Functional Research On Ssdcl1 And Identification Of Proteins Interaction With Hypovirus SsHV1 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic phytopathogenic fungus,which has a wide host range and limits crop production.Mycoviruses are ubiquitous in fungi,and some of them are related to hypovirulence on phytopathogenic fungi.Using the interaction system between the hypovirulence-associated mycoviruses and phytopathogenic fungi,it is possible to explore the host fungi antiviral defense and viruses anti-defense mechanism,analyze the pathogenic mechanism of phytopathogenic fungi,and enhance the biological characteristics cognition of mycoviruses.Mycoviruses provide theoretical support for the virocontrol sclerotinia disease by utilizing hypovirulence-associated mycoviruses.A hypovirulent strain SZ-150,harboring Sclerotinia sclerotiorum hypovirus 1(Ss HV1)and its satellite RNA(SZ-150-Sat H),exhibits obvious attenuating characteristics including less virulence and growth rate.Previous research suggested that SZ-150-Sat H,a potential silencing suppressor,interacts with Sclerotinia sclerotiorum dicer1 like protein(Ssdcl1).SZ-150-Sat H can significantly increase the accumulation of Ss HV1 due to this interaction and lead to hypovirulent properties of S.sclerotiorum.In this study,strain SZ-150 and its derivative strains were the research materials.By knocking out gene Ssdcl1 and screening interacting proteins with Ss HV1 in S.sclerotiorum,we explored the attenuating mechanism caused by Ss HV1 and SZ-150-Sat H.These findings were obtained as following:Deletion of Ssdcl1,a key gene in RNAi pathway,has no significant effect on the phenotypes of S.sclerotiorum.Two Ssdcl1 knockout transformants(?Ssdcl1-1 and?Ssdcl1-2)and a complementary transformant(?Ssdcl1-1-C)were obtained using PEGmediated protoplast transformation method.The results displayed that Ssdcl1 does not affect biological features including colony morphology,mycelial growth,sclerotia formation,and virulence of S.sclerotiorum.Ssdcl1 was confirmed to involve antiviral response of S.sclerotiorum.Mycoviruses were transmitted horizontally to ?Ssdcl1 mutants,to obtain transformants harboring diverse viruses.Analysis of biological characteristics revealed that the ?Ssdcl1 mutants that were co-infected with Sclerotinia sclerotiorum botybirnavirus 3(Ss BV3),Sclerotinia sclerotiorum mycotymovirus 1(Ss MTV1),Ss HV1 and SZ-150-Sat H exhibited a more severe hypovirulent traits than SZ-150.?Ssdcl1 mutants harboring Ss BV3,Ss MTV1,Ss HV1,and R6-Sat H showed the similar phenotypes to SZ-150,including a fanned colony,deformed,pigmentation and hypovirulence.?Ssdcl1 mutants harboring Ss BV3 shared no noticeable difference compared with the strain R59.The q RT-PCR was conducted to analyze the virus accumulation quantity in individual strain.The results showed that the deletion of Ssdcl1 resulted in a remarkable increase in the accumulation of Ss HV1,indicating that the hypovirulent characteristics of strain SZ-150 were caused by high concentration of Ss HV1 infection.This phenomenon is different from the previously reported that gene Dicer1 in fungal was not involved in RNAi antiviral process.Our study revealed that Ssdcl1 in S.sclerotiorum was involved anti Ss HV1 infection process.We identified six host proteins that interacted with Ss HV1 using yeast hybrid system and point-to-point interaction verification.Four proteins interacting with Ss HV1 UDP glucosyltransferase domain: aflatoxin B1-aldehyde reductase(Ss AKR1),histone promoter control 2(Ss HPC2),hypothetical protein(Ss GPI),putative 40 S ribosomal protein S20protein(Ss S20)were screened in S.sclerotiorum.We found two proteins interacting with the Ss HV1 helicase domain: histone promoter control 2(Ss HPC2)and a hypothetical protein(Ss GPI).Two proteins interacting with RNA-dependent RNA polymerase were obtained: ?-D-Glucosidase(Ss Glu)and a hypothetical protein(Ss HIR).In addition,Ss HV1 can suppress the expression of the genes Ss AKR1 and Ss HPC2.Furthermore,this study provides evidences that Dicer1 of S.sclerotiorum is involved in the RNAi antiviral process.Although Ssdcl1 does not affect the S.sclerotiorum biological phenotype,it is related to the quantification of Ss HV1 replication.In addition,we identified six proteins interacting with Ss HV1 conserved domains in S.sclerotiorum.This advanced research provides a reference for the analysis of fungi antiviral mechanisms and provided foundations for the study of hypovirulence-associated mycoviruses,satellite RNA,and host fungi interaction.