Jeum Kyu Hong

Expression and functional roles of the pepper pathogen-induced transcription factor RAV1 in bacterial disease resistance, and drought and salt stress tolerance

A novel pathogen-induced gene encoding the RAV (Related to ABI3/VP1) transcription factor, CARAV1, was isolated from pepper leaves infected with Xanthomonas campestris pv. vesicatoria. CARAV1 contains two distinct DNA-binding domains AP2 and B3 uniquely found in higher plants. Transient expression analysis of the smGFP:CARAV1 fusion construct in Arabidopsis protoplasts and pepper epidermal cells revealed the CARAV1 protein to be localized in the nucleus. The N-terminal region of CARAV1 fused to the GAL4 DNA-binding domain was required to activate transcription of reporter genes in yeast. In yeast one-hybrid, the recognition of CAACA and CACCTG motifs also were essential for the CARAV1 protein to bind to a specific target gene and activate the reporter gene. The expression of the CARAV1 gene was strongly induced early in pepper leaves during the pathogen infection, abiotic elicitors and environmental stresses. CARAV1 transcripts were localized in the phloem cells of leaf tissues during pathogen infection and ethylene treatment. Ectopic expression of the CARAV1 gene in transgenic Arabidopsis plants induced some PR genes and enhanced resistance against infection by Pseudomonas syringae pv. tomato DC3000 and osmotic stresses by high salinity and dehydration. The CARAV1 promoter activation was induced by P. syringae pv. tabaci, salicylic acid and abscisic acid. These data suggest that pathogen- and abiotic stress-inducible CARAV1 functions as a transcriptional activator triggering resistance to bacterial infection and tolerance to osmotic stresses.

Function of a novel GDSL-type pepper lipase gene, CaGLIP1, in disease susceptibility and abiotic stress tolerance

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 Xanthomonascampestris 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.

CAZFP1, Cys2/His2-type zinc-finger transcription factor gene functions as a pathogen-induced early-defense gene in Capsicum annuum

A pepper zinc-finger protein gene, CAZFP1, encoding the Cys2/His2-type zinc-finger transcription factor was isolated from pepper leaves inoculated with an avirulent strain Bv5-4a of Xanthomonas campestris pv. vesicatoria. The CAZFP1 protein is a nuclear targeting protein, which functions as a transcriptional regulator. The full-length CAZFP1 had no transcriptional activation activity, whereas the C-terminal region of CAZFP1 had transactivation activity. The CAZFP1 transcripts were constitutively expressed in the pepper stem, root, flower and red fruit, but were not detectable in the leaf and green fruit. The CAZFP1 transcripts accumulated earlier than the CAZFP1 (PR-1) gene in the incompatible interaction of the pepper leaves with X. campestris pv.vesicatoria. The CAZFP1 transcripts were significantly induced in the systemic, uninoculated leaf tissues early after inoculation with bacterial pathogens, but gradually declined thereafter. The CAZFP1 transcripts were localized, and confined to the phloem cells of the vascular bundle in the pepper leaf midrib in response to Colletotrichum. coccodes infection, ethylene and abscisic acid. The CAZFP1 gene was also induced much earlier by abiotic elicitors and environmental stresses, compared with the CAZFP1 gene. Overexpression of the CAZFP1 gene in the transgenic Arabidopsis plants enhanced not only the resistance against infection by Pseudomonas syringae pv. tomato, but also the drought tolerance. These results suggest that the CAZFP1 gene functions as an early-defense gene to enhance disease resistance and drought tolerance.

Promoter activation of pepper class II basic chitinase gene, CAChi2, and enhanced bacterial disease resistance and osmotic stress tolerance in the CAChi2-overexpressing Arabidopsis.

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.

A novel pepper membrane-located receptor-like protein gene CaMRP1 is required for disease susceptibility, methyl jasmonate insensitivity and salt tolerance

Plant receptor proteins are involved in the signaling networks required for defense against pathogens. The novel pepper pathogen-induced gene CaMRP1 was isolated from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). This gene is predicted to encode a membrane-located receptor-like protein that has an N-terminal signal peptide and a C-terminal transmembrane helix. A CaMRP1-GFP fusion protein localized primarily to the plasma membrane of plant cells. Strong and early induction of CaMRP1 expression occurred following exposure of pepper plants to Xcv, Colletotricum coccodes, methyl jasmonate (MeJA) and wounding stress. Virus-induced gene silencing (VIGS) of CaMRP1 in pepper conferred enhanced basal resistance to Xcv infection, accompanied by induction of genes encoding basic PR1 (CaBPR1), defensin (CaDEF1) and SAR8.2 (CaSAR82A). In contrast, CaMRP1 overexpression (OX) in transgenic Arabidopsis plants resulted in increased disease susceptibility to Hyaloperonospora parasitica infection. Arabidopsis plants overexpressing CaMRP1 exhibited insensitivity to MeJA by causing reduced expression of MeJA-responsive genes. Overexpression also resulted in tolerance to NaCl and during salt stress, the expression of several abscisic acid-responsive genes was induced. Together, these results suggest that pepper CaMRP1 may belong to a new subfamily of membrane-located receptor-like proteins that regulate disease susceptibility, MeJA-insensitivity and salt tolerance.

Temporal and subcellular localization of PR-1 proteins in tomato stem tissues infected by virulent and avirulent isolates of Phytophthora capsici.

Summary. Immunoblot analysis and immunogold labeling of PR-1 protein (pathogenesis-related protein 1) in tomato (Lycopersicon esculentum Mill.) were performed to examine the temporal and spatial expression patterns of PR-1 protein induced by Phytophthora capsici infection. Soluble proteins with molecular masses of 10, 17, 25, 27 and 75u2009kDa were induced and accumulated in P. capsici-infected stem tissues during the compatible and incompatible interactions. Western blot analysis revealed that expression of PR-1 protein (17u2009kDa), at 12 to 24u2009h after inoculation, occurred earlier in the incompatible than in the compatible interaction. Immunogold labeling of PR-1 proteins occurred over cell walls and cytoplasm of the host and the oomycete pathogen and at the interface between host and oomycete cell walls at 24u2009h after inoculation in the compatible interaction. In the incompatible interaction, numerous PR-1 proteins accumulated predominantly over oomycete cell walls and at the interface between host and oomycete cell walls. The quantity of PR-1 proteins deposited in both host and oomycete cells was much less in the compatible than the incompatible interaction. Healthy tomato stem tissue was nearly free of immunogold labeling of PR-1 proteins.

Distinct roles of the pepper pathogen-induced membrane protein gene CaPIMP1 in bacterial disease resistance and oomycete disease susceptibility.

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.