root weight ranged from 20,635 to 141,319 and reproductive indices ranged from 1.20 to 27.2 for the pepper genotypes in both tests, indicating that all eight pepper genotypes tested were susceptible to the M. incognita population used in these tests. The M. incognita population used in these studies overcame resistance conferred by the N gene in all resistant genotypes of both C. annuum and C. chinense.     

Molecular characterization of three 3-hydroxy-3-methylglutaryl-CoA reductase genes including pathogen-induced Hmg2 from pepper (Capsicum annuum)

Sesquiterpene phytoalexins, a class of plant defense metabolites, are synthesized from the cytosolic acetate/mevalonate pathway in isoprenoids biosynthetic system of plants. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the synthesis of mevalonate, which is the specific precursor of this pathway, as a multi gene family. Three kinds of cDNA clones encoding HMGR were isolated from Korean red pepper (Capsicum annuum L. cv. NocKwang) and the HMGR2 gene (Hmg2) was especially obtained from a cDNA library constructed with Phytophthora capsici-infected pepper root RNAs. The Hmg2 encoding a 604-amino-acid peptide had typical features as an elicitor-induced isoform among HMGRs on its gene structure and had a predicted amino acid sequence homology. In addition, the expression of Hmg2 was rapidly induced within 1 h in response to a fungal pathogen and continuously increased up to 48 h. Together with sesquiterpene cyclase gene that was strongly induced 24 h after pathogen-infection, the Hmg2 and farnesyl pyrophosphate synthase gene were coordinately and sequentially regulated for the biosynthesis of defense-related sesquiterpene phytoalexins in pepper.     

BAC-derived markers converted from RFLP linked to Phytophthora capsici resistance in pepper (Capsicum annuum L.)

Phytophthora capsici Leonian, an oomycete pathogen, is a serious problem in pepper worldwide. Its resistance in pepper is controlled by quantitative trait loci (QTL). To detect QTL associated with P. capsici resistance, a molecular linkage map was constructed using 100 F(2) individuals from a cross between Capsicum annuum 'CM334' and C. annuum 'Chilsungcho'. This linkage map consisted of 202 restriction fragment length polymorphisms (RFLPs), 6 WRKYs and 1 simple sequence repeat (SSR) covering 1482.3 cM, with an average interval marker distance of 7.09 cM. QTL mapping of Phytophthora root rot and damping-off resistance was performed in F(2:3) originated from a cross between resistant Mexican landrace C. annuum 'CM334' and susceptible Korean landrace C. annuum 'Chilsungcho' using composite interval mapping (CIM) analysis. Four QTL explained 66.3% of the total phenotypic variations for root rot resistance and three 44.9% for damping-off resistance. Of these QTL loci, two were located close to RFLP markers CDI25 on chromosome 5 (P5) and CT211A on P9. A bacterial artificial chromosome (BAC) library from C. annuum 'CM334' was screened with these two RFLP probes to obtain sequence information around the RFLP marker loci for development of PCR-based markers. CDI25 and CT211 probes identified seven and eight BAC clones, respectively. Nine positive BAC clones containing probe regions were sequenced and used for cytogenetic analysis. One single-nucleotide amplified polymorphism (SNAP) for the CDI25 locus, and two SSRs and cleaved amplified polymorphic sequence (CAPS) for CT211 were developed using sequences of the positive BAC clones. These markers will be valuable for rapid selection of genotypes and map-based cloning for resistance genes against P. capsici.     

Inheritance of resistance to Meloidogyne incognita race 2 in the hot pepper cultivar Carolina Cayenne (Capsicum annuum L.)

Root-knot nematodes of the genus Meloidogyne are important pathogens affecting vegetable crop production in Brazil and worldwide. The pepper species Capsicum annuum includes both hot and sweet peppers; very little emphasis has been placed on breeding sweet peppers for nematode resistance. We report on the inheritance of resistance to Meloidogyne incognita (Kofoid & White) Chitwood race 2 in the hot pepper cultivar Carolina Cayenne. The hot pepper cv. Carolina Cayenne was used as seed parent and the sweet pepper cv. Agron?mico-8 was used as pollen parent to obtain the F(1) and F(2) generations and the backcross generations BC(11) and BC(12). The plants were inoculated with M. incognita race 2 at a rate of 60 eggs/ml of substrate and, after a suitable incubation period, the numbers of root galls and egg masses per root system were evaluated on each plant. Broad- (0.77 and 0.72) and narrow-sense (0.77 and 0.63) heritability estimates were high for both root galls and egg masses, respectively. The mean degree of dominance was estimated as 0.29 and 0.25 for numbers of galls and egg masses, respectively; these estimates were not significantly different from 0, indicating a predominantly additive gene action. The results were consistent with a hypothesis of monogenic resistance in Carolina Cayenne.