Studies On The Response Of Rice To Rice Black-Streaked Dwarf Virus And Generation Of RNAi-mediated Resistance In Rice

Rice black-streaked dwarf virus(RBSDV)is a member of the genus Fijivirus in the family Reoviridae,causes rice black-streaked dwarf and maize rough dwarf diseases,which lead to severe yield losses of crops in China,Japan,Korea,and other Asian countries.RBSDV-infected plants are usually severely stunted with darkened leaves and white tumors or black-streaked swellings along the veins on the back of the leaf blades and sheaths.RBSDV is transmitted by the small brown planthopper(SBPH:Laodelphax striatellus Fallen)in a persistent circulative manner.The RBSDV virion is composed of icosahedral,two-layered particles approximately 75-80 nm in diameter and 10 segments of double-stranded genomic RNA(dsRNA),which are designed S1 to S10.Although a great deal of efforts have been made to elucidate the interactions between the virus and its host,few RBSDV proteins involved in pathogenesis have been identified,and the biological basis of disease development in rice remains largely unknown.To determine how rice transcriptome is changed in response to RBSDV infection,we carried out RNA-Seq to perform a genome-wide gene expression analysis of a suscepti-ble rice cultivar KTWYJ3.The transcriptomes of RBSDV-infected samples were compared to those of RBSDV-free(healthy)at two time points after obvious and typical disease symptoms emerged(time points are represented by group ? and ?).The results derived from the differential expression analysis in RBSDV-infected libraries vs.healthy ones in group I revealed that 102 out of total 281 significant differentially expressed genes(DEGs)were up-regulated and 179 DEGs were down-regulated.Of 2592 significant DEGs in group II,1588 DEGs were up-regulated and 1004 DEGs were down-regulated.A total of 66 DEGs were commonly identified in both groups.Of these 66 DEGs,expression patterns for 36 DEGs were similar in both groups.Further analysis demonstrated that some genes related to disease defense and stress resistance,including genes encoding flavonol synthase and rice wall-associated kinase(OsWAK32),were up-regulated while genes associated chloroplast-related genes,such as a gene for chlorophyll synthase(Os05g28200.1),were down-regulated in response to RBSDV infection.In addition,some genes associated with plant-height,such as a gene encoding pyruvate kinase(Os11g05110.1)were differentially expressed.This result indicates those genes might be involved in dwarf symptoms caused by RBSDV.Taken together,our results provide a genome-wide transcriptome analysis for rice plants in response to RBSDV infection which might contribute in the understanding of the regulatory mechanisms involved in rice-RBSDV interaction and the biological basis of RBSD disease development in rice.It has been suggested that the viral infection of plants might cause distinct disease symptoms through the inhibition or activation of host gene transcription.The knowledge about the gene expression profile changes at the early stage due to RBSDV infection may aid in the understanding of the mechanisms of symptom development and in the search for genes of resistance against RBSDV.Therefore,we carried out qRT-PCR-based analysis to elucidate time-course gene expression alterations of selected dwarf symptoms-related genes,defense-and stress-related genes,and chloroplast-and photosynthesis-related genes in rice infected by RBSDV at three time points in the early stage(3,8 and 22 days)after virus inoculation.Our results demonstrate that some resistance related rice genes and cell wall-and development-related genes,such as those for cellulose synthesis(CESA),are among the genes whose expression is altered.The dwarf symptom caused by RBSDV might be due to changes in cell wall-related structure and biosynthesis beside alternation of plant height-related genes,that possibly attributable to the decreased expression of some CESA genes,such as CESA 1 and CESA 7.Our analysis detected a significant down-regulated expression of a gene encoding pyruvate kinase at 3 days post inoculation(dpi)in RBSDV-inoculated plants.The expression of some OsWRKY genes had been changed in RBSDV-inoculated plants compared to the mock control.For instance,OsWRKY78 exhibited notable increased expression at 3 dpi,followed by significant decreased expression at 8 dpi,then again showed significant elevated expression at 22 dpi in RBSDV-inoculated plants.On the other hand,the expression of selected chloroplast-and photosynthesis-related genes was reduced at early stages of infection with RBSDV.This result is in accordance with our RNA-Seq study in which genes associated with chloroplast were down-regulated in response to RBSDV infection.MicroRNAs(miRNAs)play important roles in development,abiotic stress and responses to pathogens.Plant miRNAs are involved in virus infections and these virus-responsive miRNAs together with their target genes are always associated with viral symptoms or virus pathogenicity.A number of miRNAs involved in RBSDV infection were identified in different rice tissues and altered expression pattern for these miRNAs and their targets was noticed in RBSDV-infected rice plants.In our study,transgenic rice lines overexpressing RBSDV non structural proteins P7-1 and P9-1 namely,P7-lOvE and P9-lOvE,respectively were generated in an attempt to investigate the effect of RBSDV P7-1 and P9-1 on expression pattern of some RBSDV-responsive miRNAs target genes in rice.Our results revealed that overexpressing of P7-1 and P9-1 altered expression of some RBSDV-responsive miRNAs target genes in T3 transgenic lines.The expression of miR159 and miR169 targets,OsOlg59660 and Os03g48970,respectively,were significantly up-regulated in P7-lOvE lines.Also,lines overexpressing P9-1 showed significant up-regulated expression of miR166 targets,Os03g01890 and Os10g33960.The most significant change was noticed in P7-lOvE-overexpressed lines in which the expression of Os02g44370,a target of miR171 encodes a scarecrow-like transcription factor(SCL),was strongly up-regulated compared to the control(more than three times).Changing in the expression of RBSDV-responsive miRNAs targets indicates alternation of expression of these miRNAs which might consequently disturb its role in regulating different developmental processes.Taken together,our study provide an additional evidence of the importance of RBSDV non-structural proteins P7-1 and P9-1 in RBSD disease development and give emphasis to the significant roles of these proteins in RBSDV-host interactions.In an attempt to obtain selectable marker free RBSDV-resistant transgenic rice,we selected RBSDV non-structural protein P7-2 and P8,encoded by S7-2 and S8 gene,respectively as targets for RNA interference(RNAi).RBSDV P7-2 is a potential F-box protein and involved in the plant-virus interaction through the ubiquitination pathway while RBSDV P8 is the minor core protein that possesses potent active transcriptional repression activity.We transformed rice calli using a mini-twin T-DNA vector harboring RNAi constructs of the RBSDV genes S7-2 or S8.As a result,we obtained plants harboring the target gene constructs and the selectable marker gene,hygromycin phosphotransferase(HPT).From the offspring of these transgenic plants,we obtained HPT gene-free plants.Homozygous T5 transgenic lines which harbored either S7-2-RNAi or S8-RNAi exhibited high level resistance against RBSDV under field infection pressure from indigenous viruliferous small brown planthoppers.Our results showed that RNA interference with the expression of S7-2 or S8 genes seemed an effective way to induce high level resistance in rice against RBSD disease.