The Role Of Vesicular Protein Ss Did4 In Sexual Mating And Resistance To Stress In Sporisorium Scitamineum

The basidiomycetous fungus Sporisorium scitamineum is dimorphic pathogenic fungus,which mainly harmful to sugarcane.Only when two haploid cells from compatible mating-type fuse together and form dikaryotic hyphae（2n）can they infect the sugarcane.Vesicular transport is an important pathway for cell transport of nutrients and signal transduction.In this study,the gene Ss DID4 that involved vesicular transport was identified,and its function in the process of sexual mating and stress resistance of Sporisorium scitamineum was discussed and analyzed.The main results of this study are as follows:（1）In this study,wild-type WT17 was used as the research object.The homologous knockout mutants ssdid4Δand complementary mutant Ss DID4-COM mutants were generated by using PEG mediated protoplast transformation method.The results of FM4-64staining showed that the formation of vesicles in mutant ssdid4Δwas blocked,and recovered in complementary mutant Ss DID4-COM,which indicated that Ss Did4 was essential for vesicle formation.（2）The sexual mating between the mutants and opposite mating type wild-type WT18showed that ssdid4Δsexual compatibility was significantly decreased compared with WT17,which was restored in the complementation strains.Exogenously added sexual pheromones Mfa1 and Mfa2,it was found that both them had no effect on ssdid4Δhaploid,while Mfa2 could induce the hypha formation in both WT and complementation strains haploid cells,but had no effect on ssdid4Δhaploid.However,5 m M exogenous c AMP couldn’t recover the mating of mutant ssdid4Δ.The results of real-time PCR showed that the expression levels of Ss GPA3、Ss UAC1、Ss ADR1 in c AMP-PKA pathway had on effected,while the key genes CRK1,FUZ7,KPP2,PRF1 and downstream genes of a locus and b locus in MAPK pathway were significantly down-regulated in ssdid4Δ.It indicates that Ss DID4 may regulate the MAPK pathway by affecting the transport of pheromone signals and then regulate the sexual mating of S.scitamineum.（3）The growth curve and microscopic observation showed that the growth of ssdid4Δwas notably slower than that of WT17,and the haploid cells presented pseudomycelium-like morphology.The tolerance towards cell wall stressors showed that the mutant ssdid4Δwas hypersensitive towards Congo red（1.0 mg/ml）,but not towards CFW（0.5 mg/ml）.Besides,the tolerance of ssdid4Δtowards metal ions such as Zn²⁺（5m M）,Fe²⁺（7.5 m M）,Cu²⁺（7.5 m M）,Mn²⁺（5 m M）Mg ²⁺（800 m M）was significantly reduced compared to wild-type WT17,as well as SDS（0.004%）and EGTA（2.5 m M）.In addition,the Realtime-PCR was used to detect the beta-glucan synthesis genes,the results showed that the genes involved in the synthesis of theβ-glucan in the ssdid4Δwere significantly down-regulated.These results suggested that Ss Did4 play an important role in maintain the cell wall integrity and Metal ion homeostasis.（4）The results of oxidative stress tolerance assay showed that ssdid4Δwas hypersensitive towards H₂O₂comparied with the WT.The results of realtime-PCR showed that the expression levels of cytochrome P450 genes were significantly down-regulated.These experimental evidences indicated that Ss DID4 likely involved in the tolerance towards oxidant stress by affecting the expression of cytochrome P450 in S.scitamineum.In this study,we firstly found that the gene Ss DID4 plays important role in regulating the sexual mating,cell wall integrity,metal ion tolerance,as well as in the responses towards various stress such as high osmotic pressure and oxidative stress of S.scitamineum.The development of this project will provide a new theoretical basis for the control of sugarcane smut.