Functional Study On Sscut2 And Ssmfs1 From Sclerotinia Sclerotiorum

In this study,two genes Sscut2?Cutinase?and Ssmfs1?Major Facilitator Superfamily?of Sclerotinia sclerotiorum were cloned,and prokaryotic expression and transient expression of Ss CUT2 were conducted.At the same time,we adopted the gene knockout strategy of homologous recombination?spliting-marker method?,and PEG-mediated protoplast transformation method was used to knock out Sscut2and Ssmfs1,and obtained knockout mutations of Sscut2 and Ssmfs1 Strain.The mycelial tip purification method and single ascospore isolation were used to obtain homozygous knockout mutants.The recombinant and transient expression of Ss CUT2 can cause hyposensitiveresponse?HR?like cell deathon leaves of Nicotiana benthamiana.DAB staining revealed that a large amount of a red-brown color was produced on the necrotic leaves,speculated that Ss CUT2 could induce the reactive oxygen species burst of plant and has the function like an elicitor.the tolerance of knockout mutants of Sscut2,?Sscut2-5,Sscut2-7 and?Sscut2-17 strainsto five enviromental stress,showed that the mutant strains were more sensitive to 0.01%SDS,6 m M H₂O₂ and 1.2 M sorbitol,and their colony diameter was significantly decreased;while under the 0.5 M Na Cl and Congo red stresses,the mutant strains showed less sensitive;under the treatment of 0.3?g/m L carbendazim,the gene Sscut2 is not involved in regulating the resistance of S.sclerotiorum to fungicides.During the formation of the mycelial tip branch and the infection cushions,the hyphal tips?apical branching?of the mutants increased and shorter,the number of infection cushions showed smaller.The?Sscut2mutants showed a significantly reduced of pathogenicity on the leaves of oilseed,soybean,N.benthamiana,Arabidopsis and tomato,with the smaller lesion area than that of wild type.The tolerance of knockout mutants of Ssmfs1,?Ssmfs1-1 and?Ssmfs1-2 strainsto five enviromental stress,showed that the mutant strains were more sensitive to 0.01%SDS,and their colony diameter was significantly decreased;other 4 the mutant strains showed less sensitive;under the treatment of 0.3?g/m L carbendazim,the gene Ssmfs1 is not involved in regulating the resistance of S.sclerotiorum to fungicides.During the formation of the mycelial tip branch and the infection cushions,the hyphal tips?apical branching?of the mutants increased and shorter,the number of infection cushions showed smaller;The?Ssmfs1 of the mutants showed a significantly reduced of pathogenicity on the leaves of oilseed,soybean,N.benthamiana,Arabidopsis and tomato,with the smaller lesion area than that of wild type.In summary,the Sscut2 does not affect the growth and development of S.sclerotiorum,but participates in the infection process of S.sclerotiorum.The Ssmfs1 has no effect on the growth and development of S.sclerotiorum,but itis related to the pathogenic mechanism of S.sclerotiorum,and is a gene related to the pathogenicity of S.sclerotiorum.