Molecular And Biological Characterization Of The Mycoviruses In Sclerotinia Sclerotiorum Hypovirulent Strain AH16

Sclerotinia sclerotiorum is a notorious plant pathogenic fungus,as it can infect many economically-important oil crops,including rapeseed,soybean and sunflower.Diseases caused by this fungus lead to great losses.But there is no good method to control this kind of diseases.Using hypovirulence-associated mycoviruses is a potential biological control method and there are a few sucesseful examples.Therefore,exploitation of novel mycoviruses is valuable for control of diseases caused by S.sclerotiorum.Strain AH16 was isolated from a scleotium collected from a diseased rapeseed plant in Anhui province,China.Compared to the strain Ep-1PNA367 R,AH16 is different in some aspects: suppressed growth rate,reduced sclerotial production and decreased pathogenicity.Three ds RNA segments were detected in strain AH16.By cloning the sequences of these dsRNA segments,two mycoviruses were identified in strain AH16.SsMV4/AH16 has a typical genome of mitovirus.It is a new isolate of Sclerotinia sclerotiorum mitovirus 4?SsMV4?.The complete nucleotide sequence of SsMV4/AH16 was deposited in the GenBank database under the accession number of KT962974.The genome of SsMV4/AH16 comprises 2752 nucleotides with a low GC content?31%?.The 5' and 3'-UTRs are 471 and 85 nts long,respectively.SsMV4/AH16 contains a single ORF,and was deduced to encode a protein of RNA-dependent RNA polymerase?RdRp?when fungal mitochondrial code was applied.Multiple alignment and BLAST search revealed that SsMV4/AH16 RdRp shares high sequence identity?93%?with a strain?SsMV4/NZ1?of Sclerotinia sclerotiorum mitovirus 4?SsMV4?.Phylogenic analysis further supported that SsMV4/AH16 is closely related to members of the family Narnaviridae.SsBRV2 has a bipartite genome consisting of L1-dsRNA and L2-dsRNA.The fulllength cDNA sequences of L1-dsRNA and L2-dsRNA were found to be 6159 bp and 5872 bp,respectively,which were deposited in the Gen Bank database under the accession numbers of KT962972?for L1-dsRNA?and KT962973?for L2-dsRNA?.SsBRV2 has two ORFs,ORF1 in L1-dsRNA and ORF2 in L2-dsRNA.They were homologous to the corresponding segments of three other botybirnaviruses.ORF1,starting at nt position 413 and terminating at nt 6019,was identified on the positive strand of L1-ds RNA,and was deduced to encode a 1868-aa protein?209 kDa?.This deduced ORF1-encoded protein contains an RdRp?RdRp₄ superfamily,E-value = 1.9e-32?domain with eight conserved motifs?I to VIII?.Multiple alignment based on the RdRp conserved domain revealed that SsBRV2 was phylogenetically related to previously reported Sclerotinia sclerotiorum botybirnavirus 1?46% identities?,soybean leaf-associated botybirnavirus 1?38% identities?,Botrytis porri RNA virus 1?55% identities?.The phylogenetic tree also indicated that SsBRV2 formed a well-supported clade with SsBRV1,SlaBRV1 and BpRV1,which was distant from other known dsRNA mycoviruses.ORF2 on the positive strand of L2-dsRNA codes for a putative 1778-aa protein with unknown functions.Various methods,including virus horizontal transmission,protoplast regeneration,and virus particle-transfection were used to determine relative importance of SsBRV2 and SsMV4 in pathogenicity-attenuation in strain AH16.Both mycoviruses?SsBRV2 and SsMV4?failed to transmit horizontally from strain AH16 to strain Ep-1PNA367 R via hyphal contact.By protoplast regeneration,the two mycoviruses were not completely eliminated from strain AH16.By a PEG-mediated virus particles transfection,SsBRV2 was successfully transfected into strain Ep-1PNA367 R.Compared to AH16,the trasfectant strain Ep-1PNA367 RVT showed obvious hypovirulence-asscoiated phenotypes,including reduced mycelial growth,debilitated pathogenicity and no sclerotial production.Moreover,SsBRV2 could transmit from Ep-1PNA367 RVT to Ep-1PNA367 via hyphal contact and the newly SsBRV2-infected strain exhibited a subset of phenotypes similar to those of Ep-1PNA367 RVT.Therefore,we concluded that SsBRV2 confers hypovirulence to S.sclerotiorum.SsBRV2 and SsBRV1 are phylogenetically closely related,but they show different influence to S.sclerotiorum.Ep-1PNA367BRV2?Ep-1PNA367 infected with SsBRV2?and Ep-1PNA367BRV1?Ep-1PNA367 infected with SsBRV1?show different traits including growth rate,virulence and sclerotial production.Compared to Ep-1PNA367,Ep-1PNA367BRV2 shows more obvious hypovirulence phenotypes.But compared to Ep-1PNA367,Ep-1PNA367BRV1 does not show any obvious differences in colony morphology,growth rate and virulence.In order to investigate the different influence between SsBRV1 and SsBRV2 on strain Ep-1PNA367,the transcriptome of Ep-1PNA367,Ep-1PNA367BRV1 and Ep-1PNA367BRV2 were analysed with RNA_Seq technique.The results showed that much more differentially expressed genes?DEGs?were found in SsBRV2-infected strain Ep-1PNA367BRV2 than tin SsBRV1-infected strain Ep-1PNA367BRV1,comparing to virus-free strain Ep-1PNA367.GO analysis of differentially expressed genes between Ep-1PNA367BRV2 and Ep-1PNA367 shows that there are 45 most significantly enriched categories including oxidation-reduction process,carbohydrate metabolic process,oxidoreductase activity,metabolic process,transmembrane transport,catalytic activity,hydrolase activity and integral component of membrane.Go analysis of differentially expressed genes between Ep-1PNA367BRV1 and Ep-1PNA367 shows only 17 most significantly enriched categories.KEGG analysis of differentially expressed genes between Ep-1PNA367BRV2 and Ep-1PNA367 shows 18 most significantly enriched categories,whereas only 5 between Ep-1PNA367BRV1 and Ep-1PNA367.There are 175 common genes upregulated in Ep-1PNA367BRV2 and Ep-1PNA367BRV1,of which Go analysis shows 11 most significantly enriched categories mainly in hydrolase activity,carbohydrate metabolic process.The number of genes expressed differentially in Ep-1PNA367BRV2 VS Ep-1PNA367BRV1 is 1961,1174 upregulated only in Ep-1PNA367BRV2,679 genes downregulated only in Ep-1PNA367BRV2,86 genes upregulated in Ep-1PNA367BRV2 but downregulated in Ep-1PNA367BRV1,22 genes downregulated in Ep-1PNA367BRV2 but upregulated in Ep-1PNA367BRV1.We also found 104 genes coding for effector-like proteins expressed specifically in Ep-1PNA367BRV2,among them,59 genes up-regulated and 45 genes down-regulated.Briefly,two mycoviruses were isolated and characterized from strain AH16 of S.sclerotiorum,one is SsMV4 and the other is SsBRV2,a novel virus which is phylogenetically related to Botybirnavirus.SsBRV2 infected alone could confer hypovirulence to its host,but could not when co-infected with SsMV4.Transcriptome analysis revealed that SsBRV2 had much stronger impacts on the gene expression of host than SsBRV1 did.Our study provides a novel hypovirulence-associated mycovirus for potential biocontrol agent to control Sclerotinia disease,and it also will be important to further understand the interaction between fungal hosts and Botybirnaviruses and the pathogenicity on molecular levels.