SRBSDV-derived Small Interfering RNA Regulates Rice Gene Expression And Mediates Disease Symptoms

Southern rice black-streaked dwarf virus(SRBSDV)is a recognized species belonging to the genus Fijivirus,family Reoviradae.The virus is transmitted by the whiteback planthopper(WBPH,Sogatella furcifera)and induces infected rice shows dwarf,dark green and curly leaves,increased tillers,and high tillering position and other symptoms.Since it was first discovered in Yangxi County,Guangdong Province in 2001,the research on the genomic structure and gene function has made significant progress.In previous research in our laboratory,a large number of siRNAs derived from SRBSDV were found through sequencing of small RNAs(s RNAs),which with the sizes of 21-22 nt mainly,no strand polarization in origin,and potential function tointerfere host plant gene expression.In this study,the association analysis of small RNA and degradome sequencing data was used to screen vsiRNA that act on rice and its target genes.Molecular biological methods were used to verify the interaction between the vsiRNAs and its targets,in order to reveal the role of vsiRNA in the formation of disease symptoms,and deepen the understanding of the pathogenic mechanism of the virus.The results of this study were obtained as follows:1.Further clarified the molecular characteristics of vsiRNA derived from SRBSDV in infected rice.There were mainly 21 nt and 22 nt in length,no difference in strand polarity,and U and A preference existing in the 5’-terminal nucleotides of SRBSDV-derived vsiRNAs in 40 dpi.These characteristics were basically consistent with 14 and 28 dpi.2.The correlation analysis between vsiRNA data and degradome sequencing data revealed that three vsiRNAs were highly possible to target rice genes.Using "vsiRNA reads≥70 and its target gene degradome reads ≥10" as criteria,three vsiRNAs(vsiR-S6-10,vsiRS9-18,vsiR-S3-5)with large accumulation were screened and they may target the genes were named SAP(LOC＿Os10g13630.1),ROC1(LOC＿Os08g08820.1)and TM(LOC＿Os01g72009.1),the mRNA degradome reads of the three predicted target genes were50,33,and 14,respectively.3.The bioinformatics analysis of the three rice candidate genes targeted by vsiRNA show that vsiR-S6-10 targeted SAP gene which protein contains the SAP domain,SAP gene was predicted to be related to DNA binding and may have the function of transcription factor;the ROC1 gene targeted by vsiR-S9-18 contains the homeobox and START domains,was speculated that it may play a role in transcription regulation in rice growth and development;the TM gene targeted by vsiR-S3-5 was presumed to encode a transmembrane or transportrelated protein.4.The accumulation of three vsiRNAs and the relative expression of their target genes were detected.The results showed that vsiR-S6-10 、 vsiR-S9-18 and vsiR-S3-5 were accumulated at high levels at 20 and 40 dpi,and the corresponding target genes were downregulated significantly at one or more times of pathogenesis.5.The vsiRNAs regulated the expression of target genes were verified in rice protoplasts and the transient coexpression system of Nicotiana benthamiana.In rice protoplasts,when vsiR-S6-10 and vsiR-S9-18 were expressed,the expression of their predicted target genes SAP and ROC1 were down-regulated by 30.3% and 38.6%,respectively,but vsiR-S3-5wasn’t detected that it had a regulatory effect on the predicted target gene TM.In transient coexpression system of Nicotiana benthamiana,the expression of predicted target SAP and ROC1 but m SAP and m ROC1 with mutation in the vsiRNA matching site was significantly supressed by vsiR-S6-10 and vsiR-S9-18 respectively.But the predicted target gene TM has not yet been shown to be regulated by vsiR-S3-5.6.The subcellular localization of the target genes was observed by laser confocal microscopy.The results showed that the SAP protein was localized to the nucleus and cytoplasm,and the ROC1 protein was localized to the nucleus.7.The variation of the target gene of vsiRNA was obtained through CRISPR/Cas9,and it was confirmed that the variation of target genes SAP and ROC1 could cause dwarf,increased tiller and poorly developed roots in rice.The rice variations in two lines of the target gene SAP of vsiR-S6-10 all showed the phenotypes of dwarf and increased tiller,which similar to disease symptoms,and the variations in two lines of the target gene ROC1 of vsiRS9-18 all showed the phenotypes of poorly developed roots.In summary,this study found and basically confirmed that SRBSDV-derived vsiR-S6-10 and vsiR-S9-18 sequence-specific targeted rice genes SAP and ROC1,respectively,to degrade the gene mRNA.which played a vital role in the symptom formation of the disease.It was one of few researches on vsiRNA targeted host gene and involved in pathogenicity of plant virus so far,which expected to provide guidance for revealing the pathogenic mechanism of plant viruses and screening and evaluating plant disease resistance.