Functional Analysise Of Catalase Gene SsCat2 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is a widely distributed plant pathogenic fungus which can infect many crops like rapeseed,soybean,and vegetables,and bring serious economic losses to agricultural.The oxidative burst is an important host defensive response during the early infection of pathogens,and the tolerance to oxidative stress produced by host is critical to the successful infection of pathogens.However,the mechanism of S.sclerotiorum resistant to reactive oxygen species(ROS)is still obscure.In this research,a catalase-coding gene SsCat2 from S.sclerotiorum was studied in depth,which could provide a new idea for clarifying the detoxification mechanism of oxidative stress of S.sclerotiorum and realizing the green control.The SsCat2 protein contains 509 amino acids and has a typical catalase domain.The homologous proteins of SsCat2 are wildly distributed in Ascomycetes.The Bccat5from Botrytis cinerea has highest homology with SsCat2,and the amino acid sequence similarity reaches 94.89%.The expression level of SsCat2 was induced by exogenous H₂O₂ and host plants,suggesting that SsCat2 was closely related to pathogenic process and the response of S.sclerotiorum to oxidative stress.In this study,SsCat2 was knocked out through PEG transformation technology.There was no significant difference in the hyphae morphology and fresh weight of sclerotia between wild-type strain and SsCat2 mutants,but the number of sclerotia per plate of SsCat2 deletion strains was less than that of wild-type strain.SsCat2 deletion strains also grew slower than wild-type strain.During the stage of hyphal growth and sclerotial development,the catalase activity of SsCat2 deletion strains was significantly lower than that of wild-type strain.DAB staining showed that more H₂O₂ was accumulated in the mycelia of SsCat2 deletion strains,and the mycelial growth was more sensitive to exogenous H₂O₂,indicating that SsCat2 was closely related to the response of S.sclerotiorum to oxidative stress.The hyphal growth of SsCat2 deletion strains were more strongly inhibited on the PDA amended with NaCl,sorbitol and sodium dodecyl sulfate(SDS),suggesting that SsCat2 was associated with the integrity of hyphal cell wall.SsCat2 deletion strains showed a significant decrease in pathogenicity to both rape and tobacco,and the infected tobacco leaves accumulated more H₂O₂,indicating that SsCat2 was closely related to the pathogenicity and may be involved in the decomposition of H₂O₂ produced by the host.The SsCat2 deletion strains showed a reduced sensitivity to QoI(Quinone outside Inhibitors)fungicides such as Pyraclostrobin,Trifloxystrobin and Azoxystrobin,while the addition of hydrogen peroxide scavenger dimethylthiourea(DMTU)could help recover its sensitivity.Real-time quantitative PCR(qRT-PCR)showed that the expression of alternative oxidase Ss Aox1 in SsCat2 deletion strain was 3 times higher than that in wild-type strain,and further research showed that Ss Aox1 in S.sclerotiorum was induced by exogenous H₂O₂.These results indicated that the accumulation of H₂O₂in SsCat2 deletion strain activated the alternative oxidases pathway of S.sclerotiorum,resulting in reduced sensitivity to QoI fungicides.To further reveal the mechanism of SsCat2,proteomics sequencing was performed on SsCat2 deletion strain and wild-type strain by DIA(Data-independent Acquisition)technology.1276 proteins with different abundances between SsCat2 deletion strain and wild-type strain were identified,with 626 up-regulated and 650 down-regulated.These differentially expressed proteins were enriched in 114 metabolic pathways,including eukaryotic ribosome formation,metabolic pathways,and secondary metabolite synthesis,indicating that SsCat2 plays an important role in maintaining normal metabolism and biosynthesis of cells.The results of qRT-PCR showed that most of the differentially expressed proteins had the same trend with the transcriptional accumulation of genes.This research showed that SsCat2 was the main active catalase of S.sclerotiorum,which could affect the growth and development of mycelia and sclerotium,it also participated in the process of S.sclerotiorum responding to exogenous oxidative stress and pathogenicity,and affected the sensitivity to QoI fungicides.