Study On The Gene Functions Of SsNSRV-1 And The Interaction Mechanism With Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum,an important necrotrophic phytopathogenic ascomycete with worldwide distribution,can infect 729 plant species so far including many important crops,such as canola,soybean,and sunflower,and cause serious economic losses.Mycoviruses(or fungal viruses),the viruses infect fungi and have the potential to control the crop stem rot,are ubiquitous in S.sclerotiorum and some of them are reported to confer pathogenicity decline to their host or even cause loss of pathogenicity.Previous studies showed that Sclerotinia sclerotiorum negative-stranded virus 1(Ss NSRV-1)identified from strain AH98 is associated with the hypovirulence of its fungal host S.sclerotiorum.Ss NSRV-1 contains six linearly arranged non-overlapped open reading frame(ORF ?-VI)that encode P1,P2(N protein),P3,P4,P5(L protein),and P6 protein,respectively.The specific functions of these ORFs and their effects on their hosts are still unclear.At the same time,it was found that strain AH98 may be co-infected by multiple viruses,but the molecular characteristics of these viruses are not clear.In this study,strain AH98 and Ss NSRV-1 gene ORF I,ORF II,ORF III,ORF IV,and ORF VI overexpressing transformants in strain 1980 of S.sclerotiorum were used as research materials.The mycoviruses in strain AH98 and their molecular characteristics were determined by high-throughput sequencing.Based on the bioinformatics analysis,the structure and function of each ORF in Ss NSRV-1 were predicted.The effects of each ORF of Ss NSRV-1 on S.sclerotiorum were explored by transcriptome and metabolomic analysis,the molecular mechanism of Ss NSRV-1 inducing hypovirulence was analyzed,and the molecular network of interaction between Ss NSRV-1 and S.sclerotiorum was revealed.The results are as follows:High throughput sequencing confirmed that strain AH98 carried 6 mycoviruses,including two-ss RNA viruses(Ss NSRV-1 and Ss NSRV-5X)and four +ss RNA viruses(Bc HV1/AH98,Ss MV30/AH98,Ss MV7-A/AH98,and Ss DFV2/AH98).The genome of Ss NSRV-5X is 9993 nt with four non-overlapped ORFs(ORF I-ORF VI)that are linearly arranged in the genome.The coding strand of Ss NSRV-5X has two short untranslated regions(UTRs)of 420 nt and 168 nt in length,but no cap structure and poly A sequence at the 5'-and 3'-terminus.The ORF III(nt 2672-8553)was predicted to encode a putative large polypeptide of 1952 amino acid residues,which contains a putative mononeg?m RNA?pol domain and a mononeg?m RNAcap region.Although the sequence characteristics of Ss NSRV-5X are different from those of the currently known Sclerotimonavirus(The L protein sequence of Ss NSRV-5X only had a 17.96% identity to the L sequence of Ss NSRV-1),this virus is closely related to viruses in the family Mymonaviridae,thus it should be a new virus in this family.Ss NSRV-5X and another unreported virus(Bc NSRV-7)may represent a new genus in the family Mymonaviridae.Bc HV1/AH98 with a length of 10223 bp is associated with the reported Bc HV1(NC?037659.1)with a 98.82% identity and a 100% query cover to the Rd Rp sequence of Bc HV1(QBA69887.1).Phylogenetic analysis indicated that Bc HV1/AH98 should be classified in the genus Betahypovirus of the family Hypoviridae.ORF I,ORF II,and ORF III of Ss NSRV-1 could be recognized by fungi and spliced into small fragments in the overexpressing transformants,while ORF IV and ORF VI could not be spliced.In this study,this phenomenon was further confirmed using transcriptome sequencing analysis.By comparing the sequences of all transcripts of viral genes,the splicing sites of m RNA in the transformants were identified as the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites,and the splicing specifically recognized m RNA sequences started at the GT-rich sites and ended at the AG-rich sites.Viral ORF III was also spliced into small fragments when it was overexpressed in Fusarium graminearum and Magnaporthe oryzae,which might suggest that the viral genes may be specifically recognized and this recognition and splicing may be an extensive used antiviral mechanism in fungi.In addition,there are two normal distribution peaks in strain AH98 and overexpression transformants,which could not correspond to the two forms of N protein(p41 and p43)detected earlier,indicating that OFR II may encode other proteins.The conserved domains,subcellular localization,structural features,and possible interaction sites of proteins encoded by ORF I,ORF II,ORF III,ORF IV,and ORF VI were analyzed using bioinformatics software.Meanwhile,the transcriptome of viral ORF overexpressing transformants was performed using RNA-Seq technology and the effects of viral ORFs on S.sclerotiorum were preliminarily analyzed.Based on these results,the functions of viral genes in the interaction process between Ss NSRV-1 and S.sclerotiorum were speculated and the molecular mechanism of hypovirulence of Ss NSRV-1 was preliminarily explored.It was found that the protein P1 encoded by ORF I had the structural characteristics of negative-strand RNA virus N protein,and the possibility of homology with N protein was 82.52%.ORF I was involved in the particles formation of Ss NSRV-1 and played a role in transcriptional regulation.ORF I could control part of host genes associated with pathogenicity,transcription,transmembrane transport,protein biosynthetic,modification,metabolic process,and so on,causing the decline of growth rate and pathogenicity.Viral ORF II which encodes N protein,mainly affects the expression of ribosomal and nuclear related genes,and partially regulates the expression of host genes related to pathogenicity,transcription,transmembrane transport,metabolism,and so on,thus may reduce the growth rate and pathogenicity of transformants.The P3 protein encoded by ORF III has a certain homology with the negative-strand RNA virus glycoprotein,which may have the activity of endopeptidase.ORF III mainly affects the expression of host genes related to ribosome,proteasome and DNA-repair related genes in S.sclerotiorum,and regulates the translation and metabolism of proteins.The P4 protein encoded by ORF IV plays a role of transcriptional regulation in host,which mainly controls the expression of genes related to transcriptional regulation,redox,transmembrane transport and metabolic process by regulating mitochondrial,r RNA and ribosome related genes.The energy and metabolic processes were seriously affected that may cause the decline of ORF IV overexpression transformants.The P6 protein encoded by ORF VI may be a nucleoporin complex,which participates in nuclear cytoplasmic transport and plays an important role as a transcription regulator.The overexpression of ORF VI mainly affected the genes related to transcriptional regulation,transmembrane transport and metabolic process.ORF VI could partially regulate the expression of genes related to r RNA and ribosome,which seriously affected the carbohydrate and nucleotide metabolism.The metabolites produced by S.sclerotiorum after the overexpression of the viral ORF I,ORF II,ORF III,ORF IV,and ORF VI were identified,the most significant differential metabolites were preliminarily identified,and the possible pathways influenced by these metabolites were analyzed.The results showed that ORF I,ORF II,and ORF III had a greater impact on the metabolism of S.sclerotiorum and the differential metabolites were similar among the transformants of the three genes.The marker metabolites response to the overexpression of ORF I,ORF II,and ORF III were ketones(up-regulated)and amides(down-regulated).ORF IV and ORF VI had little effect on the metabolism of S.sclerotiorum,and the marker metabolites response to the overexpression of ORF IV were phospholipids and amides(down-regulated),while the marker metabolites response to the overexpression of ORF VI were phospholipids(up-regulated).There are 253 common differential metabolites in ORF I,ORF II,and ORF III overexpression transformants,which were involved in the biosynthesis of unsaturated fatty acids,amino acid metabolism,riboflavin metabolism,pentose phosphate pathway,aminoacyl t RNA biosynthesis,steroid biosynthesis,and other KEGG pathways.There are 76 differential metabolites in ORF IV overexpression transformants,which were involved in sterols biosynthesis,cysteine biosynthesis/homocysteine degradation(trans-sulfuration)and other pathways.There are 50 differential metabolites(45 up-regulated and 5 down-regulated)in ORF IV overexpression transformants,which were involved in dopamine degradation,biotin carboxyl carrier protein assembly,initiation of fatty acid biosynthesis,uracil degradation and palmitic acid biosynthesis.In this study we found that strain AH98 of S.sclerotiorum is co-infected by six mycoviruses,among which,Ss NSRV-5 may represent a new genus of the Mymonaviridae.We also found that there was an unknown antiviral mechanism in fungi which splices the m RNA of viral genes using eukaryotic spliceosome.By combining transcriptome and metabonome analysis of vrial gene transformants,we investigated the functions of viral genes and the potential debilitation mechanisms of S.sclerotiorum caused by Ss NSRV-1.Thus,the findings in this study provide new ideas and materials for studying the diversity of negative-strand RNA virus,the interaction between negative-strand RNA virus and host,and the control of Sclerotinia diseases.