The Study Of Gpa3 Regulates Sexual Mating And Stress Tolerance Via CAMP Signalling In Sporisorium Scitamineum

Sugarcane is main raw material of sugar industry and significant biomass energy material.Sugarcane diseases seriously affect yield of sugarcane.Sugarcane smut is one of the most significant diseases.The monophyletic group of smut fungus Sporisorium scitamineum is nonpathogenic.Two haploid cells from compatible mating-type fuse together and form dikaryotic hyphae(2n)to infect the sugarcane.In this study,a T-DNA random insertional mutant library of wild type haploid strain WT17 was established,and sexual mating-defective mutants were screened.HiTAIL-PCR was used to identify the mutant genes and in-depth research on one of the sexual mating-related gene Gpa3 was conducted.Finding of the proposed study are as follows:1)A total 23,000 T-DNA random insertional mutants were generate by Agrobacterium tumefaciens-mediated transformation(ATMT).Fifteen sexual mating-defective mutants were selected among them eight mating-reduced mutants and seven mating-defective mutants.Southern blotting stud ies were showing that all of these mutants have single copy T-DNA insertion.HiTAIL-PCR confirms the presence of Prf1,Gpa3,Rop1 genes and other several hypothetical proteins.2)Further study on the role of Gpa3 gene in sexual mating,homologous recombination knockout Gpa3 gene mutant △g5892 was generated.In which sexual mating was significantly decreased,sharing the consistent phenotype with T-DNA insertion mutant 9W65.3)The results of Realtime-PCR showed that expression of pheromone precursor gene(Mfa1),pheromone receptor gene(Pra1)and c AMP synthetic gene(Uac1,encoding adenylyl cyclase)was significantly reduced in the mutant △g5892,which indicates that,Gpa3 gene is located upstream of these genes.4)Sexual mating of mutant △g5892 and 9W65 was restored after addition of 10 mM exogenous c AMP,resulting white hyphae formed indicating that Gpa3 gene could regulate the sexual mating of S.scitamineum by enhancing the regulation of cAMP signal.5)The pathogenicity of inoculation indicated that the morbidity in the mutant△g5892 was significantly reduced(4.68%)when compared to wild type control(86.49%).6)Growth and sexual mating of S.scitamineum was inhibited by antioxidant glutathione(GSH),high concentration sucrose,glucose and sodium chloride.Mutant △g5892 was more sensitive to these chemicals as compared to wild type.Exogenous addition of c AMP could improve the ability of anti-adversity and restore the ability of sexual mating.The results of Realtime-PCR showed that the expression of glutathione peroxidase gene(GPX2),glutathione-s-transferase gene(GST)and glutathione reductase gene(GXR5)of wild type WT17 and mutant △g5892 was significantly increased in the presence of 10 mM GSH,and it was significantly restored on addition of10 mM c AMP.In this study,a number of sexual mating defective mutants were generated by T-DNA insertional mutagenesis.The intensive research of Gpa3 gene(G protein alpha subunit gene,g5892)showed that Gpa3 regulate the ability of sexual mating and anti-adversity of S.scitamineum by regulating c AMP signal.The development of this study will provide theoretical basis for sugarcane smut prevention and control technology.