Isolation And Characterization Analysis Of Pathogen-related CaWRKY Transcription Factors In Hot Pepper

Plants have evolved excellent defense mechanisms to protect themselves from biotic and abiotic stresses. One common feature of active plant responses to environmental stresses is transcriptional activiation or repression of specific sets of genes. A key step in induced plant resistance is the timely recognition of invading pathogen, which can trigger a lot of transduction pathways and subsquently induce multiple pathogenesis-related (PR) genes to enhance resistance to a broad spectrum of virulent pathogens. In this reaction, transcription factors play a critical role with related proteins to activate or repress the expressions of defense genes. Overexpression of one defense gene will improve the resistance to specific pathogen, whereas overexpression of a transcription factor will activate the expression of multiple downstream PR genes. Therefore, a better understanding of the function of stress-inducible transcription factors has fundamental importance in dissecting the molecular basis of interactions between plants and environment. It is a novel strategy to breed stress-resistant crops.The WRKY proteins comprise a superfamily of transcription factors that are unique to plant kingdom. Family members appear to be involved in the regulation of various physiological programs, including leaf senescence, trichome development, embryogenesis and seed development in plant, and particularly in biotic or abiotic stresses responses.The material of this study is the pepper cultivar, SCM334 which is resistant to Phytophthora capsici Leon. Using degenerate PCR primers that were designed from the highly conserved DNA-binding domain and other conserved motifs of WRKY proteins, six CaWRKY fragments were isolated from cDNA of pepper leaves that are treated with salicylic acid. Blast results indicate that five of them are new CaWRKY genes in pepper. CaWRKY2 contains two WRKY domains and others contain one. The phylogenetic trees indicate that CaWRKY1,2,3,4,6 are phylogenetically closer than CaWRKY5. The Solanaeae WRKY protein, group III of Arabidopsis WRKY sequences and six Capsicum annuum WRKY partial amino acid sequences were analyzed together. The resulting phylogenetic tree showed that the evolution relationship of six CaWRKY partial sequences is closer to Solanaeae WRKY protein than Arabidopsis WRKY group III members. CaWRKY2,3,4,6 amino acid sequences are similar to NtWRKY1,2,4. Data indicate that NtWRKY1,2,4 possiblely involve in elicitor-responsive transcription of defense genes in tobacoo. Thereby, we presume that CaWRKY2,3,4,6 play similar roles as NtWRKY1,2,4 in defense responses.After phylogenetic analysis, we chose CaWRKY2 for further analysis. We obtained partial 3'cDNA sequence using the experiment of Rapid Amplification of cDNA Ends. It is 1265bp, and the open reading frame of CaWRKY2 cDNA encoded a polypeptide of 290 amino acid residues. CaWRKY2 contains one WRKY domain and a C₂H₂ zinc-finger-like motif at its N-terminal end and C-terminal end respectively. Therefore, we suggest that CaWRKY2 belongs to WRKY family group I .To determine whether elicitors induce expression of CaWRKY2 gene, we analyzed the transcript levels of CaWRKY2 gene induced in SA, BABA and MeJA-treated pepper plants respectively. Theresults indicated that CaWRKY2 induced strongly in SA and BABA-treated pepper leaves, on the contrary, it wasn't induced in MeJA-treated pepper leaves. Thereby, this data show that CaWRKY2 may take part in SA signal transduction.We also have studied whether CaWRKY2 is induced during the infection of Phytophthora capsici Leon and root knot nematode. Northern blotting analysis showed that mRNA for CaWRKY2 were expressed constitutively at a low level in Phytophthora capsici Zeow-treated pepper plants;by contrast, the transcript of CaWRKY2 were accumulated strongly in nematode -treated pepper plants. Therefore, this data indicated that CaWRKY2 may not play a specific function to Phytophthora capsici Leon infection, and should play an significant role in resistance responses to nematode.In conclusion, CaWRKY2 may play an important role in regulation of gene expression in pepper defense responses. Pepper is an important economic vegetable in our country. However, lots of pathogens such as Phytophthora capsici Leon and root knot nematode will decrease the production of hot peppers dramatically. Based on the above, it is a potential way to improve the pepper resistance by overexpression ofCaWRKY2. Meanwhile, to reveal the molecular mechanisms of pathogen resistance in pepper plants is much important.