Research On The Biological Functions And Application Of Sumoylation Pathway In Botrytis Cinerea

Small ubiquitin-related modifiers are one kind of ubiquitin-like proteins.Sumoylation pathway regultes the localization and biological function of target proteins,which is a reversible process.SUMO is processed by cysteine-specific SUMO proteases(ULPs/SENPs),then exposes a C-terminal Gly-Gly motif.The matured SUMO is adenylated by the E1 SUMO activating enzyme(Aos1 and Uba2)and transferred to the catalytic Cys of the Uba2 subunit.Through thioester linkage,SUMO is then transferred to catalytic Cys of the E2 SUMO conjugating enzyme(Ubc9).E3 SUMO ligases enzyme can facilitate transfer of SUMO from the E2 conjugating enzyme to the targets.Sumoylated proteins can be desumoylated also by cysteine-specific SUMO proteases.To data,the studies of the sumoylation pathway mainly focus on yeast and mammalian cells.Although some studies were performed in Candida albicans,Aspergillus nidulans and Aspergillus flavus,the regulatory mechanism of sumoylation is not clear.Therefore,we explored the functions of sumoylation pathway in B.cinerea,which will help to understand the biological functions of sumoylation pathway in fungi.In this study,we characterized the functions of key components(Bc Ulp1,Bc Smt3,Bc Uba2,Bc Aos1 and Bc Ubc9)in sumoylation pathway of B.cinerea by the approaches of gene deletion and complementation.Our study found that 1)Compared to the wild type,the mutants ?Bc Ulp1,?Bc Smt3,?Bc Uba2,?Bc Aos1 and ?Bc Ubc9 exhibits reduced mycelial growth and dramatically decreased conidiation.The growth defects of ?Bc Uba2,?Bc Aos1 and ?Bc Ubc9 were more serious than those in ?Bc Ulp1 and ?Bc Smt3.Moreover,?Bc Uba2,?Bc Aos1 and ?Bc Ubc9,but not ?Bc Ulp1 and ?Bc Smt3 displayed decreased pigment synthesis.2)All the mutants exhibited reduced virulence in comparison with the wild type.The growth defects of ?Bc Uba2,?Bc Aos1 and ?Bc Ubc9 displayed even lower virulence than ?Bc Ulp1 and ?Bc Smt3.3)All mutants showed supersensitivity to DNA damage and low temperature stresses,and increased sensitivity to antifunal compounds fludioxonil and rapamycin.4)Using the affinity capture assay,we identified two Bc Ubc9-interacting proteins Bc Cof A and Bc Hsp70.Further,we confirmed the interaction with the two-yeast hybrid assays,and found that the mutants of Bc Cof A and Bc Hsp70 displayed increased sensitivity to low temperature stress compared with the wild type.Taken together,our study found sumoylation pathway regulates mycelial growth,conidiation,virulence and responses to low temperature and DNA damage stresses,which would extend the understanding of sumoylation pathway in fungi.