Identification Of A Novel Rice Virus RCDaV And Study Of The Rice LncRNA-mRNA Regulatory Network In Response To RBSDV Infection Based On RNA-seq

Rice(Oryza sativa)is one of the most important food crops in China and occupies an essential position in agricultural production.However,rice production has been seriously threatened by rice viral diseases for decades.The identification and discovery of new rice viruses and the study of the defense mechanism of rice host in response to virus infection are of great importance to ensure rice production.To this end,this study identified a new picornavirus infecting rice plants usingRNA-seq technique,and further analyzed the function and cleavage sites of the virus-encoded protease and the evolutionary relationship of this new virus.The study also analyzed the transcriptional regulation of long non-coding RNA(lncRNA)in rice in response to RBSDV infection throughRNA-seq,and investigated the regulatory network of lncRNA-mRNA upon RBSDV infection.The results are as following: 1.Identification of rice curl dwarf-associated virus(RCDaV)and the functional study of RCDaV 3C proteaseA novel rice-infecting picornavirus was identified from the rice plants,which showed dwarfing and curling symptoms,through transcriptomeRNA-seq followed by bioinformatics analysis.The virus was tentatively named as rice curl dwarf-associated virus(RCDaV).RCDaV genome is a positive-single strandedRNA of 8,987 nucleotides with a poly(A)tail and encodes two large polyproteins.Using in vitro cleavage assays,we identified the cis and trans cleavage activities of RCDaV 3C protease(3Cpro),and deciphered the cleavage sites catalyzed by the 3Cpro.The cleavage processing of the two polyproteins of RCDaV by 3Cpro yields 12 mature proteins.It's noteworthy that we discovered that RCDaV 3Cpro recognizes and cleaves the conserved EPT/S,which is completely different from 3Cpros of other viruses that cleave the conserved Q(E)/G(S)dipeptides as previously reported.Our findings show that RCDaV 3Cpro remarkable distinguishes from the canonical3 Cpros of other picornaviruses and indicate that RCDaV 3Cpro is a novel virus-encoded serine protease.Phylogenetic analysis showed that RCDaV was clustered into a new clade in the order Picornavirales,together with seven other unclassified picornaviruses.In addition,sequence analysis showed that all the clustered viruses have similar genome organizations,as well as the same type of 3Cpros with a key Gx SG motif and the conserved EPT/S cleavage sites.Based on the results of phylogenetic analysis,similar genome organization,low viral protein sequence identity with known viruses in eight official families,and the novel type of 3Cpro with the distinct cleavage site(EPT/S),we propose that RCDaV and these seven unclassified picornaviruses should group together to form a new picornavirus family in the order Picornavirales.2.Study of the rice lncRNA-mRNA regulatory network in response to rice black-streaked dwarf virus infectionThe plant genome can produce long non-codingRNAs(lncRNAs),some of which have been identified as important regulators of gene expression.To better understand the response mechanism of rice plants to rice black-streaked dwarf virus(RBSDV)infection,we performedRNA-seq and a comparative transcriptome analysis between the RBSDV-infected and non-infected rice plants.A total of 1,342 mRNAs and 22 lncRNAs were identified to be differentially expressed in response to RBSDV infection.A network of differentially expressed lncRNAs(DElncRNAs)and mRNAs(DEmRNAs)was then constructed.Most differentially expressed transcripts involved in the plant-pathogen interaction pathway were upregulated after RBSDV infection,indicating the activation of rice defense response by RBSDV.Among them,there were 56 plant-pathogen interaction-related DEmRNAs co-expressing with 20 DElncRNAs,suggesting these DElncRNAs and DEmRNAs may play roles in rice immunity against RBSDV.To verify the predicted regulatory relationship of DElncRNA-DEmRNA,some of the lncRNA-mRNA regulatory relationships were experimentally verified in rice calli by a quick and effective method established in this study.Three DElncRNAs and seven predicted correlative DEmRNA were selected,and found that four mRNAs were regulated by these three DElncRNAs.These results will provide new ideas and means to reveal the molecular interaction mechanism between rice and RBSDV.