Molecular Characterizations Of RNA Viruses And The Biological Function Of The Satellite RNA SatH In The Hypovirulent Strain SZ-150 Of Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum?Lib.?de Bary,an important necrotrophic phytopathologenic fungus with worldwide distribution,has a wide range of hosts and causes significant yield loss on crops.Many mycoviruses have been characterized and some of them could confer hypovirulence on S.sclerotiorum.Those hypovirulence-associated mycoviruses not only have the potential to develop virocontrol agents to combat sclerotinia disease,but also will supply new insights on understanding the molecular mechanism of pathogenicity of S.sclerotiorum and biological function of mycoviruses.A hypovirulent strain SZ-150 of S.sclerotiorum was previously reported to be coinfected by Sclerotinia sclerotiorum hypovirus 1?SsHV1?and its related satellite RNA?SatH?.It is interesting that strains infected with SsHV1 but lacking SatH shows normal phenotypes.Based on the previous research,three new conclusions were obtained in the present research:?1?mycoviruses were finally determined in strain SZ-150 via deep sequencing of small RNAs,?2?the biological function of satellite RNA SatH was explorated by the techniques of transgenic and protein interaction,and?3?the molecular mechanism of hypovirulence of strain SZ-150 was analyzed by m RNA-seq.Two novel mycoviruses and mycovirus-derived small RNAs were determined by deep sequencing of small RNAs in strain SZ-150,and the effects of the mycoviruses on the biological characteristics of the host were also analyzed.Previously,we reported that the hypovirulent strain SZ-150 is coinfected by SsHV1/SZ-150 and its related satellite RNA?SatH?.Here,we detected two other mycoviruses,Sclerotinia sclerotiorum botybirnavirus 3?Ss BV3/SZ-150?and Sclerotinia sclerotiorum mycotymovirus 1?Ss MTV1/SZ-150?,coinfecting strain SZ-150,by deep sequencing and assembly of mycovirus-derived small RNAs.The genome of Ss BV3 was found to be composed of two linear ds RNA segments,6,212 bp,and 5,880 bp in size,respectively.Each ds RNA segment of Ss BV3 contains a large open reading frame?ORF?encoding RNA-dependent RNA polymerase?Rd Rp?and a hypothetical protein with unknown function.The whole genome of Ss BV3 shares more than 95%sequence identity with Botrytis porri botybirnavirus 1?Bp BV1?at the nucleotide?nt?or amino acid?aa?level.Thus,Ss BV3 was assumed to be a strain of Bp BV1.The genome of Ss MTV1 consists of 6,391 nt excluding the poly?A?tail.Ss MTV1 was predicted to contain a large ORF that encodes a putative replication-associated polyprotein?RP?with three conserved domains of viral RNA methyltransferase?Mtr?,viral RNA helicase?Hel?,and Rd Rp.Phylogenetic analyses suggested that Ss MTV1is related,albeit distantly,to members of the family Tymoviridae.Analysis of the small RNAs derived from Ss BV3 and Ss MTV1 revealed that small-RNA lengths mainly range from 20 to 24 nt,with a peak at 22 nt,and the most abundant5'-terminal nucleotide is uridine,suggesting that the Dicer 2 and Argonaute 1,two key components in the RNA inference pathway,may play important roles in the resistance to mycoviral infection in S.sclerotiorum.Combined with the RT-PCR results,the biological characteristics of strains carrying different virus combinations were analyzed,suggesting that Ss BV3 had no significant effect on host biological characteristics,while Ss MTV1 has limited impact on host.Those resluts suggested that SsHV1 and SatH are key factors related to abnormal colony morphology and hypovirulence in strain SZ-150.The satellite RNA SatH was confirmed to play important roles in hypovirulence on S sclerotiorum.The transformants?R6-SatH and A6-SatH?that were overexpressed SatH gene in strains R6?infection with SsHV1?and A6?without mycovirus?,respectively,were obtained by constructing the full-length c DNA infectious clone vector of SatH.There were no significant differences between transformants A6-SatH and its parental strain A6 in colony morphology,growth rate,and virulence,but the sclerotium produced by transformants A6-SatH are smaller,suggesting that only SatH without SsHV1 could not cause hypovirulence on S.sclerotinia.Compared to the R6 strain,transformants R6-SatH grew slowly on PDA plates,and developed abnormal colony morphology with many sectors at the colony margin,and had lower virulence on its host.Moreover,three transformants R6-SatH-1,R6-SatH-4,and R6-SatH-5 have no significant difference to strain SZ-150 in colony morphology,growth rate,and virulence.These results suggested that SatH is an important factor related to the abnormal colony morphology and hypovirulence in strain SZ-150.The accumulation of Ss BV3 and Ss MTV1 in strain SZ-150 was significantly lower than that in strain R6,whereas the accumulation of SsHV1 in strain SZ-150 was significantly higher than that in strain R6,suggesting that SatH can significantly increase the accumulation of its helper virus SsHV1.These results supported that the hypovirulent traits of strain SZ-150 are caused by coinfection with SsHV1 and SatH.Yeast hybrid and pull-down experiments confirmed that SatH could interact with the Dicer1 protein and suppress the expression of Dicer1 in S.sclerotinia.Thus,SatH interacts with the Dicer1,and results in suppressing RNAi anti-virus response in strain SZ-150,which can increase the accumulation of its helper virus SsHV1 in strain SZ-150,which causes the phenotype abnormality and hypovirulence of strain SZ-150.The molecular mechanism of hypovirulence of strain SZ-150 was investigated.Compared to the R6 strain,the hyphae of strain SZ-150 is dense and has more branches.Most of the organelles were seriously damaged,the cell wall thickened,and the cells were surrounded by particulate substance.Virulence assay revealed that SZ-150 failed to form infection cushions on rapeseed leaves.The oxalic acid yield produced by SZ-150 is reduced and the time is delayed compared to strain R6.The expression levels of the whole transcripts of strains SZ-150 and R6 cultured on PDA for 12 h and 24 h were obtained by high-throughput sequencing.The results showed that 2210 genes in total were differentially expressed when cultured on PDA for 12 h,of which 1140 genes were up-regulation and 1070 genes were down-regulation,respectively.2238 genes were differentially expressed when cultured on PDA for 24 h,of which 1343 genes were significant up-regulation and 995 genes were significant down-regulation,respectively.These differentially expressed genes are mainly involved in oxidation-reduction process,metabolic processes,transmembrane process,transcription and translation of S.sclerotinia.G protein signal pathway,Ca²⁺signal pathway and MAPK pathway in strain SZ-150 can be affected by SsHV1 and its satellite RNA SatH.In summary,we showed that strain SZ-150 is finally confirmed to be coinfected by Ss BV3/SZ-150,Ss MTV1/SZ-150,SsHV1/SZ-150 and its satellite RNA.SsHV1/SZ-150 and SatH are nessary for conferring abnormal colony morphology and hypovirulence in strain SZ-150.SatH and SsHV1 co-infections can affect the expression of many genes in strain SZ-150,and SatH interacts with the Dicer1 of S.sclerotinia,then suppress the RNAi in strain SZ-150,increase the accumulation of SsHV1/SZ-150 in strain SZ-150.