Research On Novel RNA Viruses Of Sclerotinia Sclerotiorum

The fungus Sclerotinia sclerotiorum（Lib.） de Bary is an important plant pathogen worldwide, with a broad range of hosts and natural distribution. Mycoviruses or fungal viruses are widely present in fungi, and hypovirulence-associated mycoviruses are potential to be exploited as biological control agents. There are many and diverse mycoviruses in the fungus S. sclerotiorum. Mycoviruses discovery from this fungus contributes to look insight into the interactions between mycoviruses and fungi, virus classification and evolution, and provides useful tools for the biological control of sclerotial disease.We have isolated 1967 strains of S. sclerotiorum collected from Hunan province and Anhui province, and obtained 38 strains with an abnormal colony morphology, of which there were 29 strains from Hunan province and 9 strains from Anhui province. Based on the colony morphology, we have classified these strains into 4 types. Type I, 7 strains had an abnormal colony morphology but restored a normal colony morphology during subcultured on PDA. Type II, 9 strains had a normal colony morphology without sclerotium. Type III, 13 strains had an abnormal colony morphology without sclerotium. Type IV, 9 strains have an abnormal colony morphology with a few sclerotia. Agarose gel-electrophoresed analysis of ds RNA and DNA isolated from these strains showed that there were multiple ds RNA or extrachromosomal DNA segments. PCR detection for the Sclerotinia sclerotiorum associated-hypovirulent DNA virus 1（Ss HADV-1） revealed that Ss HADV-1 was present in a few strains and thus, was widely spread in the natural environment. Virulence test on the detached leaves of rapeseed showed that 28 strains were hypovirulent.Two viruses were identified from strain AH98 which was collected from Feidong county, Anhui province. One was the Sclerotinia sclerotiorum hypovirulent virus 1（Ss HV1） and the other was a novel negative-stranded RNA（-ss RNA） virus named as Sclerotinia sclerotiorum negative-stranded RNA virus 1（Ss NSRV-1）. The full-length of Ss NSRV-1 genome was 10002 nt（Gen Bank accession, KJ186782） with 6 non-overlapped genes（ORF I-VI） which were linearly arranged in the genome. ORF V encoded a large protein（L protein） with 1934 amino acids and comprised a conserved mononegaviral Rd Rp domain Mononeg_RNA_pol（pfam00946） which was closely related to mononegaviruses, while other ORFs didn’t have a significant sequence similarity with any sequence in the NCBI database. Conserved sequences（A/U）（U/A/C）UAUU（U/A）AA（U/G）AAAACUUAGG（A/U）（G/U） were identified among each ORF, which is the “Gene-junction sequences” of Ss NSRV-1. The sequence “AAAACUUAGG” was the transcriptional stop signal of the upstream ORF and “UAUUUAAUAAAACU” was the transcriptional start signal of the downstream ORF. “Gene-junction sequences” are widely present in mononegaviruses and is the unique property of Mononegaviruses. 5’ RACE, 3’ RACE and Northern blot analysis revealed that all the six ORFs can be transcribed independently. Ss NSRV-1 may have enveloped virions which were observed both in the purified virion preparations and ultrathin hyphal sections. The nucleocapsids are long, flexible, helical, and are 22 nm in diameter and 200–2,000 nm in length. SDS/PAGE showed that the nucleocapsid possibly contained two nucleoproteins with different molecular masses, 43 k Da（p43） and 41 k Da（p41） both of which were translated from ORF II. Northern blot analysis confirmed Ss NSRV-1 genome released from the virions and indicated the presence of defective RNA. We have cloned the sequences of the defective RNAs and the results showed that there were different sequence-deleted types in ORF I and ORF II while a short sequence was deleted in the 5’ termini. Phylogenetic analysis based on Rd Rp showed that Ss NSRV-1 is a member of Mononegavirales and clustered with viruses of Nyamiviridae and Bornaviridae. Ss NSRV-1 was different from Nyamiviruses and Bornaviruses in genome organization and may represent a new evolutionary lineage. Purified Ss NSRV-1 virions can successfully transfect the protoplasts of S. sclerotiorum and lead to debilitating symptoms in the fungal host characterized by lower growth rate, abnormal colony morphology, less pathogenicity and curled hyphal tips. Moreover, Ss NSRV-1 is widely distributed in the natural envirionment, thus we have firstly found a-ss RNA virus that can infect the fungal host and demonstrated that the fungal-ss RNA virus was present in the natural environment.Two novel ds RNA viruses were identified from strain SCH941 which was collected from Yingjing county, Sichuan province. One was a bipartite ds RNA virus named as Sclerotinia sclerotiorum botybirnavirus 1（Ss BRV1） and the other is a novel reovirus named as Sclerotinia sclerotiorum reovirus 1（Ss Re V1）. The genome Ss BRV1 comprised two ds RNA segments（ds RNA1 and ds RNA2）, with sizes of 6457 bp（Gen Bank accession, KP774592） and 5965 bp（Gen Bank accession, KP774593）, respectively. One large ORF was found in ds RNA1, which encoded a protein with 1925 amino acids and contained a conserved Rd Rp domain（Rd Rp₄, Pfam02123）. One large ORF was also found in ds RNA2, which encoded a protein with 1757 amino acid whose function was unclear. A satellite-like RNA（Satl RNA） was also found with 1647 bp in size（Gen Bank accession, KP774594） and didn’t have any significant senquence similarity with ds RNA1 or ds RNA2. A hormone receptor binding domain（GHBP, Pfam12772） was identified in ORF2（nt 2650-2953）. There was a region between ORF 1 and ORF 2 which shared a significant sequence similarity with E value 4e-10. Ss BRV1 had spherical virions with 38 nm in diameter, and three ds RNA segments corresponding to ds RNA1, ds RNA2 and Satl RNA of Ss BRV1. Three structural proteins were identified with masses of 120 KDa（p120）, 100 KDa（p100） and 80 KDa（p80） which were translated from ORF 2 and ORF1, respectively. Virions of Ss BRV1 can successfully transfect the protoplasts of S. sclerotiorum with PEG-mediated method while Satl RNA can be eliminated in some transfectants. Ss BRV1 carrying Satl RNA can significantly reduce the growth and virulence of S. sclerotiorum while had little effect on the colony morphology. Ss BRV1 lost Satl RNA had no obvious effects on the biological properties of S. sclerotiorum. Phylogenetic analysis revealed that Ss BRV1 had a close relationship with bipartite ds RNA virus Bp RV1 and monopartite ds RNA viruses Um V-H1, Sp FV1 and Ci TV1. Ss BRV1 may belong to the virus family Botybirnaviridae proposed by Wu et al. 2013.The genome of Ss Re V1 encompassed 11 ds RNA segments（S1-S11） which was confirmed by Northern blot analysis. The full-length genome of Ss Re V1 was 28055 bp with 42.6% GC content and had yet not been submitted in the NCBI database. Conserved 5’-terminal sequences “5’-GAGWUKK-3’” and 3’-terminal sequences “5’-UGCAGUC-3’” were found in each ds RNA segment and the nucleotides underlined was strictly conserved（W, U or A; K, U or G）. There was only one ORF in each ds RNA segment except ds RNA6 and ds RNA11 in which there were two ORFs. Corresponding to ds RNA1-ds RNA11, the ORFs were named as ORF1-ORF11 whose proteins were subsequently named VP1-VP11. Protein VP1 was predicted to be the Rd Rp of Ss Re V1 responsible for virus replication and maintenance. VP2 was likely to be viral methyltransferase, and VP8 was likely to be viral kinase and helicase, while the roles of other viral proteins were unkown. There was a conserved Double-stranded RNA binding motif（DSRM, pfam00035） in VP6-1 which shared a significant sequence similarity with the RNase III of many bacteria. There was also a conserved UV radiation resistance protein and autophagy-related subunit 14（Atg14, pfam10186） in VP7 which shared a significant sequence similarity with a conserved hypothetical protein widely spread in fungi. Ss Re V1 had spherical virions with 65 nm in diameter which had no obvious protuberant structures on the surface. Ss Re V1 virions had 6 protein components named as p165, p145, p65, p63, p57 and p30 corresponding to the molecular weight of these proteins. Polypeptides mass finger-Mass spectrum（PMF-MS） analysis showed that the proteins p165, p145 and p57 were VP1, VP2 and VP9 encoded by ORF1, ORF2 and ORF9, respectively. The other proteins p65, p63 and p30 were encoded by ORF5, ORF8 and ORF9, respectively. Ss Re V1 virions can successfully transfect the protoplasts of S. sclerotiorum with PEG-mediated method. Ss Re V1 infection was cryptic and had no obvious effects on the biological properties of the fungal host. Phylogenetic analysis based on the Rd Rp sequence revealed that Ss Re V1 was not a member of Mycoreovirus and more closely related to animal viruses Coltiviruses. Potential interactions may be present between Ss BRV1 and Ss Re V1.Two viruses were identified from strain HN138 which was collected from Anxiang county, Hunan province. One was the DNA virus Ss HADV-1 and the other was a ds RNA virus named as Sclerotinia sclerotiorum botybirnavirus 3（Ss BRV3）. Ss BRV3 genome also encompassed two ds RNA segments, ds RNA1 and ds RNA2 with sizes of 6213 bp and 5878 bp, respectively. The genome sequence of Ss BRV3 has yet not been submitted to the NCBI database. Between Ss BRV3 and Bp RV1, there were 92% and 97% sequence similarity at nucleotide level, and 97% and 99% sequence similarity at amino acid level. Therefore, Ss BRV3 and Bp RV1 should be different strains of the same virus. Ss BRV3 had no obvious effects on the biological properties of S. sclerotiorum and Botrytis cinerea, while Bp RV1 conferred debilitating symptoms to its fungal host. Between the ORF1-encoded polypeptides of Ss BRV3 and Bp RV1, there were 53 different amino acids, of which 23 amino acids were changed in chemical properties. There was a proline deleted in the ORF1-encoded polypeptide of Ss BRV3. Between the ORF2-encoded polypeptides of Ss BRV3 and Bp RV1, there were 15 different amino acids, of which 4 amino acids were changed in chemical properties. Strain HN138 had more aerial mycelia and lost sclerotial productivity which were distinct from that of Ss HADV-1-infected strains. Therefore, potential interactions may be present between Ss BRV3 and Ss HADV-1.We have firstly used metavirome to discover viruses from 50 strains of S. sclerotiorum and obtained 36 viruses-like sequences including 24 +ss RNA viruses, 8 ds RNA viruses, 4 DNA viruses including Ss HADV-1 while there were another 108 sequences with no information. Therefore, Metavirome is a powerful tool to discover fungal viruses and reveals that there are many and diverse mycoviruses in the fungus S. sclerotiorum.