Pathogenic Function Analysis Of 2-Oxoglutarate Dehydrogenase Gene And Patatin-Like Phospholipase Gene In Verticillium Dahliae

Verticillium dahliae is a soil-borne plant vascular fungal pathogen,which can attack cotton and cause Vericillium wilt.Functional analysis of growth-and pathogenicity-related genes has become a current research hotspot.2-oxoglutarate dehydrogenase(OGDH)is the key enzyme that catalyzes the oxidation of ?-oxoglutarate in the tricarboxylic acid cycle.Phospholipase hydrolyzes phospholipids into fatty acid and other lipophilic substances.Potential roles of OGDH and patatin-like phospholipase involved in development and pathogenicity of V.dahliae remains unknown.In this study,the function of OGDH and PLP in V.dahliae is characterized by generating gene knockout(?Vd OGDH and ?Vd PLP)and complementary mutants(?Vd OGDH-C and ?Vd PLP-C).Functional characterizations were studied by measuring phenotypic identification,pathogenicity and detecting the transcriptional level of some metabolic pathways related genes.Obtained the following results:1.For OGDH gene analysis: Compared with Vd-wt and ?Vd OGDH-C strains,the growth rate of ?Vd OGDH on Czapek Dox medium contained different carbon sources showed significant decrease.At the same time,mycelium of the ?Vd OGDH mutants expanded and had no typical round branch shape.The transcriptional expression level of TCA related genes(Vd ICL,Vd ICDH,Vd MDH,and Vd PDH)in ?Vd OGDH mutants were significantly increased.?Vd OGDH showed hypersensitive to reactive oxygen stress,the sensitivity increased by about 54%.Cotton plants inoculated with ?Vd OGDH exhibited less disease symptoms than Vd-wt and ?Vd OGDH-C strains.The disease index also decreased significantly.Fungal biomass of cotton seedlings infected with ?Vd OGDH mutants decreased significantly.The results demonstrate that OGDH gene mainly participates in the regulation of the metabolism of carbon source and the vegetative growth of mycelium,and regulates the adaptability of active oxygen stress,thus affecting the pathogenicity of V.dahliae.3.For PLP gene analysis: Compared with Vd-wt and ?Vd PLP-C strains,?Vd PLP mutants were obviously impaired in hyphal growth under the different carbon source culture conditions.The conidium production decreased significantly in ?Vd PLP mutants.?Vd PLP mutants were more sensitive to cell-wall-perturbing agents.Meanwhile,the expression levels of Vd MKK1,Vd MK1 and chitin synthase gene(Vd Chi1,Vd Chi3,Vd Chi4 and Vd Chi7)in the cell wall integrity pathway were downregulated in ?Vd PLP mutants.?Vd PLP cannot penetrate and cross the cellophane membrane.?Vd PLP strains showed distinct impairment in their virulence and ability to colonize Nicotiana benthamiana roots.N.benthamiana plants inoculated with ?Vd PLP mutants showed decreased in disease index and less disease symptoms than Vd-wt and ?Vd PLP-C strains,and the fungal biomass in the tobacco also decreased significantly.PLP regulates hyphal growth and conidial production and is involved in stabilizing the cell wall;PLP mediates the penetration,thus mediating the pathogenicity of V.dahliae.The roles of OGDH and PLP genes in the pathogenicity of V.dahliae were evaluated,providing novel insight into the molecular pathogenic mechanisms of V.dahliae.