Identification Of Four Candidate Genes Of Sclerotinia Sclerotiorum Related To Mycovirus Synergism

Sclerotinia sclerotiorum,cause crop rot disease,is a typical necrotrophic plant pathogenic fungus,and results in crop quality reduction and serious yield loss.Mycoviruses are ubiquitous in fungi populations,and multiple viruses infecting a single strain is a common phenomenon in nature.The complex interaction between viruses will affect the pathogenicity of host fungi,antagonism interaction will lead to hypervirulence,while synergism interaction will cause more serious hypovirulence.The previously found that the hypovirulent strain SCH941 harbors six RNA mycoviruses.The hypovirulent strain T3 was obtained by protoplast regeneration of strain SCH941 and co-infected by three mycoviruses including Sclerotinia sclerotiorum botybirnavirus 1(Ss BRV1),Sclerotinia sclerotiorum botybirnavirus 3(Ss BRV3),Sclerotinia sclerotiorum reovirus 1(Ss Re V1),and Sclerotinia sclerotiorum yado-kari virus 1(Ss YKV1),and there was a synergism effect among Ss BRV1,Ss Re V1,Ss YKV1 viruses.Based on previous transcriptome sequencing analysis,we selected four S.sclerotiorum genes of Ss Nmr A(sscle?13g092100),Ss ZFH2(sscle?15g104540),Ss Pho A(sscle?11g084560),and Ss RFX1(sscle?03g025950)that were significantly different in the process of three mycoviruses synergism.In this research,the mycovirus-free strain SCH941A1 and strain T3 were used as research materials to explore the biological function of the four candidate genes of S.sclerotiorum and their roles in the process of viruses synergism.The biological functions of two genes,Ss Nmr A and Ss ZFH2,that were up-regulated during viruses synergism were analyzed.Ss Nmr A encodes a hypothetical protein containing304 amino acids and consists of a conserved domain,PCBER-SDR,that belongs to the REDOX enzyme superfamily.The Ss Nmr A gene knockout transformant was obtained using a gene knockout strategy.The knockout transformant has no significant difference with its paternal strain SCH941A1 in colony morphology and virulence.The knockout transformant was induced three mycoviruses,including Ss BRV1,Ss YKV1,and Ss Re V1,and showed the similar biological phenotypes with the T3 strain,and there was no significant difference in the abundance of virus accumulation,suggesting that the deletion of the Ss Nmr A gene did not affect the synergism effect among Ss BRV1,Ss YKV1 and Ss Re V1.The gene Ss ZFH2 encodes a 361 amino acids hypothetical protein with a C2H2 zinc finger conserved domain at the position of 294-316 amino acids,and is a negative transcriptional regulator.Although the gene of Ss ZFH2 knockout transformant showed no significant difference from SCH941A1 strain in colony morphology and pathogenicity,the knockout transformant co-infected by Ss BRV1,Ss YKV1,and Ss Re V1 showed normal phenotypes,including growth rate and virulence.Moreover,the abundance of mycoviruses was significantly lower than that of the T3 strain.The results showed that the Ss ZFH2 gene was associated with the synergism of the mycoviruses.The biological functions of two genes,Ss Pho A and Ss RFX1,that were down-regulated during viruses synergism were analyzed.Ss Pho A encodes a hypothetical protein containing413 amino acids and consisting of a conserved domain acidic phosphatase(ALP-like).The Ss Pho A gene knockout transformant was obtained using a gene knockout strategy.The knockout transformant has no significant difference with its paternal strain SCH941A1 in colony morphology and virulence.The knockout transformant was induced three mycoviruses,including Ss BRV1,Ss YKV1,and Ss Re V1,and showed similar biological phenotypes with the T3 strain,and there was no significant difference in the abundance of virus accumulation,the results showed that the deletion of the Ss Pho A gene did not affect the synergism effect among Ss BRV1,Ss YKV1,and Ss Re V1.The Ss RFX1 gene encodes a hypothetical protein with a full-length of 821 amino acids,and has a conserved RFX DNA binding domain,which is a candidate transcription factor.The knockout transformants reduced growth rate,pathogenicity,the compound appressoria forming is delayed,is a new S.sclerotiorum disease-causing genes,but the role of this gene in the process of viral cogeneration needs to be further verified.In summary,the biological functions of four genes that up-regulated or downregulated during the synergism of Ss BRV1,Ss Yk V1,and Ss Re V1 were studied in this research.The RFX DNA binding transcription factor gene Ss RFX1 was found to be related to virulence of S.sclerotiorum.The C2H2 type zinc finger protein transcription factor gene Ss ZFH2 plays an important role in promoting viral replication and in virus synergism.These results not only found new genes related to the virulence of S.sclerotiorum,which provided a new genetic resource for control of sclerotinia disease,but also identified gene promoting virus replication in the process of virus co-infection,which provided an important reference for the analysis of the synergism mechanism.