Molecular Characteristics Of Two Rhabdoviruses That Confer Hypervirulence In Rhizoctonia Solani And Their Impacts On Sclerosphere Microbiota

Rice sheath blight,caused by Rhizoctonia solani,is considered to be one of the most destructive rice diseases in the world.Mycovirus exists widely in Rhizoctonia solani,and some mycoviruses cause changes in host phenotype,such as slowing down the growth rate of host fungi or enhancing the pathogenicity of the host.The study of mycovirus provides more resources for the classification and evolution of viruses,and is of great significance for the interaction between viruses and host fungi.This paper mainly focuses on three parts,firstly,the molecular characteristics of two new rhabdoviruses in R.solani strain XY175;second,the interaction mechanism between these two rhabdoviruses and the host;third,the changes of two viruses on the microorganisms around sclerotia of R.solani.The main research contents are shown bellow:XY175 was infected by six different mycoviruses,including four +ss RNA viruses and two-ss RNA viruses.The full-length genomic sequences of six viruses were obtained,and the genomic information of six viruses was analyzed.It was found that six viruses belong to three virus families,Endornaviridae,Mitoviridae and Rhabdoviridae.Two endornaviruses are named Rhizoctonia solani endornavirus 1(Rs EV1/XY175)and Rs EV9,respectively.Rs EV1/XY175 shares 97% identities to Rs EV1,and these two viruses are different strains of the same virus species.Rs EV9,sharing 36% identity to Ceratobasidium endornavirus F,is a new endornavirus infecting R.solani.Two mitoviruses are named Rhizoctonia solani mitovirus 40(Rs MV40)and Rs MV41,respectively.Rs MV40 has 51% identity to Rhizoctonia solani mitovirus 25,and Rs MV40 has 53% identity to Alternaria alternate mitovirus 1.Rs MV40 and Rs MV41 are new mitoviruses that are found in R.solani.According to the genome structure and phylogenetic analysis,the two-ss RNA viruses are new rhabdoviruses,named Rhizoctonia solani rhabdovirus 1(Rs Rh V1)and Rs Rh V2,respectively.Rs Rh V1 and Rs Rh V2 contain five ORFs encoding different proteins,among which ORF Ⅰ,ORF Ⅳ,and ORF Ⅴ,respectively,encode nucleocapsid protein,glycoprotein,and replicase-related protein.Unlike non-fungal host rhabdoviruses,ORF Ⅱ and ORFⅢ of Rs Rh V1 and Rs Rh V2 encode unknown functional proteins.Only the structure of the gene junction between ORFI and ORFII of Rs Rh V1 and Rs Rh V2 is similar to the classical rhabdovirus.The law of gene spacer between other ORFs of Rs Rh V1 and Rs Rh V2 is different from non-fungal host rhabdovirus,which is also similar to the characteristics of Ss Rh V1 found in Sclerotinia sclerotiorum.Three proteins,including N,G,and L protein,of Rs Rh V1 and Rs Rh V2 have less than 35% homology with non-fungal rhabdovirus,but 37%,45% and57% homology with the corresponding proteins encoded by a fungal rhabdovirus Ss Rh V1(Sclerotinia sclerotiorum rhabdovirus 1),respectively.Phylogenetic analysis based on L protein showed that Rs Rh V1 and Rs Rh V2 were closely clustered with members of the genera Sripuvirus.Rs Rh V1 and Rs Rh V2 are clustered into a distinct monophyletic group with Ss Rh V1.Antibodies anti-N1 and anti-N2 were prepared based on the N protein of Rs Rh V1 and Rs Rh V2.The flexuous rods virions of Rs Rh V1 and Rs Rh V2 are decorated by polyclonal antibodies anti-N1 and anti-N2.This result proves that the virus particles of rsrhv1 and rsrhv2 are flexuous rods virions that are different from other known rhabdoviruses.This study found that Rs Rh V1 and Rs Rh V2 can enhance the virulence of R.solani strain,which is confirmed by the experiments on virus elimination from rhabdovirus-infected strains and horizontal transmission to rhabdovirus-free strains.We proposed to establish a new genus,Mycorhabdovirus,to accommodate fungi host rhabdoviruses,including Rs Rh V1 and Rs Rh V2 infecting R.solani,and Ss Rh V1 infecting S.sclerotiorum.The correlation between two fungal rhabdoviruses and the enhancement of pathogenicity of R.solani was clarified,and its potential molecular mechanism was preliminarily analyzed by RNA-seq.We obtained the global transcripts expression level of XY175 and three protoplast-derived isolates induced by inoculation on rice leaves for24 hours based on high-throughput RNA sequencing,and to preliminarily explain the potential mechanism of Rs Rh V1 and Rs Rh V2 related to the enhancement of pathogenicity of R.solani.Through the analysis of enriched GO terms of DEGs Rs Rh V1 and Rs Rh V2 affect oxidoreductase activity,cellular aromatic compound metabolic process,RNA metabolic process,amino acid metabolic process,ion transport,oxidoreductase activity,hydrolase activity,lyase activity of host cells.Rs Rh V1 infects host fungi,resulting in the downregulation of host transmembrane transporter activity-related genes.Rs Rh V2 infection affected the up-regulation of host transmembrane transporter activity genes and the down-regulation of defense response-related genes.The expression of cazymes-related genes increased when Rs Rh V1 and Rs Rh V2 infected strain of R.solani.When Rs Rh V2 infects the strain of R.solani,the expression of GH13(AG1IA＿03311),PL4(AG1IA＿01129),pectinesterases(AG1IA＿02891 and AG1IA＿07743),and polygalacturonase(AG1IA＿04727 and AG1IA＿02200)related genes are significantly up-regulated.When infected with Rs Rh V1,GH5(AG1IA＿08772 and AG1IA＿02923),pectinesterases(AG1IA＿02891 and AG1IA＿07743)and laccase(AG1IA＿04672、AG1IA＿04673、AG1IA＿06531、AG1IA＿08834and AG1IA＿08835)related genes of the host R.solani were significantly up-regulated.When Rs Rh V1 and Rs Rh V2 infected the host R.solani,the expression of cell death-related effectors(AG1IA＿05142 and AG1IA＿05500)genes increased.It is speculated that Rs Rh V1 and Rs Rh V2 may enhance the ability of host R.solani to invade plants by regulating the expression of cell wall degrading enzyme-related genes,and causing the up-regulation of R.solani effectors to trigger plant cell death and enhance the parasitic ability of host R.solani.This study creatively explored the changes in the sclerosphere microorganism of R.solani when the strain of R.solani was infected by Rs Rh V1 and Rs Rh V2.It was found that the bacteria with high content in the sclerosphere microorganism of R.solani belonged to Proteobacteria,actinobacteria,Bacteroidetes,and the fungi with high content belong to Ascomycota,Basidiomycota,and Mortierella respectively.After Rs Rh V1 and Rs Rh V2 infection,the richness of R.solani sclerosphere microorganism decreased.The abundance of bacteria such as Streptomyces,Agromyces,Sphingomonas,Caulobacter,Rhizobium and biocontrol potential fungi such as Trichoderma and Talaromyces decreased after Rs Rh V1 and Rs Rh V2 infection.The interaction network of 805 ASVS was constructed.The interaction network analysis of sclerosphere microbiome of R.solani shows the microorganism with positive and negative correlation with R.solani.Aspergillus and Tausonia have a positive correlation with R.solani,Solitalea has a negative correlation with R.solani.Solitalea may be a potential biocontrol factor of R.solani.It is speculated that Rs Rh V1 and Rs Rh V2 may reduce the number of microorganism with biocontrol effect and recruit the pathogenic fungi of rice.In this study,we found that strain XY175 strain was co infected by six ss RNA viruses,two of them were new rhabdoviruses.The two rhabdoviruses can enhance the pathogenicity of the host R.solani.And the particle of these two rhabdoviruses are flexuous rod virions,different from the known rhabdoviruses.The mechanism of the effect of Rs Rh V1 and Rs Rh V2 on host pathogenicity was preliminarily explained at the gene level by RNA-seq analysis.Innovatively explore the relationship between mycovirus,R.solani,and sclerosphere microbiome of R.solani,and explain the effects of Rs Rh V1 and Rs Rh V2 on the changes in sclerosphere microbiome.This study provides more resources for the classification and evolution of mycoviruses and different insight into the interaction between mycoviruses and hosts fungi.At the same time,it also provides a new research direction for how mycoviruses affect sclerosphere microbiome and promote the survival of fungal sclerotia.