Biological Function Of Interaction Between Maize Cysteine Protease ZmSee1 And Sugarcane Mosaic Virus-encoded VPg

Maize dwarf mosaic disease induces severe losses in several maize major production regions of China.Sugarcan mosaic virus(SCMV)is the main pathogen in China.SCMV,a member of genus Potyvirus,possess a single-stranded,positive-sense RNA genome encoding 11 proteins.VPg,a viral protein genome-linked,is covalently attached to the 5' terminus of the genomic RNA.As a multiple-functional protein,VPg has been explored in involvement in viral RNA replication and translation,and also in virus movement.Previous study identified a maize cysteine protease ZmSeel that interacted with VPg in yeast.ZmSeel is a member of the papain-like cysteine proteases family,which is reported to be involved in plant response to abiostresses and biostresses.This study aimed to investigate the biological function of the interaction between SCMV VPg and ZmSeel in SCMV infection and pathogenicity.Firstly,the specific interaction between VPg and ZmSeel was verified with Y2H and GST pull-down in vitro.Then the interaction was confirmed in vivo by coimmunoprecipitation(co-IP).Studies on tissue-specific expression pattern of ZmSeel revealed that its mRNA level was much higher in the leaf than that of root and leaf sheath.To investigate the changes of ZmSeel expression responding to SCMV infection,the levels of transcription and translation were measured.At the same time,the transcriptional levels of PR1 and PR5 were detected.The results showed there was no difference on the ZmSeel transcript level in the inoculated leaf and upper systemically-infected leaves between mock and SCMV-infected maize Va35 seedlings at 3 days post inoculation(dpi),while the PR1 and PR5 increased significantly in SCMV infected leaves.In SCMV-infected inbred line Zheng58,the protein level of ZmSeel mature protease increased significantly at 24 hpi and 72 hpi,and there was no change in SCMV-infected inbred lines Chang7-2,Huangzao4,B73,Mol7 and Zong31.Transient expression of ZmSeel:mRFP in epidermal cells of Nicotiana benthemiana,healthy and SCMV-infected maize leaves showed ZmSeel:mRFP located in apoplastic space and cytoplasm,but not in the nucleus.Co-expression of ZmSeel:mRFP and SCMV VPg:GFP did not change their subcellular localization,and VPg did not interfere with post-translational maturation of ZmSeel in N.benthamiana.Interestingly,transient expression of SCMV VPg in N.benthamiana inhibited the enzymatic activity of ZmSeel.Knocking down the expression of ZmSeel using both Brome mosaic virus-and Cucumber mosaic virus-based gene silencing vectors in maize significantly decreased the transcriptional and protein level of ZmSeel.The SCMV mRNA and CP significantly increased to approximately 2-4 fold in ZmSeel-silenced seedlings compared with that of nonsilenced seedlings.Overexpression and stem-loop mediated RNAi transgenic maize plants were developed to study the function of ZmSeel in SCMV infection.The ratio of leaf width/length of RNAi transgenic maize seedlings was significantly increased compared with that of nontransgenic seedlings.The SCMV mRNA and CP significantly increased in the systemically infected ZmSeel-RNAi transgenic seedlings at 5 dpi compared with that of nonsilenced seedlings.For ZmSeel overexpression transgenic plants,the growth phenotype was similar to nontransgenic plants,while the SCMV mRNA and CP decreased significantly in the systemically infected seedlings at 5 dpi compared with that of nonsilenced seedlings.