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Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBs T-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants. [ABSTRACT FROM AUTHOR]

Formate dehydrogenase (FDH; EC 1.2.1.2) is an NAD-dependent enzyme that catalyzes the oxidation of formate to carbon dioxide. Here, we report the identification and characterization of pepper (Capsicum annuum) mitochondrial FDH1 as a positive regulator of cell death and defense responses. Transient expression of FDH1 caused hypersensitive response (HR)-like cell death in pepper and Nicotiana benthamiana leaves. The D-isomer-specific 2-hydroxyacid dehydrogenase signatures of FDH1 were required for the induction of HR-like cell death and FDH activity. FDH1 contained a mitochondrial targeting sequence at the N-terminal region; however, mitochondrial localization of FDH1 was not essential for the induction of HR-like cell death and FDH activity. FDH1 silencing in pepper significantly attenuated the cell death response and salicylic acid levels but stimulated growth of Xanthomonas campestris pv vesicatoria. By contrast, transgenic Arabidopsis (Arabidopsis thaliana) overexpressing FDH1 exhibited greater resistance to Pseudomonas syringae pv tomato in a salicylic acid-dependent manner. Arabidopsis transfer DNA insertion mutant analysis indicated that AtFDH1 expression is required for basal defense and resistance gene-mediated resistance to P. syringae pv tomato infection. Taken together, these data suggest that FDH1 has an important role in HR-like cell death and defense responses to bacterial pathogens. [ABSTRACT FROM AUTHOR]

Xanthomonas campestris pv vesicatoria type III effector protein, AvrBsT, triggers hypersensitive cell death in pepper (Capsicum annuum). Here, we have identified the pepper SGT1 (for suppressor of the G2 allele of skp1) as a host interactor of AvrBsT and also the pepper PIK1 (for receptor-like cytoplasmic kinase1). PIK1 specifically phosphorylates SGT1 and AvrBsT in vitro. AvrBsT specifically binds to the CHORD-containing protein and SGT1 domain of SGT1, resulting in the inhibition of PIKl-mediated SGT1 phosphorylation and subsequent nuclear transport of the SGT1-PIK1 complex. Liquid chromatography-tandem mass spectrometry of the proteolytic peptides of SGT1 identified the residues serine-98 and serine-279 of SGT1 as the major PIK1-mediated phosphorylation sites. Site-directed mutagenesis of SGT1 revealed that the identified SGT1 phosphorylation sites are responsible for the activation of AvrBsT-triggered cell death in planta. SGT1 forms a heterotrimeric complex with both AvrBsT and PIK1 exclusively in the cytoplasm. Agrobacterium tumefaciens-mediated coexpression of SGT1 and PIK1 with avrBsT promotes avrBsT-triggered cell death in Nicotiana benthamiana, dependent on PIK1. Virus-induced silencing of SGT1 and/or PIK1 compromises avrBsT-triggered cell death, hydrogen peroxide production, defense gene induction, and salicylic acid accumulation, leading to the enhanced bacterial pathogen growth in pepper. Together, these results suggest that SGT1 interacts with PIK1 and the bacterial effector protein AvrBsT and promotes the hypersensitive cell death associated with PIK1-mediated phosphorylation in plants. [ABSTRACT FROM AUTHOR]

Plants produce various proteinaceous inhibitors to protect themselves against microbial pathogen attack. A xyloglucan-specific endo-β-1,4-glucanase inhibitorl gene, CaXEGIP1, was isolated and functionally characterized in pepper (Capsicum annuum) plants. CaXEGIP1 was rapidly and strongly induced in pepper leaves infected with avirulent Xanthomonas campestris pv vesicatoria, and purified CaXEGIP1 protein significantly inhibited the hydrolytic activity of the glycoside hydrolase74 family xyloglucan-specific endo-β-l,4-glucanase from Clostridium thermocellum. Soluble-modified green fluorescent protein-tagged CaXEGIP1 proteins were mainly localized to the apoplast of onion (Allium cepa) epidermal cells. Agrobacterium himefaciens-mediated overexpression of CaXEGIP1 triggered pathogen-independent, spontaneous cell death in pepper and Nicotiana benthamiana leaves. CaXEGIP1 silencing in pepper conferred enhanced susceptibility to virulent and avirulent X. campestris pv vesicatoria, accompanied by a compromised hypersensitive response and lowered expression of defense-related genes. Overexpression of dexamethasone: CaXEGIP1 in Arabidopsis (Arabidopsis thaliana) enhanced resistance to Hyaloperonospora arabidopsidis infection. Comparative histochemical and proteomic analyses revealed that CaXEGIP1 overexpression induced a spontaneous cell death response and also increased the expression of some defense-related proteins in transgenic Arabidopsis leaves. This response was also accompanied by cell wall thickening and darkening. Together, these results suggest that pathogen-inducible CaXEGIPl positively regulates cell death-mediated defense responses in plants. [ABSTRACT FROM AUTHOR]

Ubiquitination is essential for ubiquitin/proteasome-mediated protein degradation in plant development and defense. Here, we identified a novel E3 ubiquitin ligase RING1 gene, CARING1, from pepper (Capsicum anmtum). In pepper, CARING1 expression is induced by avirulent Xanthomonas campestris pv vesicatoria infection. CaRINGI contains an amino-terminal transmembrane domain and a carboxyl-terminal RING domain. In addition, it displays in vitro E3 ubiquitin ligase activity, and the RING domain is essential for E3 ubiquitin ligase activity in CARING1. CARING1 also localizes to the plasma membrane. In pepper plants, virus-induced gene silencing of CARING1 confers enhanced susceptibility to avirulent X. campestris pv vesicatoria infection, which is accompanied by compromised hypersensitive cell death, reduced expression of PATHOGENESIS- RELATED1, and lowered salicylic acid levels in leaves. Transient expression of CARING1 in pepper leaves induces cell death and the defense response that requires the E3 ubiquitin ligase activity of CaR1NG1. By contrast, overexpression of CARING1 in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to hemibiotrophic Pseudomonas syringae pv tomato and biotrophic Hyaloperonospora arabidopsidis infections. Taken together, these results suggest that CARING1 is involved in the induction of ceil death and the regulation of ubiquitination during the defense response to microbial pathogens. [ABSTRACT FROM AUTHOR]

Plant mannose-binding lectins (MBLs) are crucial for plant defense signaling during pathogen attack by recognizing specific carbohydrates on pathogen surfaces. In this study, we isolated and functionally characterized a novel pepper (Capsicum ammunt) MBL gene, CaMBL1, from pepper leaves infected with Xanthomonas campestris pv vesicatoria (Xcv). The CaMBL1 gene contains a predicted Galanthus nivalis agglutinin-related lectin domain responsible for the recognition of high-mannose N-glycans but lacks a middle S-locus glycoprotein domain and a carboxyl-terminal PAN-Apple domain. The CaMBL1 protein exhibits binding specificity for mannose and is mainly localized to the plasma membrane. Immunoblotting using a CaMBL1-specific antibody revealed that CaMBL1 is strongly expressed and accumulates in pepper leaves during avirulent Xcv infection. The transient expression of CaMBL1 induces the accumulation of salicylic acid (SA), the activation of defense-related genes, and the cell death phenotype in pepper. The G. nivalis agglutinin-related lectin domain of CaMBL1 is responsible for cell death induction. CaMBL1-silenced pepper plants are more susceptible to virulent or avirulent Xcv infection compared with unsilenced control plants, a phenotype that is accompanied by lowered reactive oxygen species accumulation, reduced expression of downstream SA target genes, and a concomitant decrease in SA accumulation. In contrast, CaMBL1 overexpression in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to Pseudomonas syringae pv tomato and Alternaria brassicicola infection. Together, these data suggest that CaMBL1 plays a key role in the regulation of plant cell death and defense responses through the induction of downstream defense-related genes and SA accumulation after the recognition of microbial pathogens. [ABSTRACT FROM AUTHOR]

Lipoxygenases (LOXs) are crucial for lipid peroxidation processes during plant defense responses to pathogen infection. A pepper (Capsicum annuurn) 9-LOX gene, CaLOX1, which encodes a 9-specific lipoxygenase, was isolated from pepper leaves. Recombinant CaLOX1 protein expressed in Escherichia coli catalyzed the hydroperoxidation of linoleic acid, with a Km value of 113. 9 μM Expression of CaLOX1 was differentially induced in pepper leaves not only during Xanthoinonas campestris pv vesicatoria (Xcv) infection but also after exposure to abiotic elicitors. Transient expression of CaLOX1 in pepper leaves induced the cell death phenotype and defense responses. CaLOX1-silenced pepper plants were more susceptible to Xcv and Colletotrichum coccodes infection, which was accompanied by reduced expression of defense-related genes, lowered lipid peroxidation, as well as decreased reactive oxygen species and lowered salicylic acid accumulation. hifection with Xcv, especially in an incompatible interaction, rapidly stimulated LOX activity in unsilenced, but not CaLOX1-silenced, pepper leaves. Furthermore, overexpression of CaLOX1 in Arabidopsis (Arabidopsis thaliana) conferred enhanced resistance to Pseudomonas syringae pv tomato, Hya/operonospora arabidopsidis, and Alternaria brassicicola. In contrast, mutation of the Arabidopsis CaLOX1 ortholog AtLOX1 significantly increased susceptibility to these three pathogens. Together, these results suggest that CaLOX1 and AtLOX1 positively regulate defense and cell death responses to microbial pathogens. [ABSTRACT FROM AUTHOR]

Pathogen-inducible antimicrobial defense-related proteins have emerged as key antibiotic peptides and enzymes involved in disease resistance in plants. A novel antimicrobial protein gene, CaAMP1 (for Capsicum annuum ANTIMICROBIAL PROTEIN1), was isolated from pepper (C. annuum) leaves infected with Xanthomonas cam pestris pv vesicatoria. Expression of the CaAMP1 gene was strongly induced in pepper leaves not only during pathogen infection but also after exposure to abiotic elicitors. The purified recombinant CaAMP1 protein possessed broad-spectrum antimicrobial activity against phytopathogenic bacteria and fungi. CaAMP1:smGFP fusion protein was localized mainly in the external and intercellular regions of onion (A/hum cepa) epidermal cells. The virus-induced gene silencing technique and gain-of-function transgenic plants were used to determine the CaAMP1 gene function in plant defense. Silencing of CaAMP1 led to enhanced susceptibility to X. cam pestris pv vesicatoria and Cohletotrichum coccodes infection, accompanied by reduced PATHOGENESIS-RELATED (PR) gene expression. In contrast, overexpression of CaAMP1 in Arabidopsis (Arabidopsis thahiana) conferred broad-spectrum resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora parasitica, and the fungal necrotrophic pathogens Fusarium oxysporum f. sp. matthiobae and Alternaria brassicicola. CaAMP1 overexpression induced the salicylic acid pathway-dependent genes PR1 and PR5 but not the jasmonic acid-dependent defense gene PDF1.2 during P. syringae pv tomato infection. Together, these results suggest that the antimicrobial CaAMP1 protein is involved in broad- spectrum resistance to bacterial and fungal pathogen infection. [ABSTRACT FROM AUTHOR]

Plants elaborate a vast array of enzymes that synthesize defensive secondary metabolites in response to pathogen attack. Here, we isolated the pathogen-responsive CaMNR1 [menthone: (+)-(3S)-neomenthol reductase] gene, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, from pepper (Capsicum annuum) plants. Gas chromatography-mass spectrometry analysis revealed that purified CaMNR1 and its ortholog AtSDR1 from Arabidopsis (Arabidopsis thaliana) catalyze a menthone reduction with reduced nicotinamide adenine dinucleotide phosphate as a cofactor to produce neomenthol with antimicrobial activity. CaMNR1 and AtSDR1 also possess a significant catalytic activity for neomenthol oxidation. We examined the cellular function of the CaMNR1 gene by virus-induced gene silencing and ectopic overexpression in pepper and Arabidopsis plants, respectively. CaMNRTI-silenced pepper plants were significantly more susceptible to Xantho,nonas cam pestris pv vesicatoria and Colletotrichum coccodes infection and expressed lower levels of salicylic acid-responsive CaBPR1 and CaPRIO and jasmonic acid-responsive CaDEF1. CaMNRI-overexpressing Arabidopsis plants exhibited enhanced resistance to the hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000 and the biotrophic pathogen Hyaloperonospora parasitica isolate Noco2, accompanied by the induction of AtPR1 and AtPDFTL2. In contrast, mutation in the CaMNR1 ortholog AtSDRI significantly enhanced susceptibility to both pathogens. Together, these results indicate that the novel menthone reductase gene CaMNR1 and its ortholog AtSDR1 positively regulate plant defenses against a broad spectrum of pathogens. [ABSTRACT FROM AUTHOR]

Ubiquitination is essential for ubiquitin/proteasome-mediated protein degradation in plant development and defense. Here, we identified a novel E3 ubiquitin ligase RING1 gene, CARING1, from pepper (Capsicum anmtum). In pepper, CARING1 expression is induced by avirulent Xanthomonas campestris pv vesicatoria infection. CaRINGI contains an amino-terminal transmembrane domain and a carboxyl-terminal RING domain. In addition, it displays in vitro E3 ubiquitin ligase activity, and the RING domain is essential for E3 ubiquitin ligase activity in CARING1. CARING1 also localizes to the plasma membrane. In pepper plants, virus-induced gene silencing of CARING1 confers enhanced susceptibility to avirulent X. campestris pv vesicatoria infection, which is accompanied by compromised hypersensitive cell death, reduced expression of PATHOGENESIS- RELATED1, and lowered salicylic acid levels in leaves. Transient expression of CARING1 in pepper leaves induces cell death and the defense response that requires the E3 ubiquitin ligase activity of CaR1NG1. By contrast, overexpression of CARING1 in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to hemibiotrophic Pseudomonas syringae pv tomato and biotrophic Hyaloperonospora arabidopsidis infections. Taken together, these results suggest that CARING1 is involved in the induction of ceil death and the regulation of ubiquitination during the defense response to microbial pathogens. [ABSTRACT FROM AUTHOR]