The Mechanism For The Regulation Of Rice Stripe Virus Infecting Nicotiana Benthamiana By Ubiquitination

Rice stripe disease caused by rice stripe virus（RSV）infection has caused serious damage to rice production in China and even in Asia.It is of great significance to study the pathogenic mechanism of RSV and find disease-resistant genes for the prevention and control of RSV.The ubiquitination is widely involved in various processes of RSV infection in host plants and vector insects.Studying the mechanism of ubiquitination in response to RSV infection can help us to understand RSV pathogenesis.In this study,we systematically profiled the global changes of protein ubiquitination in N.benthamiana during RSV infection by omics approaches,and the mechanism of the host factor sterol methyltransferase 2（SMT2）regulating RSV infection as well as the ubiquitination and degradation of NbSMT2 mediated by RSV infection were investigated.We combined the ubiquitomics approach with the label-free quantitative proteomics approach to investigate potential ubiquitination status changes of N.benthamiana infected with RSV.399 ubiquitinated lysine（Kub）sites that showed significant changes upon RSV infection were identified.Bioinformatics analysis showed that the ubiquitination levels of Kub sites in several ribosome-related proteins were significantly up-regulated during RSV infection,suggesting that the ribosome function was seriously disturbed.Tobacco rattle virus（TRV）based gene silencing was used to investigate the potential influence of proteins with differentially ubiquitinated sites on RSV infection.Silencing of 3-isopropyl malate dehydratase large subunit（Leu C）inhibited RSV infection,while silencing of m RNA decapping enzyme-like protein（DCP1）promoted RSV symptom development in the late stage of RSV infection.In addition to regulating the ubiquitination of host proteins in response to RSV infection,N.benthamiana also ubiquitinated RSV-encoded proteins.Kub sites were identified in all seven RSV-encoded proteins.Three Kub sites were identified and significantly up-regulated in NbSMT2 and the protein level of NbSMT2 was down-regulated during RSV infection in the omics data.This interested us to investigate if NbSMT2 could regulate RSV infection.TRV-based gene silencing and transgenic overexpression assays showed that NbSMT2positively regulated RSV infection.Additionally,it was found that silencing of NbSMT2 significantly inhibited the formation of RSV infection foci in inoculated leaves,whereas transgenic overexpression of NbSMT2 promoted the spread of RSV infection foci in inoculated leaves.Furthermore,the effect of NbSMT2 on RSV cell-to-cell movement was investigated.The results showed that NbSMT2 positively regulates the cell-to-cell movement capacity of RSV movement protein（MP）,and further analysis of the effects of other genes in the sterol biosynthesis pathway on the cell-to-cell movement capacity of RSV MP showed that Nb SMO2 and Nb DWF5displayed similar function to NbSMT2.During RSV infection,the transcript level of NbSMT2 was significantly up-regulated,the protein level was significantly down-regulated and the ubiquitination level was significantly up-regulated.Protein stability assay revealed that NbSMT2could be rapidly degraded in RSV-infected N.benthamiana leaves and 26S proteasome pathway inhibitor MG132 was able to inhibit the degradation of NbSMT2,which indicated that RSV infection mediated the ubiquitination and degradation of NbSMT2.RSV infection could induce endoplasmic reticulum stress（ER stress）,combined with the localization of NbSMT2 which was localized on the endoplasmic reticulum membrane,we speculated that the degradation of NbSMT2 might be a concomitant behavior of ER stress caused by RSV infection.The mechanism of ubiquitination and degradation of NbSMT2 mediated by RSV infection was investigated.Agents that could induce ER stress were used to treat NbSMT2 and it was found that infiltrating of H₂O₂ in N.benthamiana leaves could effectively promote the degradation of NbSMT2.Meanwhile,high levels of reactive oxygen species（ROS）were found to exist in N.benthamiana leaves infected with RSV,which means that the leaves were under oxidative stress.Treatment of RSV-infested leaves with the reducing agent was able to inhibit the up-regulation of NbSMT2 transcription mediated by RSV infection.Extra reducing agents（dithiothreitol and N-Acetyl-L-cysteine）in semi-in vivo degradation experiments effectively inhibited the protein degradation of NbSMT2,which means that ROS induced by RSV infection mediated the polyubiquitination and degradation of NbSMT2.In RSV-infected leaves,transiently expressed NbSMT2 tended to form aggregates through intermolecular disulfide bonds,while mutant NbSMT2C133/176/180/203S only form dimers.The protein stability of NbSMT2C133/176/180/203S was significantly lower and the ubiquitination level was significantly higher than those of NbSMT2 in RSV-infected leaves,which indicated that NbSMT2 protects itself from degradation mediated by ROS through the forming of disulfide bonds.In summary,this paper analyzed the global changes of the ubiquitination in N.benthamiana during RSV infection by proteomics and ubiquitomics approaches.NbSMT2 was found to positively regulate the cell-to-cell movement capacity of RSV MP to promote RSV infection,while ROS induced by RSV infection in turn mediated the degradation of NbSMT2 through ubiquitin-26S proteasome pathway for inhibiting RSV infection.