Genome Organizations And Functional Analyses Of Two Partitiviruses From Rhizoctonia Solani AG-1 Ia

Rice sheath blight caused by Rhizoctonia solani Kühn AG-1 IA is one of the most important diseases of rice(Oryza sativa L.).In recent years,with the development of mycovirus research,the mycoviruses in rice sheath blight fungus have been gradually discovered and studied.Most mycoiruses reported in rice sheath blight fungus are double-stranded RNA(dsRNA)mycoviruses,including partitiviruses,endornaviruses,unclassified mycoviruses and so on.Previous studies have shown that some mycoviruses have certain effects on biological characteristics of their host fungi.However,recently,there are few researches on mycoviruses and their original host fungi.In order to discover more new mycoviruses and to clarify the effects of some mycoviruses on their host fungi,13 mycovirus-containing strains of R.solani AG-1 IA stored in our laboratory were used as materials,and a series of research work on the screening of new mycoviruses,the genome organizations and functional analyses of mycoviruses,as well as the effects of mycoviruses on host fungi were carried out in this study.The main results are as follows:1.Reverse transcription PCR(RT-PCR)method was used to search for novel mycoviruses from 13 mycovirus-containing strains screened in our laboratory previously,the results showed that: strains B240 and B261 were infected by both segments of Rhizoctonia solani dsRNA virus 1(Rs V1)but only Rhizoctonia solani partitivirus 1segment 1 for strain A111 indicating its independent infectivity.Strains A5 and B261 were infected by Rhizoctonia solani partitivirus 2(Rs PV2).Whereas strains A82、A102、A105、A106、A154、A182、D122、D168 and Y1 were infected by unknown myviruses that different from either Rs V1 or Rs PV2.Strains A105 and D122 were chosen for further study according to the agarose gel electrophoresis bands of nucleic acid in these 9 strains.2.Rhizoctonia solani dsRNA virus 3(RsRV3),a novel mycovirus,was isolated fromthe strain A105 of the rice sheath blight pathogen R.solani AG-1 IA.The RsRV3 genome consists of two segments of dsRNA(dsRNA1,1,890 bp and dsRNA2,1,811 bp).Ds RNA1 has a single open reading frame(ORF)with a putative conserved RNA-dependent RNA polymerase(Rd Rp)domain,and dsRNA2 comprises a single ORF,was predicted to encode a coat protein.Purified viral particles of RsRV3 were isometric and meatured approximately 20 nm in diameter by negative-stain transmission electron microscope(TEM).Phylogenetic analyses indicated that RsRV3 is highly similar to those mycoviruses in the genus Alphapartitivirus of family Partitiviridae.Taken together,the integrative analyses of viral genomic organization,amino acid sequence alignments and phylogenetics have clearly demonstrated that RsRV3 isolated from R.solani AG-1 IA strain A105 is a new member of the genus Alphapartitivirus in the family Partitiviridae.3.Rhizoctonia solani dsRNA virus 5(RsRV5),a novel mycovirus,was isolated from the strain D122 of rice sheath blight pathogen R.solani AG-1 IA.The RsRV5 genome consists of two segments of dsRNA(dsRNA1,1,894 bp and dsRNA2,1,755 bp).Ds RNA1 has a single open reading frame(ORF)with a putative conserved RNA-dependent RNA polymerase(Rd Rp)domain,and dsRNA2 comprises a single ORF,was predicted to encode a coat protein.Purified viral particles of RsRV5 were isometric and meatured approximately 20 nm in diameter by negative-stain transmission electron microscope(TEM).Phylogenetic analyses indicated that RsRV5 is highly similar to those mycoviruses in the genus Gammapartitivirus of family Partitiviridae.Taken together,the integrative analyses of viral genomic organization,amino acid sequence alignments and phylogenetics have clearly demonstrated that RsRV5 isolated from R.solani AG-1 IA strain D122 is a new member of the genus Gammapartitivirus in the family Partitiviridae.4.Mycovirus removal methods including hyphal tipping,virus inhibitors treating,mycelium fragment method and protoplast regeneration method were tried for curing strain D122.The mycovirus RsRV5 was successfully removed from strain D122 by method protoplast reproduction regeneration and a derivative isogenic mycovirus-free strain D122-P was obtained with reduced mycelial growth,increased pigmentation sclerotia production and hypovirulence to rice leaves compared with D122 after 7 days of culture in darkness.5.To compare the differences between the two isogenic samples in transcriptional levels transcriptome analysis was performed using Illumina Hiseq2000 system.Bioinformatics analysis showsTranscriptome sequencing(RNA-seq)and bioinformatics analysis of the wild-type strain D122(RsRV5-containing)and the isogenic strain D122-P of R.solani were carried out to compare the differences in transcription levels between the two samples so as to understand the effects of mycoviruses on the host fungal transcriptome.The results showed that the effective sequence number of sequencing data were 127380018 and the number of sequences that can be compared to the reference genome were 96371422 with percentage ratio above 70%.65,535 variable splicing events were identified and the proportion of intron retention was the largest,followed by skipped exon.Differential gene expression(DEGs)results showed that gene rna6273 and rna6673 coding hypothetical protein of unknown function up-regulated and gene rna9529 coding protein kinase inhibitor(PKI)domain-containing protein was down-regulated compared with mycovirus-containing strain D122.In summary,this study elucidated the relationship between the genome organizations and functions of two newly discovered partitiviruses(RsRV3 and RsRV5),and preliminarily clarified the effects of mycovirus RsRV5 on the biological characteristics and transcriptome of host fungus.