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Plant cytochrome P450 enzymes are involved in a wide range of biosynthetic reactions, leading to various fatty acid conjugates, plant hormones, or defensive compounds. Herein, we have identified the pepper cytochrome P450 gene CaCYP450A, which is differentially induced during Xanthomonas campestris pv. vesicatoria ( Xcv) infection. CaCYP450A contains a heme-binding motif, PXFXXGXRXCXG, located in the C-terminal region and a hydrophobic membrane anchor region at the N terminal. Knock-down of CaCYP450A by virus-induced gene silencing (VIGS) led to increased susceptibility to Xcv infection in pepper. CaCYP450A-overexpressing Arabidopsis plants exhibited lower pathogen growth and reduced disease symptoms, and they were more resistant to Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis than wild-type plants. Overexpression of CaCYP450A also enhanced HO accumulation and cell death. However, CaCYP450A Arabidopsis ortholog CYP94B3 mutants showed enhanced susceptibility to virulent Pst DC3000, but not to avirulent Pst DC3000 avrRpm1 or virulent H. arabidopsidis infection. Taken together, these results suggest that CaCYP450A is required for defense responses to microbial pathogens in plants. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number HM581974. [ABSTRACT FROM AUTHOR]

Analysis of the promoters of defense-related genes is valuable for determining stress signaling and transcriptional activation during pathogen infection. Here, we have isolated and functionally characterized the promoter region of the pepper ( Capsicum annuum) pectin methylesterase inhibitor 1 ( CaPMEI1) gene in transiently transformed tobacco plants and stably transformed Arabidopsis plants. Among four 5′ deletion constructs analyzed, the −958-bp CaPMEI1 promoter induced a high level of GUS reporter activity in tobacco leaf tissue, driven by pathogen infection as well as by ethylene and methyl jasmonate (MeJA) treatment. The 204-bp region from −958 bp to −754 bp of the CaPMEI1 promoter is responsible for the stress-responsive expression. In addition, the pepper transcription factor CARAV1 activated the CaPMEI1 promoter in tobacco leaves, whereas the transcription factor CAbZIP1 did not. In the transgenic Arabidopsis plants, the −958 bp CaPMEI1 promoter was functionally regulated by developmental cues, bacterial and oomycete pathogen infections, and treatment with ethylene and MeJA. Histochemical GUS staining analyses of Arabidopsis tissues revealed that the CaPMEI1 promoter was mainly activated in leaf veins in response to various biotic and abiotic stimuli. Together, these results suggest that CaPMEI1 promoter activation may be a critical molecular event for host defense response and ethylene- and MeJA-mediated CaPMEI1 gene expression. [ABSTRACT FROM AUTHOR]

Biotic signaling molecules including abscisic acid (ABA) are involved in signal transduction pathways that mediate the defense response of plants to environmental stresses. The antimicrobial protein gene CaAMP1, previously isolated from pepper ( Capsicum annuum), was strongly induced in pepper leaves exposed to ABA, NaCl, drought, or low temperature. Because transformation is very difficult in pepper, we overexpressed CaAMP1 in Arabidopsis. CaAMP1-overexpressing (OX) transgenic plants exhibited reduced sensitivity to ABA during the seed germination and seedling stages. Overexpression of CaAMP1 conferred enhanced tolerance to high salinity and drought, accompanied by altered expression of the AtRD29A gene, which is correlated with ABA levels and environmental stresses. The transgenic plants were also highly tolerant to osmotic stress caused by high concentrations of mannitol. Together, these results suggest that overexpression of the CaAMP1 transgene modulates salt and drought tolerance in Arabidopsis through ABA-mediated cell signaling. [ABSTRACT FROM AUTHOR]

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-β-glucuronidase ( GUS) gene fusion, serially 5′-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The −1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The −417- and −593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than −793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, β-amino- n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection. [ABSTRACT FROM AUTHOR]

Plant integral membrane proteins have essential roles in diverse internal and external physiological processes as signal receptors or ion transporters. The pepper CaPIMP1 gene encoding a putative integral membrane protein with four transmembrane domains was isolated and functionally characterized from pepper leaves infected with the avirulent strain Xanthomonas campestris pv. vesicatoria ( Xcv). CaPIMP1-green fluorescence protein (GFP) fusions localized to the plasma membrane in onion cells, as observed by confocal microscopy. CaPIMP1 was expressed in an organ-specific manner in healthy pepper plants. Infection with Xcv induced differential accumulation of CaPIMP1 transcripts in pepper leaf tissues during compatible and incompatible interactions. The function of CaPIMP1 was examined by using the virus-induced gene silencing technique in pepper plants and by overexpression in Arabidopsis. CaPIMP1-silenced pepper plants were highly susceptible to Xcv infection and expressed lower levels of the defense-related gene CaSAR82A. CaPIMP1 overexpression ( CaPIMP1-OX) in transgenic Arabidopsis conferred enhanced resistance to P. syringae pv. tomato infection, accompanied by enhanced AtPDF1.2 gene expression. In contrast, CaPIMP1-OX plants were highly susceptible to the biotrophic oomycete Hyaloperonospora parasitica. Taken together, we propose that CaPIMP1 plays distinct roles in both bacterial disease resistance and oomycete disease susceptibility. [ABSTRACT FROM AUTHOR]

Pectin is one of the main components of the plant cell wall that functions as the primary barrier against pathogens. Among the extracellular pectinolytic enzymes, pectin methylesterase (PME) demethylesterifies pectin, which is secreted into the cell wall in a highly methylesterified form. Here, we isolated and functionally characterized the pepper ( Capsicum annuum L.) gene CaPMEI1, which encodes a pectin methylesterase inhibitor protein (PMEI), in pepper leaves infected by Xanthomonas campestris pv. vesicatoria ( Xcv). CaPMEI1 transcripts are localized in the xylem of vascular bundles in leaf tissues, and pathogens and abiotic stresses can induce differential expression of this gene. Purified recombinant CaPMEI1 protein not only inhibits PME, but also exhibits antifungal activity against some plant pathogenic fungi. Virus-induced gene silencing of CaPMEI1 in pepper confers enhanced susceptibility to Xcv, accompanied by suppressed expression of some defense-related genes. Transgenic Arabidopsis CaPMEI1-overexpression lines exhibit enhanced resistance to Pseudomonas syringae pv. tomato, mannitol and methyl viologen, but not to the biotrophic pathogen Hyaloperonospora parasitica. Together, these results suggest that CaPMEI1, an antifungal protein, may be involved in basal disease resistance, as well as in drought and oxidative stress tolerance in plants. [ABSTRACT FROM AUTHOR]

GDSL-type lipase is a hydrolytic enzyme whose amino acid sequence contains a pentapeptide motif (Gly-X-Ser-X-Gly) with active serine (Ser). Pepper GDSL-type lipase ( CaGLIP1) gene was isolated and functionally characterized from pepper leaf tissues infected by Xanthomonas campestris pv. vesicatoria ( Xcv). The CaGLIP1 protein was located in the vascular tissues of Arabidopsis root. The CaGLIP1 gene was preferentially expressed in pepper leaves during the compatible interaction with Xcv. Treatment with salicylic acid, ethylene and methyl jasmonate induced CaGLIP1 gene expression in pepper leaves. Sodium nitroprusside, methyl viologen, high salt, mannitol-mediated dehydration and wounding also induced early and transient CaGLIP1 expression in pepper leaf tissues. Virus-induced gene silencing of CaGLIP1 in pepper conferred enhanced resistance to Xcv, accompanied by the suppressed expression of basic PR1 ( CaBPR1) and defensin ( CaDEF1) genes. The CaGLIP1 lipase produced in Escherichia coli hydrolyzed the substrates of short and long chain nitrophenyl esters. The CaGLIP1-overexpressing Arabidopsis exhibited enhanced hydrolytic activity toward short and long chain nitrophenyl ester, as well as enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato and the biotrophic oomycete Hyaloperonospora parasitica. SA-induced expression of AtPR1 and AtGST1, also was delayed in CaGLIP1-overexpressing plants by SA application. During seed germination and plant growth, the CaGLIP1 transgenic plants showed drought tolerance and differential expression of drought- and abscisic acid (ABA)-inducible genes AtRD29A, AtADH and AtRab18. ABA treatment differentially regulated seed germination and gene expression in wild-type and CaGLIP1 transgenic Arabidopsis. Overexpression of CaGLIP1 also regulated glucose- and oxidative stress signaling. Together, these results indicate that CaGLIP1 modulates disease susceptibility and abiotic stress tolerance. [ABSTRACT FROM AUTHOR]

A Capsicum annuum hypersensitive induced reaction protein1 (CaHIR1) was recently proposed as a positive regulator of hypersensitive cell death in plants. Overexpression of CaHIR1 in transgenic Arabidopsis plants conferred enhanced resistance against the hemi-biotrophic Pseudomonas syringae pv. tomato ( Pst) and the biotrophic Hyaloperonospora parasitica. Infection by avirulent Pseudomonas strains carrying avrRpm1 or avrRpt2 caused enhanced resistance responses in transgenic plants, suggesting that CaHIR1 is involved in basal disease resistance in a race-nonspecific manner. H. parasitica exhibited low levels of asexual sporulation on CaHIR1 seedlings. In contrast, transgenic plants were susceptible not only to the necrotrophic fungal pathogen Botrytis cinerea but were also sensitive to osmotic stress caused by high salinity and drought. To identify proteins whose expression was altered by CaHIR1 overexpression in Arabidopsis leaves, a quantitative comparative proteome analysis using two-dimensional gel electrophoresis coupled with mass spectrometry was performed. Of about 400 soluble proteins, 11 proteins involved in several metabolic pathways were up- or down-regulated by CaHIR1 overexpression. Genes encoding glycine decarboxylase (At2g35370) and an unidentified protein (At2g03440), which were strongly upregulated in CaHIR1-overexpressing Arabidopsis, were also differentially induced at the transcriptional level by Pst infection. Arabidopsis carbonic anhydrase (At3g01500), highly similar to tobacco salicylic acid-binding protein 3, was up-regulated by CaHIR1 overexpression. The activity of an anti-oxidant enzyme, cooper/zinc superoxide dismutase (At2g28190), was also attenuated in transgenic Arabidopsis by CaHIR1 overexpression. Together, these results suggest that CaHIR1 overexpression in Arabidopsis mediates plant responses to biotrophic, hemi-biotrophic and necrotrophic pathogens, as well as to osmotic stress in different ways. [ABSTRACT FROM AUTHOR]

Transcription factors often belong to multigene families and their individual contribution in a particular regulatory network remains difficult to assess. We identify and functionally characterize the pepper bZIP transcription factor CAbZIP1 gene isolated from pepper leaves infected with Xanthomonas campestris pv. v esicatoria. Transient expression analysis of the CAbZIP1–GFP fusion protein in Arabidopsis protoplasts revealed that the CAbZIP1 protein is localized in the nucleus. The N-terminal region of CAbZIP1 fused to the GAL4 DNA-binding domain is required to activate transcription of reporter genes in yeast. The CAbZIP1 transcripts are constitutively expressed in the pepper root and flower, but not in the leaf, stem and fruit. The CAbZIP1 gene is locally or systemically induced in pepper plants infected by either X. campestris pv. vesicatoria or Pseudomonas fluorescens. The CAbZIP1 gene is also induced by abiotic elicitors and environmental stresses. The CAbZIP1 transgenic Arabidopsis exhibits a dwarf phenotype, indicating that CAbZIP1 may be involved in plant development. The CAbZIP1 overexpression in the transgenic Arabidopsis plants confers enhanced resistance to Pseudomonas syringae pv. tomato DC3000, accompanied by expression of the AtPR-4 and AtRD29A. The transgenic plants also exhibit increased drought and salt tolerance during all growth stages. Moreover, the transgenic plants are tolerant to methyl viologen-oxidative stress. Together, these data suggest that the CAbZIP1 transcription factor function as a possible regulator in enhanced disease resistance and environmental stress tolerance. [ABSTRACT FROM AUTHOR]

The activation of the CAChi2 promoter as the result of bacterial infection and osmotic stresses was examined using the Agrobacterium-mediated transient expression assay. Several stress-related cis-acting elements were revealed within the upstream genomic sequence of the CAChi2 gene. In tobacco leaf tissues transiently transformed with the CAChi2 promoter-β-glucuronidase (GUS) gene, the CAChi2 promoter was up-regulated by Pseudomonas syringae pv. tabaci infection. The CAChi2- GUS activation was closely related to osmotic stresses, including treatment with mannitol and NaCl. The −378 CAChi2 promoter was sufficient for the CAChi2 gene induction by salicylic acid treatment. CAChi2 overexpression in the transgenic Arabidopsis plants enhanced bacterial disease resistance against Pseudomonas syringae pv. tomato infection. CAChi2-overexpressing Arabidopsis plants also exhibited increased tolerance to NaCl-induced osmotic stresses during seed germination and seedling growth. CAChi2 overexpression induced the expression of the NaCl stress-responsive gene RD29A in the absence of NaCl stress. The CAChi2-overexpressing transgenic plants exhibited increased sensitivity to abscisic acid during seed germination. [ABSTRACT FROM AUTHOR]

The inoculation of primary pepper leaves with an avirulent strain of Xanthomonas campestris pv. vesicatoria induced systemic acquired resistance (SAR) in the non-inoculated, secondary leaves. This SAR response was accompanied by the systemic expression of the defense-related genes, a systemic microoxidative burst generating H2O2, and the systemic induction of both ion-leakage and callose deposition in the non-inoculated, secondary leaves. Some defense-related genes including those encoding PR-1, chitinase, osmotin, peroxidase, PR10, thionin, and SAR8.2 were markedly induced in the systemic leaves. The conspicuous systemic accumulation of H2O2 and the strong increase in peroxidase activity in the pepper leaves was suggested to play a role in the cell death process in the systemic micro-hypersensitive responses (HR), leading to the induction of the SAR. Treatment of the primary leaves with diphenylene iodinium (DPI), an inhibitor of oxidative burst, substantially reduced the induction of some of the defense-related genes, and lowered the activation of the oxidative bursts in the systemic leaves distant from the site of the avirulent pathogen inoculation and subsequently SAR. Overall, these results suggest that the induction of some defense-related genes as well as a rapid increase in oxidative burst is essential for establishing SAR in pepper plants. [ABSTRACT FROM AUTHOR]

The 5' flanking region of theCALTPIgene, which encodes a basic lipid transfer protein, was isolated and characterized from the genomic DNA ofCapsicum annuum. Four different regions of the promoter sequence of theCALTPIgene were fused to the ß-glucuronidase (GUS) coding region. In anAgrobacterium-mediated transient expression assay, the transcriptional activations of the promoter deletions were examined in tobacco leaves after infection withPseudomonas syringaepv.tabaci, and treatment with ethylene and salicylic acid. The -808 bp region of theCALTPIgene promoter sequence exhibited full promoter activity. The W-box and ERE-box elements, which are essential for induction by all signals, were localized in the region between -555 bp and -391 bp upstream of the translation initiation site. ACALTPItransgene was then introduced under the control of the 35S promoter into theArabidopsisecotype Col-0. TransgenicArabidopsislines expressing theCALTPIgene developed rapidly compared to the wild-type plants, indicating thatCALTPImay be involved in plant development. Overexpression of theCALTPIgene enhanced the resistance against infection byP. syringaepv.tomatoandBotrytis cinerea. The transgenic plants expressing theCALTPIgene also showed high levels of tolerance to NaCl and drought stresses at various vegetative growth stages. No transcription of thePR-1,PR-2,PR-5, thionin, andRD29Agenes was observed in untreated leaf tissues of the transgenic plants. The enhanced resistance to pathogen and environmental stresses in transgenicArabidopsiscorrelated with the enhanced expression of theCALTPIgene. [ABSTRACT FROM AUTHOR]

Pepper (Capsicum annuum L.) SAR8.2 genes, designated CASAR82A, B and C, which are induced by all the biotic and abiotic stresses, were isolated from a pepper cDNA library constructed with the mRNAs from pepper plants infected with Xanthomonas campestris pv. vesicatoria. The pepper CASAR82A, B and C gene products, which are very similar to each other in amino acid sequences, have 43–50% homology with those of tobacco SAR8.2 genes. The CASAR8.2 genes were not constitutively expressed in any of the organs of healthy pepper plants. In contrast, the CASAR82A gene was locally or systemically induced in pepper plants infected by X. campestris pv. vesicatoria, Colletotrichum coccodes or Phytophthora capsici. Strong induction of the CASAR82A gene also was found in pepper leaves treated with ethylene, methyl jasmonate, indole-3-acetic acid, abscisic acid, salicylic acid, benzothiadiazole, DL-β-n-amino butyric acid or hydrogen peroxide. Interestingly, the transcription of the CASAR82A gene was rapidly triggered by high salinity, drought or low-temperature stresses, but not by mechanical wounding. In situ hybridization results revealed that the CASAR82A mRNAs were localized in phloem and epidermal cells of pepper leaf and stem tissues infected by C. coccodes and P. capsici, or treated with salicylic acid. These results thus suggest that pepper SAR8.2 genes may be valuable as a molecular marker for the detection of various pathogen infections, abiotic elicitors and environmental stresses. [ABSTRACT FROM AUTHOR]