Expression And Functional Analysis Of Two WRKY Genes Encoding Transcription Factors Isolated From Powdery Mildew-resistant Chinese Wild Vitis Pseudoreticulata 'Baihe-35-1'

Disease responses of plants are regulated by multiple signaling pathways. Transcription control of the expression of disease-resistance genes is a crucial part of plant response to a range of disease. WRKY transcription factors are a large family which is mainly involved in transcriptional regulation associated with immune response. Cultivated grapevine (Vitis vinifera) is susceptible to many pathogens, while Chinese wild grapevines are often resistant to pathogens. To utilize the valuable wild recourse, exploring for promising genes from wild grapevines is the first step for generating transgenic resistant varieties of V. vinifera. In this research, two genes encoding WRKY transcription factors were isolated from Chinese wild V. pseudoreticulata W. T. Wang'Baihe-35-1'. The expression and function of the two genes were studied. The main results were as follows:1. Full lengths of the two WRKY genes were cloned from Chinese wild V. pseudoreticulata W. T. Wang'Baihe-35-1'by using RACE technique based on the EST sequences, and were designated as VpWRKY1 (GenBank accession no. GQ884198) and VpWRKY2 (GenBank accession no. GU565706). VpWRKY1 cDNA was 1157 bp, encoding a polypeptide of 322 amino acids, while VpWRKY2 was 1607 bp, encoding a polypeptide of 499 amino acids. Phylogenetic analysis showed that VpWRKY1 and VpWRKY2 belong to group III and I, respectively, of the WRKY superfamily.2. Results of qRT-PCR showed that expression of VpWRKY1 and VpWRKY2 was induced by Erysiphe necator infection during 6 to 12 hpi in eleven grapevine (The five E. necator-resistant grapevines are Chinese wild V. pseudoreticulata W. T. Wang'Baihe-35-1','Baihe-13','Baihe-13-1','Guangxi-1', and'6-12-6'that is a cross between Chinese wild V. pseudoreticulata W. T. Wang'Baihe-35-1'and V. vinifera L. cv. Carignane. The six E. necator-susceptible grapevines were V. vinifera L. cv. Carignane, Chinese wild V. pseudoreticulata W. T. Wang'Guangxi-2','Hunan-1','Shangnan-2','Baihe-35-2', and'6-12-2'that is another cross between Chinese wild V. pseudoreticulata W. T. Wang'Baihe-35-1'and V. vinifera L. cv. Carignane). In addition, basal VpWRKY1 transcript level in'Baihe-35-1'was not significantly induced by Eth and MeJA but was slightly induced by SA at 3 hpt (Fig. 1c, d, e). In contrast, VpWRKY2 was induced rapidly by these three signaling molecules with SA as the strongest inducer. Furthermore, results of semi-quantitative RT-PCR showed that expression of VpWRKY1 was strong in stems, followed by in tendrils and leaves, and was almost undetectable in roots, while expression of VpWRKY2 was also strong in stems, but followed by in leaves, and was weak in roots and tendrils.3. Transient expression of VpWRKY1 and VpWRKY2 showed that both proteins are localized in the nucleus of onion epidermal cells by using particle bombardment. Besides, VpWRKY1 and VpWRKY2 can activate reporter GUS expression by binding to the 140-bp promoter fragment that contains three W-boxes by using Agra-bacteria infiltration. Moreover, VpWRKY1 can activate reporter genes expression in yeast by using yeast one hybrid.4. Ectopic over-expression of VpWRKY1 or VpWRKY2 in Arabidopsis results in enhanced resistance to Erysiphe cichoracearum. What's more, ectopic over-expression of VpWRKY2 in Arabidopsis results in enhanced tolerance to Phytophthora parasitica.5. Over-expression of VpWRKY1 and VpWRKY2 regulate the expression of defense maker genes AtPR1,AtPR10,AtNPR1,AtCOR1 and AtPDF1.2 in Arabidopsis, and transient expression of VpWRKY1 and VpWRKY2 regulate the expression of defense maker genes VpPR1,VpPR10 and VvNPR1 in grapevine leaves, suggesting that expression of defense maker genes is regulated by VpWRKY1 and VpWRKY2.6. Promoter regions of VpWRKY1 and VpWRKY2 were cloned from Chinese wild V. pseudoreticulata W. T. Wang'Baihe-35-1'. The sequences were designated as PvpWRKY1 (GenBank accession no. GU565705) and PvpWRKY2 (GenBank accession no. HQ174899). PvpWRKY1 was 694bp in length, while PvpWRKY2 was 643bp in length. The activity of PvpWRKY1 and PvpWRKY2 was induced by E. necator in leaves of Chinese wild V. pseudoreticulata W. T. Wang'Baihe-35-1'.