Identification And Functional Analysis Of Key Genes For Tomato Resistance To Powdery Mildew

Tomato powdery mildew caused by Oidium neolycopersici is a kind of fungus with a single-celled conidium produced on conidiosphore, which is a worldwide plant fungal disease distributed in Europe, African, South and North America and Asian. Tomato powdery mildew is responsible for a remarkable reduction in quality and yield of tomato, while insecticide spraying to stop the disease results in environmental contamination. The most effective way to prevent this disease is resistant breeding, which depends on the resistant materials and resistance genes. In the paper, virus induced gene silencing(VIGS)as a functional genomic research platform established was used to identify and functional analyze key genes for tomato resistance to powdery mildew, and resistance mechanism of tomato to powdery mildew was studied in the cellular and genetic levels, which provided theoretical basis for tomato resistant breeding. The main results were as follows:1. The O. neolycopersici Shangqiu isolate was identified as the causual agent of tomato powdery mildew spreading in Shangqiu, which was the first report of O. neolycopersici in China. The pathogen of tomato powdery mildew was studied based on colony morphology, histological structure and internal transcribed spacer (ITS) sequence of rDNA. It indicated that the pathogen was O. neolycopersici. The wild types and cultivated varieties of tomato were inoculated with the fungi for disease test. Tomato cultivated varieties (Solanum lycopersicum) were susceptible to the fungi, while the tomato wild types S. habrochaites G1.1560 (carrying Ol-1 gene)and S. arcanum LA2172 (carrying Ol-4 gene)were highly resistant.2. Active oxygen accumulation, physiological and biochemical changes in different tomato species to powdery mildew fungi were examined in the cellular level. It indicated that the slow accumulation speed and low quantity of active oxygen resulted in incompletely inhibiting the growth of powdery mildew in susceptive tomato, while the fast accumulation and high quantity of active oxygen can inhibit the growth of powdery mildew in resistant tomato successfully, in which the pathogen can’t finish it life cycle. The tomato resistance responses to powdery mildew mediated by Ol-1 or Ol-4 gene were involved with fast hypersensitive reaction. Systemic acquired resistance of tomato induced by unspecialized elicitor– riboflavin was studied by analyzing enzyme activities related to resistant pathway. The results suggested that riboflavin induced physiological changes including H2O2 accumulation, lipid peroxidation, POD and PAL could play a role in the increase of tomato resistance to O. neolycopersici.3. VIGS system was used to analyze the function of the up-regulated candidate gene sequences associated with tomato resistance to powdery mildew. It was found silencing of candidate gene sequences resulted in resistance-breaking phenotype of different degrees in S. habrochaites G1.1560(carrying Ol-1 gene). Microscopic observation showed that O. neolycopersici can finish its whole life cycle and a slow hypersensitive reaction was displayed in epidermal cells of silenced plant leaves compared to fast hypersensitive reaction in control plant. Based on the results of VIGS analysis and microscopic observation, four genes ShGSTU1, ShME1, ShMADSTF and ShORR-1 were identified as key genes for tomato resistance response to powdery mildew. By BLAST, the putative encoding proteins of these genes were predicted, ShGSTU1 encoding glutathione transferases, ShME1 encoding NADP-malic enzyme, ShMADSTF encoding MADS box transcription factor, and ShORR-1 encoding an unknown function protein. Primers for obtaining complete sequences of ShGSTU1、ShME1 and ShORR-1 were designed based on gene walking in tomato genome sequence. PCR products of ShGSTU1、ShME1 and ShORR-1 amplified from S. habrochaites G1.1560 genemic DNA were sequenced to get the complete sequence. The GeneBank code of ShGSTU1 was JF957860, ShME1 was JF957861.