Role of the pepper cytochrome P450 gene CaCYP450A in defense responses against microbial pathogens

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 H₂O₂ 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.     

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.     

Molecular and biochemical characterization of the<Emphasis Type="Italic"> Capsicum annuum calcium-dependent protein kinase 3</Emphasis> (<Emphasis Type="Italic">CaCDPK3</Emphasis>) gene induced by abiotic and biotic stresses

The isolated full-length Capsicum annuum calcium-dependent protein kinase 3 (CaCDPK3) cDNA clone was selected from the chili pepper expressed sequence tag database (). 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.     

Characterization of a stress-responsive ankyrin repeat-containing zinc finger protein of Capsicum annuum (CaKR1)

We isolated many genes induced from pepper cDNA microarray data following their infection with the soybean pustule pathogen Xanthomonas axonopodis pv. glycines 8ra. A full-length cDNA clone of the Capsicum annuum ankyrin-repeat domain C(3)H(1) zinc finger protein (CaKR1) was identified in a chili pepper using the expressed sequence tag (EST) database. The deduced amino acid sequence of CaKR1 showed a significant sequence similarity (46%) to the ankyrin-repeat protein in very diverse family of proteins of Arabidopsis. The gene was induced in response to various biotic and abiotic stresses in the pepper leaves, as well as by an incompatible pathogen, such as salicylic acid (SA) and ethephon. CaKR1 expression was highest in the root and flower, and its expression was induced by treatment with agents such as NaCl and methyl viologen, as well as by cold stresses. These results showed that CaKR1 fusion with soluble, modified green fluorescent protein (smGFP) was localized to the cytosol in Arabidopsis protoplasts, suggesting that CaKR1 might be involved in responses to both biotic and abiotic stresses in pepper plants.     

The pepper extracellular peroxidase CaPO2 is required for salt, drought and oxidative stress tolerance as well as resistance to fungal pathogens

In plants, biotic and abiotic stresses regulate the expression and activity of various peroxidase isoforms. Capsicum annuum EXTRACELLULAR PEROXIDASE 2 (CaPO2) was previously shown to play a role in local and systemic reactive oxygen species bursts and disease resistance during bacterial pathogen infection. Here, we report CaPO2 expression patterns and functions during conditions of biotic and abiotic stress. In pepper plants, CaPO2 expression was strongly induced by abscisic acid, but not by defense-related plant hormones such as salicylic acid, ethylene and jasmonic acid. CaPO2 was also strongly induced by abiotic and biotic stress treatments, including drought, cold, high salinity and infection by the hemibiotrophic fungal pathogen Colletotrichum coccodes. Loss-of-function of CaPO2 in virus-induced gene silenced pepper plants led to increased susceptibility to salt- and osmotic-induced stress. In contrast, CaPO2 overexpression in transgenic Arabidopsis thaliana plants conferred enhanced tolerance to high salt, drought, and oxidative stress, while also enhancing resistance to infection by the necrotrophic fungal pathogen Alternaria brassicicola. Taken together, these results provide evidence for the involvement of pepper extracellular peroxidase CaPO2 in plant defense responses to various abiotic stresses and plant fungal pathogens.     

PAD4 functions upstream from salicylic acid to control defense responses in Arabidopsis.

The Arabidopsis PAD4 gene was previously shown to be required for synthesis of camalexin in response to infection by the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 but not in response to challenge by the non-host fungal pathogen Cochliobolus carbonum. In this study, we show that pad4 mutants exhibit defects in defense responses, including camalexin synthesis and pathogenesis-related PR-1 gene expression, when infected by P. s. maculicola ES4 326. No such defects were observed in response to infection by an isogenic avirulent strain carrying the avirulence gene avrRpt2. In P. s. maculicola ES4 326-infected pad4 plants, synthesis of salicylic acid (SA) was found to be reduced and delayed when compared with SA synthesis in wild-type plants. Moreover, treatment of pad4 plants with SA partially reversed the camalexin deficiency and PR-1 gene expression phenotypes of P. s. maculicola ES4 326-infected pad4 plants. These findings support the hypothesis that PAD4 acts upstream from SA accumulation in regulating defense response expression in plants infected with P. s. maculicola ES4 326. A working model of the role of PAD4 in governing expression of defense responses is presented.