Cioning And Functional Analysis Of Isochorismatase Hydrolase Candidate Gene In Verticillium Dahliae From Cotton

Verticillium wilt caused by Verticillium dahliae is one of the most destructive diseases in cotton. Host resistance to V. dahliae is unavailable in most crops, and microsclerotia produced by V. dahliae as survival structures can survive for dozens years in soil, make it difficult to control this pathogen. Little is known about the molecular basis of Verticillium wilt and the molecular mechanisms that V. dahliae employ to cause this disease. Therefore, the study of V. dahliae interactions with host would provide more insights into management of Verticillium wilt. Isochorismatase hydrolase, which was found only in the highly aggressive isolate of V. dahliae, hydrolyzes isochorismate into2,3-dihydroxybenzoate and pyruvate and may inhibit salicylic acid formation, which is important for plant defense response signaling. Thus, the isochorismatase hydrolase gene (VdISO) may act as a plant-defense suppressor in V. dahliae and may inhibit the production of salicylic acid in host after V. dahliae infection. In this study, we cloned the VdISO gene from a highly aggressive isolate of V. dahliae V991and analyzed the functions of the VdISO gene through overexprssion the VdISO gene in Nicotiana tabacum as well as deletion and complementation of VdISO gene in V991.The VdISO gene was cloned from a highly aggressive isolate of Verticillium dahliae V991by RT-PCR and a plant overexpression vector containing VdISO gene was constructed. Tobacco TO plants were obtained through Agrobacterium-mediated transformation with leaf disc. T1plants resulted from selfing were subjected to tobacco mosaic virus (TMV) for analysis of local resistance and systemic acquired resistance. Transgenic plants overexpressing VdISO gene become more susceptible to TMV with more lesions on inoculation leaves than the wild type. The biosynthesis of salicylic acid was compared between the transgenic tobacco and the wild type. The content of salicylic acid in transgenic tobacoo was found only0-16.5%of that in the wild type. Thouth the TMV infection increased the biosynthesis of pathogen-induced salicylic acid, the content of salicylic acid in the transgenic tobacco was no more than40-45%of the wild type at48h after TMV inoculation. RT-PCR revealed that the transcription level of pathogenesis-related (PR) protein genes, including PR-1a, PR-2, PR-3, PR-5and basic PR-1genes, was decreased significantly in transgenic tobacco at48h after TMV inoculation, with a level of8-33%in the wild type. These results suggested that VdISO gene may reduce the biosynthesis of pathogen-induced salicylic acid through the hydrolyzation of isochorismate by isochorismatase hydrolase and inhibit the expression of PR genes in the VdISO gene overexpressing tobacco, resulted to a more susceptible plant.Gene knockout construct and complementary construct for VdISO gene were prepared and used to Agrobacterium tumefaciens mediated transformation of the highly aggressive isolate of Verticillium dahliae and its derived mutant. Two knockout transformats (Aiso12and Aisol8) and the complementary transformats of Aiso12(EctAiso) were obtained and identified by PCR and RT-PCR, respectively. When compared to the wild type, Aiso12and Aiso18bared different colony characteristics with a light yellow colore, less aerial hyphae and11-fold increasing in conidia production, while the complementary transformat EctAiso recovered normal white colore and normal conidia production as that in the wild type. In the aspect of pathogenicity tested in cotton, the knockout transformants Aiso12showed a weaker ability in pathogenicity at the early stage of inoculation (14dpi,21dpi) and a comparable pathogenicity at the later stage of inoculation (30dpi) when compared with the wild type, in contrast to the complementary transformat EctAiso with unaffected pathogenicity as that showed the wild type. The results showed that VdISO gene played a part in pathogenicity of Verticillium dahliae.