Doil Choi

Only limited information has been published to date on the similarities and differences between climacteric and non-climacteric fruit ripening on transcriptional level. To address this issue, we performed a direct comparative transcriptome analysis between tomato and pepper fruits using heterologous microarray hybridization. Given the significant differences in the morphological, physiological, and biochemical characteristics of pepper and tomato fruits, the existence of extensive common regulons is surprising. This finding suggests the conservation of ripening mechanisms in climacteric and non-climacteric fruits. However, disparate expression profiles were also observed in both fruits. This study revealed that a gene that encodes an enzyme that converts lycopene to downstream carotenoids is induced in pepper but not in tomato. Most of the genes that encode ribosomal proteins are only induced in early fruit-stage pepper fruit and show rapidly diminishing expression in the later developmental stages. The genes involved in ethylene biosynthesis were not induced in pepper fruit. However, the EIL-like genes, ethylene-mediated signaling components, were induced in pepper fruit. Divergent types of transcription factors were expressed in ripening tomato and pepper fruits, suggesting they may be key factors that differentiate these distinct ripening processes.

Plants protect themselves against pathogens using a range of response mechanisms. There are two categories of nonhost resistance: Type I, which does not result in visible cell death; and Type II, which entails localized programmed cell death (or hypersensitive response) in response to nonhost pathogens. The genes responsible for these two systems have not yet been intensively investigated at the molecular level. Using tobacco plants (Nicotiana tabacum), we compared expression of 12 defense-related genes between a Type I (Xanthomonas axonopodis pv. glycines 8ra) nonhost interaction, and two Type II (Pseudomonas syringae pv. syringae 61 and P. syringae pv. phaseolicola NPS3121) nonhost interactions, as well as those expressed during R gene-mediated resistance to Tobacco mosaic virus. In general, expression of most defense-related genes during R gene-mediated resistance was activated 48 h after challenge by TMV; the same genes were upregulated as early as 9 h after infiltration by nonhost pathogens. Surprisingly, X. axonopodis pv. glycines (Type I) elicited the same set of defense-related genes as did two pathovars of P. syringae, despite the absence of visible cell death. In two examples of Type II nonhost interactions, P. syringae pv. phaseolicola NPS3121 produced an expression profile more closely resembling that of X. axonopodis pv. glycines 8ra, than that of P. syringae pv. syringae 61. These results suggest that Type I nonhost resistance may act as a mechanism providing a more specific and active defense response against a broad range of potential pathogens.

SGT1 associates with suppressor of kinetochore protein (Skp1)‐Cullin‐F‐box (SCF)–ubiquitin‐ligase complexes playing important roles in controlling developmental processes and defense responses in plants, yeast, and human. In this study, full‐length cDNAs ofSgt1andSkp1orthologues were isolated from pepper (Capsicum annuumL.) to characterize their functions. Protein sequences of CaSgt1 and CaSkp1 showed high degrees of similarities with their homologues in other plant species. Southern blot analyses revealed thatCaSgt1was a single copy gene in the pepper genome, whereasCaSkp1corresponded to multi copy genes. Levels ofCaSgt1andCaSkp1mRNAs increased in pepper leaves in response to incompatible pathogen challenge or salicylic acid treatment. Silencing ofCaSgt1orCaSkp1using Tobacco rattle virus‐based virus‐induced gene‐silencing (VIGS) system resulted in severe dwarfism and final damping‐off symptom in the greenhouse. To identify factors determining damping‐off symptom inCaSgt1‐ orCaSkp1‐silenced plants, we employed VIGS under sterile conditions. Under such conditions, damping‐off symptom was not observed suggesting thatCaSgt1andCaSkp1play an essential role in plant growth and development as well as basal disease resistance in pepper plant.

We aim to isolate genes in chili pepper that are regulated during the hypersensitive response to infection by nonhost pathogens, with a view to elucidating the defense responses against pathogen attack. Among the 90 transcription factors initially characterized by reverse RNA gel blot analysis, a cDNA clone, CaNAC1 (Capsicum annuum NAC1) containing the plant-specific NAC domain motif was further characterized. Expression of the CaNAC1 gene was rapidly and specifically induced during incompatible interactions between pepper and bacterial or viral pathogens. Additionally, this gene was strongly induced by exogenously applied salicylic acid and ethephon, whereas methyl jasmonate only had a transient effect. A CaNAC1-smGFP (soluble modified green fluorescent protein) fusion protein localized to the nucleus following transfection into the epidermis of onion. Using the yeast system, we further disclose that the transcription activation domain of CaNAC1 is located in the C-terminal half of the protein. Our results collectively suggest that the plant-specific NAC domain protein, CaNAC1, may play a role in the regulation of defense responses in plants.

SUMMARY: To understand better the defence responses of plants to pathogen attack, we challenged hot pepper plants with bacterial pathogens and identified transcription factor‐encoding genes whose expression patterns were altered during the subsequent hypersensitive response. One of these genes, CaPIF1 ( Capsicum annuum Pathogen‐Induced Factor 1 ), was characterized further. This gene encodes a plant‐specific EPF‐type protein that contains two Cys ₂ /His ₂ zinc fingers. CaPIF1 expression was rapidly and specifically induced when pepper plants were challenged with bacterial pathogens to which they are resistant. In contrast, challenge with a pathogen to which the plants are susceptible only generated weak CaPIF1 expression. CaPIF1 expression was also strongly induced in pepper leaves by the exogenous application of ethephon, an ethylene‐releasing compound, and salicylic acid, whereas methyl jasmonate had only moderate effects. CaPIF1 localized to the nuclei of onion epidermis when expressed as a CaPIF1–smGFP fusion protein. Transgenic tobacco plants over‐expressing CaPIF1 driven by the CaMV 35S promoter showed increased resistance to challenge with a tobacco‐specific pathogen or non‐host bacterial pathogens. These plants also showed constitutive up‐regulation of multiple defence‐related genes. Moreover, virus‐induced silencing of the CaPIF1 orthologue in Nicotiana benthamiana enhanced susceptibility to the same host or non‐host bacterial pathogens. These observations provide evidence that an EPF‐type Cys ₂ /His ₂ zinc‐finger protein plays a crucial role in the activation of the pathogen defence response in plants.

The isolated full-length Capsicum annuum calcium-dependent protein kinase 3 (CaCDPK3) cDNA clone was selected from the chili pepper expressed sequence tag database (http://www.pdrc.re.kr/ks200201/pepper.html). Phylogenetic analysis based on the deduced amino acid sequence of CaCDPK3 cDNA revealed significant sequence similarity to the winter squash (Cucurbita maxima) CmCPK2 gene (81% identity). Genomic gel blot analysis disclosed that CaCDPK3 belongs to a multigene family in the pepper genome. CaCDPK3 expression was root tissue-specific, as shown by Northern blot data. The gene was rapidly induced in response to various osmotic stress factors and exogenous abscisic acid application in pepper leaves. Moreover, CaCDPK3 RNA expression was induced by an incompatible pathogen and by plant defense-related chemicals such as ethephon, salicylic acid and jasmonic acid. The biochemical properties of CaCDPK3 were investigated using a CaCDPK3 and glutathione S-transferase (GST) fusion protein. The recombinant proteins retained calcium-binding ability, and displayed autophosphorylation activity in vitro in a calcium-dependent manner. Further transient-expression studies showed that CaCDPK3 fused with soluble modified green fluorescent protein (smGFP) localized to the cytosol in chili pepper protoplasts. We propose that CaCDPK3 is implicated in biotic and abiotic stresses in pepper plants.

An ERF/AP2-type transcription factor (CaPF1) was isolated by differential-display reverse transcription-PCR, following inoculation of the soybean pustule pathogen Xanthomonas axonopodis pv glycines 8ra, which induces hypersensitive response in pepper (Capsicum annuum) leaves. CaPF1 mRNA was induced under conditions of biotic and abiotic stress. Higher levels of CaPF1 transcripts were observed in disease-resistant tissue compared with susceptible tissue. CaPF1 expression was additionally induced using various treatment regimes, including ethephon, methyl jasmonate, and cold stress. To determine the role of CaPF1 in plants, transgenic Arabidopsis and tobacco (Nicotiana tabacum) plants expressing higher levels of CaPF1 were generated. Gene expression analyses of transgenic Arabidopsis and tobacco revealed that the CaPF1 level in transgenic plants affects expression of genes that contain either a GCC or a CRT/DRE box in their promoter regions. Furthermore, transgenic Arabidopsis plants expressing CaPF1 displayed tolerance against freezing temperatures and enhanced resistance to Pseudomonas syringae pv tomato DC3000. Disease tolerance was additionally observed in CaPF1 transgenic tobacco plants. The results collectively indicate that CaPF1 is an ERF/AP2 transcription factor in hot pepper plants that may play dual roles in response to biotic and abiotic stress in plants.

Large-scale single-pass sequencing of cDNA libraries and microarray analysis have proven to be useful tools for discovering new genes and studying gene expression. As a first step in elucidating the defense mechanisms in hot pepper plants, a total of 8,525 expressed sequence tags (ESTs) were generated and analyzed in silico. The cDNA microarray analysis identified 613 hot pepper genes that were transcriptionally responsive to the non-host soybean pustule pathogen Xanthomonas axonopodis pv. glycines (Xag). Several functional types of genes, including those involved in cell wall modification/biosynthesis, transport, signaling pathways and divergent defense reactions, were induced at the early stage of Xag infiltration. In contrast, genes encoding proteins that are involved in photosynthesis, carbohydrate metabolism and the synthesis of chloroplast biogenetic proteins were down-regulated at the late stage of Xag infiltration. These expression profiles share common features with the expression profiles elicited by other stresses, such as fungal challenge, wounding, cold, drought and high salinity. However, we also identified several novel transcription factors that may be specifically involved in the defense reaction of the hot pepper. We also found that the defense reaction of the hot pepper may involve the deactivation of gibberellin. Furthermore, many genes encoding proteins with unknown function were identified. Functional analysis of these genes may broaden our understanding of non-host resistance. This study is the first report of large-scale sequencing and non-host defense transcriptome analysis of the hot pepper plant species. (The sequence data in this paper have been submitted to the dbEST and GenBank database under the codes 10227604–10236595 and BM059564–BM068555, respectively. Additional information is available at http://plant.pdrc.re.kr/ks200201/pepper.html).