Functional Analysis Of MAPK Signaling Pathway And Autophagy In Sporisorium Scitamineum Mating/Filamentation

The basidiomycetous fungus Sporisorium scitamineum causes sugarcane smut that leads to severe economic losses in the major sugarcane growing areas in China,India and Brazil.However,the molecular mechanism of S.scitamineum pathogenicity is poorly understood.In this study,we investigated the functions of two important cellular pathway: MAPK signal transduction and autophagy,in S.scitamineum mating/filamentation,as follows:1.We identified the S.scitamineum MAP kinase Kpp2 by using the Candida albicans Cek1 protein sequence,named the coding gene as SsKPP2.Alignment of the SsKpp2 with it’s ortholous proteins demonstrated that there is an invariant TXY motif among the MAP kinases,which has been shown to be phosphorylated upon stimulation through its MAPK signaling cascade.2.We generated SsKPP2 deletion mutants and found that mating of sskpp2Δ × WT(wild-type)sporidia was suppressed,and sskpp2Δ × sskpp2Δ could not mate at all.The result reveals that SsKPP2 regulates S.scitamineum mating/filamentation.Addition of cAMP could effectively restore mating between WT and sskpp2Δ or between two sskpp2Δ mutants,indicating a partial overlap between cAMP/PKA and MAPK signaling pathways in fungal mating/filamentation.Additiong of the established fungal quorum-sensing molecule tryptophol could fully restore mating between WT and sskpp2Δ but it could not restore mating between two sskpp2Δ mutants.Further more,we found that tryptophol could suppress sporidial growth of the sskpp2Δ mutnat.Overall,our results showed that SsKPP2 MAPK signaling pathway may regulate S.scitamineum growth and mating/filamentation likely through modulating tryptophol metabolism.3.We identified the S.scitamineum ATG8 gene,named SsATG8,by TBLASTN using the S.cerevisiae Atg8 protein sequence as a bait.Alignment of the SsAtg8 protein with its othorlogs in other smut fungi and model organisms showed a high degree of conservation in their amino acid sequence,including the conserved C-terminal glycine residue subject to post-translational modification,which is essential for autophagy induction.4.We generated SsATG8 deletion mutants by complete replacement of the SsATG8 gene with the recombinant hygromycin(HPH)-resistant selection marker.Mating/filamentation was not affected in the ssatg8Δ mutants.We also found the sporidial growth was not affected by loss of SsATG8 function.However,we noticed that ssatg8Δ sporidial colony appeared wrinkle,less smooth compared to the wild type,when cultivated on solid medium in an extended period(> 7 d).The microscopic observation showed that newly formed sporidia budding was defective in fission from it’s mother cell,thus causing the formation of elongated pseudohyphae.Therefore we conclude that SsATG8 affects S.scitamineum morphogenesis.Eventually,we tested the stress tolerance of the mutants,and found that mutants were hypersensitive to nitrogen starvation and oxidative stress,demonstrating that SsATG8 plays an important role in stress resistance.