The Secretory Factor Antigen1 Influences Microsclerotial Formation And Mycelial Growth In Verticillum Dahliae

Verticillium dahliae Kleb.a phytopathogenic fungus that causes wilt diseases in hundreds of dicotyledonous plant species leading to enormous economic losses,especially in cotton production.V.dahliae mainly infect the vascular bundle,causing them turned grey or brown,leading the transfusio n tissue disruption.The leaves turn yellow,wilt and boll shedding,causing death and yield loss of the cotton.So far there is no effective drug control of the Verticillium wilt.Resistant varieties breeding is the main means to control Verticillium wilt at present.At present,the related research on the molecular genetic basis and pathogenic mechanism of V.dahliae is insufficient.Therefore,in-depth study of this pathogen and its pathogenic mechanism finding out the key pathogenic genes is the key to control Verticillium Wilt.The mainly studied genes related to pathogenicity are concentrated on the secreted proteins which are reacted with hosts.These protein factors of V.dahliae have played an important role in many aspects,such as assisting pathogens into host cells,controlling the immune defense reaction of host cells and so on.Eukaryotic microbial pathogens such as pathogenic fungi,when infecting host,they will secrete a large mount of effector proteins functioning in the host infection process.The function of these protein factors have been gradually clarified with further researches.For V.dahliae,studies have shown that the secreted proteins are the main reason caused the biological toxicity.Thorough researching all secretory protein of V.dahliae genome scale,it will reveal the pathogenic mechanism for the development of searching new agents,the new controlling method and laying the foundation for breeding resistant cultivars.With the development of Verticillium dahliae genome sequencing,researchers began using bioinformatics method to predict genome-wide secreted protein,and analysis of its characteristics and function,provides a solid foundation for the further study on the pathogenic mechanism of V.dahliae.In this study,by predicting a V.dahliae secreted protein,and studied the pathogenicity,physiological and biochemical aspects,we discovered that this protein has nothing to do with its pathogenicity,but it is related with microsclerotia formation and hyphal morphology.V.dahliae produces a melanized resting structure,known as microsclerotia,which is important for this pathogen's long-term survival and primary infection.To explore the molecular mechanisms involved in the development of the important structure,we have identified a secretory factor protein gene antigen1.We construct the ?antigen1 knockout mutant and then studied its function.The main results of this research were showed in following:1.antigen1 gene knockout vector pzptk 8.10-antigen1 was achieved using the fusion PCR;Four antigen1 transformants were achieved by Agrobacterium-mediated T-DNA insertion in V.dahliae V991.The transformants were screened through hygromycin resistance and used for PCR and RT-PCR detection.2.Complementary vector pNTP-antigen1 was achieved and its selected marker is spectinomycin resistance gene.Several positive transformants were obtained by Agrobacterium-mediated T-DNA insertion in antigen1 deleted mutant.According to colony morphology,growth rate,pathogenicity and genetic stability,wild-type similar transformants were screened.The geneticstability,pathogenic ability and phenotypic features of the complemented mutant are quite with wild type.3.By observation of colony morphology,mycelial growth rate,pathogenicity,pressure sensitivity,microsclerotial production and so on,the antigen1 is related with microscertial production and hyphea growth,but has nothing to do with pathogenicity.