| Late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is an economically important disease of tomato. The most effective way to prevent this disease is resistant breeding, which depends on understand interactions mechanism between tomato and P. infestans. WRKY transcription factors are key regulatory components of plant responses to biotic and abiotic stresses. Only a few members of the WRKY transcription factors from tomato have hitherto been functionally characterized, the function of the majority tomato WRKY members remains unknown. There have been no reports to date investigating the biological functions of tomato WRKYs directly involved in the tomato plant-P. infestans interaction. Solanum pimpinellifolium L3708 has been identified as a particular source of late blight resistance in tomato. In the present study, we isolated some WRKY genes during the incompatible interaction between S. pimpinellifolium L3708 and P. infestans, and then investigated their expression patterns as well as function in Solanum pimpinellifolium L3708 and susceptible cultivars Solanum lycopersicum Zaofen NO.2. The main results are as follows:(1) Three P. infestans-induced WRKY genes, designated as SpWRKYl, SpWRKY2 and SpWRKY3, were isolated from late blight resistant tomato varieties S. pimpinellifolium L3708 using in silico cloning strategies and RT-PCR methods. These genes encoded 535,508 and 460 amino acids, respectively. There were hydrophilic proteins, no transmembrane regions and signal peptide, contain multiple phosphorylation sites. A subcellular localization analysis predicted that they may exist in the nucleus. Domain prediction of the full-length deduced protein clearly showed that they contained two typical WRKY domains and two zinc finger motifs and belonged to group I WRKY transcription factors.(2) The expression pattern of three SpWRKY genes was characterized. SpWRKYl, SpWRKY2 and SpWRKY3 showed low expression in root and stem, whereas relatively high expressions were observed in leaf. The results of qRT-PCR analysis indicated that the transcript of three SpWRKY genes could be induced by P. infestans, and their transcript induction in tomato wild species resistant S. pimpinellifolium L3708 were higher than that in susceptible cultivars S. lycopersicum Zaofen NO.2 in all the inoculated time points. Furthermore, the peak expressions of SpWRKYl and SpWRKY3 were higher than SpWRKY2. SpWRKY genes transcript were also rapidly and significantly increased after salt and drought treatment, but the peak expression of SpWRKYl was higher than SpWRKY2 and SpWRKY3. Their expressions were also significantly induced following treatment with salicylic acid (SA), methyl jasmonate (MeJA) and abscisic acid (ABA).(3) The functions of SpWRKYl and SpWRKY3 in defense responses to pathogen were confirmed. Virus induced gene silence (VIGS) studies showed that silence of SpWRKY1 or SpWRKY3 decreased resistance to P. infestans infection. The silenced plants exhibited reduced mRNA accumulation of several pathogenesis-related (PR) genes. Overexpression of SpWRKY1 and SpWRKY3 in susceptible cultivars tomato S. lycopersicum cv. Zaofen NO.2 results in transgenic tomato plants markedly increased resistance to P. infestans than untransformed wild-type plant, mainly demonstrated by lower H2O2 and superoxide anion radicals (O2-) accumulation, malonaldehyde (MDA) content and relative electrolyte leakage (REL); and higher peroxidase (POD), superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL), chlorophyll content and photosynthetic rate. This resistance was also coupled with enhanced the expression of PR genes, antioxidant enzyme-related genes and salicylic acid (SA)/jasmonic acid (JA) biosynthesis-related genes. Heterologous expression of SpWRKYl in tobacco conferred transgenic plants greater resistance to Phytophthora nicotianae infection.(4) The functions of SpWRKY1 in defense responses to salt and drought were confirmed. Transgenic tomato also displayed an enhanced tolerance to salt and drought stress by decreasing reactive oxygen species (ROS) generation, improving POD, SOD and proline content, keeping the relative water content and chlorophyll content, and protecting photosynthetic rate, accompanied by enhanced expression of some stress-related genes in response to salt and drought stress. In addition, transgenic tobacco plants also displayed an enhanced tolerance to salt and drought stress.In conclusion, overexpression of SpWRKY1 gene positively regulates tomato resistance to P. infestans infection and tolerance to salt and drought stress, through ROS scavenging, facilitating osmotic adjustment, maintaining higher activities of defense-related enzymes, and involving different signaling pathways and defense-related genes regulation. |
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