Functional Analysis Of Tomato SpWRKY6 Transcription Factor In Disease Resistance

Late blight caused by the oomycete pathogen Phytophthora infestans, which causes a major threat to tomato production, and it always led to severe economic loss. The predominant method to control for late blight is through frequent use of fungicides and traditional breeding method. However, fungicides application is costly and has a negative impact on environmental safety and human health. The traditional breeding method cultivates resistant varieties, possessing some drawbacks such as cycle long. As the development of biotechnology, the improvement of tomato is performed by genetic engineering method. Obtaining excellent resistance varieties has become the novel strategy and method of tomato molecular breeding. In the previous study, WRKY transcription factors play vital roles in plant disease resistance. However, research on the tomato is rarely reported. Screening of tomato WRKY transcription factors involving in disease resistance explore it functional mechanisms which provide the candidate genes for cultivate new varieties tomato of disease resistance and provide the practical evidence for further work of molecular breeding.In our laboratory, we have obtained WRKY gene, named SpWRKY6. Bioinformatics results predicated that the estimated molecular weight was 182.98 kDa. Its isoelectric point was 4.90. It was unstable hydrophobic protein composed by 739 amino acids. It did not harbor transmembrane region and signal peptide. A subcellular localization analysis predicated that the protein may exist in the nucleus. The SpWRKY6 protein contained two WRKY domains and C2H2-type zinc finger motifs. Therefore, it was classified as a group ? WRKY transcription factors. The promoter analysis showed that SpWRKY6 has biotic and abiotic stress responsive elements as well as genevestigator online software predicted that it might be affected by biotic and abiotic stress. Performing analysis of expression patterns by qRT-PCR, the results suggested that the gene expressed in the root, stem and leaf. However, the highest expression was appeared in leaf. Phytophthora infestans, Botrytis cinerea, salicylic acid, methyl jasmonate, abscisic acid, salt, drought, heat, cold and HgCl2 can easily induce its expression, which is in accordance with the bioinformatics results.To further investigate its function, SpWRKY6 was firstly silenced in the late blight resistant tomato varieties Solanum pimpinellifolium L3708 by virus induced gene silencing method. The results showed that silencing tomato plants attenuated resistance to P. infestans.The expression levels of pathogenesis-related genes were reduced in silencing tomato plants compared with control plants. Secondly, constructing plant pBI121-SpWRKY6 expression vector, the fused construct of pBI121-SpWRKY6 was transformed into the leaf disk of cultivated tomato Solanum lycopersicum cv. Zaofen NO.2 by Agrobacterium tumefaciens strain GV3101, obtaining transgenic tomato plants. After inoculation with P. infestans, the results suggested that transgenic plants resulted in markedly increased resistance to P. infestans, mainly demonstrated by lower H2O2 and O2- production, MDA content and REL, but higher POD, SOD, PAL and chlorophyll content. This resistance was also coupled with enhanced expression of some defense-related genes. Heterologous expression of SpWRKY6 in tobacco also exhibited increased resistance to Phytophthora nicotianae.