Auxin Regulation In The Interaction Between Rice Stripe Virus And Host Rice

Rice stripe virus(RSV) is the causal agent of rice stripe disease which is one of the most serious rice diseases in temperate and subtropical China,and transmitted in a persistent,propagative manner by the small brown planthopper(Laodelphax striateUus).Planting susceptible varieties in large scale is one of the major factors contributed to the RSV epidemic of unprecedented magnitude.Now,the lack of resistant varieties for RSV is an important key without control this disease,further investigation for the interaction between RSV and rice will great help solve this question.The differences of gene expression when Wuyujing 3(susceptible variety) and KT95-418(resistant variety) infected by RSV were investigated by microarray, and revealed that auxin signal transduction involved the interaction between RSV and its host.Here,this study use genetic approaches to reveal the function of OsSAuRs of rice resistant variety KT95-418 on virus infection and replication in tobacco based on the analysis of microarray between RSV and rice.The results are summarized as follows:Firstly,the detection of RSV coding seven genes by real-time RT-PCR were established.And the expression differences of these seven genes in the course of RSV infection and replication in rice suspension cells were analysed by real-time RT-PCR. The result indicated that the RNA expression ofRdRp gene reached its peak at 12 h after RSV infection,the others genes of RSV reached their peaks at 48 h post-infection.The variabilities of RSV 7 genes in the course of RSV replication were analyzed via t-test,disclosed that the negative sense coding genes(RdRp,NSvc2 and NSvc4) showed more difference with significance except for CP gene in the RSV replication course.RdRp gene showed most difference with significance,and the value of t-test was 2.67(P=0.05?0.025).However,CP gene showed most difference without significance,the value of t-test was 1.35(P=0.40?0.20).All of positive sense coding genes(NS2?NS3 and SP) were proved more difference without significance. This study also screened and selected three types of L.striatellus that were significantly different in RSV transmission competence:highly efficient transmitting population(HETPs),lowly efficient transmitting population(LETPs) and non-transmissible viruliferous population(NTVPs),representing the inoculation rates of 95%,5%,and 0.4%,respectively.RNA expressions of RSV coding seven genes from individual female L.striatellus with distinct virus transmission competence were determined using real-time reverse transcription PCR.Results showed that the RSV transmission competence of L.striatellus might be inherited and regulated by the great selection pressure of RSV,and that the expressions of NSvc2 and NSvc4 genes were associated with the RSV transmission competence.In addition,the SP gene expressions of HETPs and LETPs were significantly different from that of NTVPs. NS3 gene was the most stable expression among the three different vector types. Results from this study are believed to be beneficial for future Tenuivirus transmission and pathogenesis studies.The different resistance against Rice stripe virus(RSV) between rice Wuyujing 3 and KT95-418 was investigated via detecting the replication cycle and relative amount of RSV in two varieties rice suspension cells by real-time RT-PCR assay and conventional RSV infected assay.The expression differences of some genes related auxin signaling pathway that involved in interaction between RSV and rice using microarray analysis were validated by real-time RT-PCR and bioinformatics method.The result revealed that four auxin early response-gene showed more significant difference in resistant variety KT95-418 infected by RSV,including OsSAuR1(LOC_Os01g56240)?OsSAuR39 (LOC_Os09g37330)?OsSAuR53(LOC_Os09g37480) and OsIAA31 (LOC_Os12g40900).And five genes expressed great differently in diseased plants of susceptible variety Wuyujing 3,including OsIAA6(LOC_Os01g53880)?OsIAA9 (LOC_Os02g56120)?OsIAA18(LOC_Os05g44810)?OsIAA31 (LOC_Os12g40900) and GH3-5(LOC_Os05g50890).OsIAA9 and OsIA431 were failed to be detected in any tissues of rice plant,including RSV diseased plants.This could be due to without transcription or because of their low aboundance.The other six genes(OsIAA6?OsIAA18?OsGH3-5?OsSAuR1?OsSAuR39 and OsSAuR53) showed a dynamic process during RSV infection and replication in Wuyujing 3 and KT95-418 palnts.The expressions of these genes were up-regulated in rice suspension cells infected by RSV,whereas,only a transient up-regulation at 4?8 d after RSV infection in rice plants and the expression profile at 16 d after RSV infection were consistent with the result of microarray analysis.The samples investigated in microarray were collected at about 16 d after RSV infection.In order to confirm auxin involved in the course during RSV infection and replication,the expression of YUCAA1 gene and the amount of endogenous IAA in rice plants and rice suspension cells infected by RSV were investigated by using real-time RT-PCR and high performance liquid chromatography,respectively.The expression of YUCAA1 gene and the amount of endogenous IAA increased at various times(16 h?32 h?48 h and 64 h) after post-infection by RSV in rice suspension cells. In rice plants infected by RSV,the expression of YUCAA1 gene and the amount of endogenous IAA increased at 4 d?8 d after post-infection as comparison with that of healthy rice plants.However,which decreased at 12 d and 16 d.These results indicated that RSV infection could regulate auxin biosynthesis in rice.Meanwhile,the amount of endogenous IAA and the mRNA expression of YUCCA1 gene in KT95-418 diseased plants were more higher than that of Wuyujing 3 diseased plants. That suggest KT95-418 could improve resistance through repressing auxin level in plant.Addtionally,the expression of RSV CP increased 2.9 times in rice plants after it was treated with KPSC buffer to deplete the endogenous auxins,and decreased 45% after 30?M IAA treatment.All of these results suggested that the auxin might play a role among RSV replication in rice plant.In order to confirm auxin receptors that located in upstream of auxin signaling pathway involved in the course during RSV infection and replication,because auxin receptor eonding SCF^TIR1 complex could regulate the stability of auxin early response gene,such as OsIAA6?OsIAA9?OsIAA18?OsIAA31?OsGH3-5 and OsSAuR53.and the expression of these genes were down-regulation at 16 d after RSV infection by real-time RT-PCR and microarray analysis.At the same time,the changes of the expression of YUCAA1 gene and the amount of endogenous IAA in rice plants and rice suspension cells were involved in the course of RSV infection.It is necessary to study the auxin receptor weather plan a rule or not when RSV infection.Three auxin receptor proteins containing F-box domain in Orazy sativa were determined based on the amino acid sequences of four auxin receptor proteins(TIR1?AFB1?AFB2 and AFB3) in Arabidopsis thaliana.The phylogenetic analysis of these seven auxin receptor protein sequences revealed that OsTIR1?TIR1 and AFB1 were clustered a group,showed more highly relationship,OsAFB1?OsAFB2?AFB2 and AFB3 showed more highly homology as one group.In additional,the mRNA expression of rice auxin receptor genes after infection with RSV and Rice dwarf virus(RDV) were investigated by using real-time RT-PCR.The results indicated that the transcript abundances of OsTIR1 and OsAFB1 increased after post-infection by RSV and RDV. Meanwhile,decrease was observed for OsAFB2 in diseased rice plants as comparison with healthy ones.To determine the functions office OsSAUR1?OsSAUR39 and OsSAUR53 genes, three genes were cloned by RT-PCR and used for construction of prokaryotic expression vectors.The result indicated that the cDNA sequences of OsSAUR1?OsSAUR39 and OsSAUR53 genes were 276 bp?516 bp and 435 bp respectively,and coding 93?173 and 145 amino acid residue.And the GST-OsSAUR1?GST-OsSAUR39 and GST-OsSAUR53 fusion proteins were expressed in E.coli BL21 after being induced with IPTG.The purified fusion proteins were used to immunize mice to gain polyclonal antibody,the specificity and immunizing potence of polyclonal antibody were evaluated by Western-blot and ELISA.These antibodies showed high specific and more immunizing potence.However,OsSAUR1?OsSAUR39 and OsSAUR53 were failed to detect in healthy rice plants and RSV diseased plants that revealed these proteins had low expression or had little stabilization in plants.Whereas,the antibodies raised against GST-OsSAUR1?GST-OsSAUR39 and GST-OsSAUR53 fusion proteins could detect the OsSAUR1?OsSAUR39 and OsSAUR53 proteins in the plant extract only in the presence of a proteasome inhibitor,MG132.These results disclosed that SCFTIR1 possibly modulated the stability of OsSAUR proteins.To further disclosed the functions of rice OsSAUR1?OsSAUR39 and OsSAUR53 proteins,fusion gene of these proteins and enhanced green fluorescent (EGFP) were inserted into a pEGAD vector for constructing the plant expression vector pEGAD-OsSAUR1?pEGAD-OsSAUR39 and pEGAD-OsSAUR53.Then,the recombinant vector were transformed into onion epidermal ceils through particle bombardment.The study revealed that rice OsSAUR1?OsSAUR39 and OsSAUR53 proteins,were exclusively localized in the nuclear under blue light excitation by fluorescence microscopy.Meanwile,leaf discs of Nicotiana tobacco were infected by Agrobacterium tunefaciens strain EH105 containing these plant expression vectors, including PKYLX-OsSAUR1?PKYLX-OsSAUR39 and PKYLX-OsSAUR53.Kan resistant plants from individual transformation events were obtained and PCR checking and GUS staining confirmed the integration of foreign genes.In total,33 OsSAUR1 overexpression lines?36 OsSAUR39 overexpression lines and 21 OsSAUR53 overexpression lines were obtained in this study.But none of them showed visible phenotypes changes among OsSAUR1 overexpression lines and OsSAUR39 overexpression lines.These suggested that OsSAUR1 and OsSAUR39 are redundant in tobacco development.However,the overexpression of OsSAUR53 functioned in yellowing leaf?rolled leaf and slow growth determination of four transgenic plants among 21 transgenic plants.Further analysis of influence for Tobacco mosaic virus(TMV) infection and replication in transgenic plants revealed no significant difference were observed after TMV infected.It suggested that overexpression of OsSAUR1?OsSAUR39 and OsSAUR53 didn't influence the TMV infection and spread in tobacco.In this study,the expression differences and functions of some genes of auxin signaling pathway that involved the interaction between RSV and rice were investigated by genetic approach.And disclosed auxin signal transduction involved in the course of RSV infection and replication in rice plant,and the resistant variety KT95-418 could restrain endogenous IAA level to improve plant resistance for RSV. However,three specific genes(OsSAUR1?OsSAUR39 and OsSAUR53) significantly down-regulated in RSV diseased KT95-418,these overexpression in transgenic tobacco plant could not influence the TMV infection and replication in tobacco.All together,this study will help to discover the mechanism of RSV pathogenicity and variety resistance.