Studies On The Interaction Between Maize Violaxanthin De-epoxidase And Sugarcane Mosaic Virus HC-Pro

Sugarcane mosaic virus(SCMV)is the major causal agent of maize dwarf mosaic disease in China.SCMV belongs to the genus Potyvirus.The helper component-proteinase(HC-Pro)encoded by potyviruses is a viral RNA silencing suppressor and is also a multifunctional protein,which plays important roles during virus infection.Virus diseases are considered as the result of the interactions between viruses and their host plants,therefore,it has important scientific significance to identify virus-interacting host factors and analyze their roles during virus infection.To this purpose,the students from our laboratory have identified the maize(Zea mays L.)violaxanthin de-epoxidase(ZmVDE)as an interactor with SCMV HC-Pro through a yeast two-hybrid(Y2H)screen of a maize cDNA library by using SCMV HC-Pro as a bait.ZmVDE is a nuclear-encoded chloroplast protein,whose mRNA is translated as a transit peptide-containing precursor protein in the cytoplasm,and which is ultimately transported into the thylakoid lumen after cleavage of its transit peptide.However,only the mature ZmVDE protein(ZmVDEm)was shown to interact with SCMV HC-Pro in previous co-transformation assay.In addition,the mRNA transcript and protein expression levels of ZmVDE were up-regulated at 10 days post inoculation with SCMV,and the accumulation of SCMV RNA was significantly increased when Zm VDE was transiently silenced.In this study,the interaction between SCMV HC-Pro and ZmVDE was further studied and the significance of their interaction was also explored.Y2H and in vivo co-immunoprecipitation(Co-IP)together with the subcelluar localization assay in maize protoplasts demonstrated that both ZmVDE precursor(ZmVDEp)and ZmVDEm(from ZmVDEp processing)interacted with SCMV HC-Pro and ZmVDEm was the crucial region essential for this interaction.Among SCMV-encoded proteins,only HC-Pro was detected to interact with ZmVDEm by using Y2H assay.However,HC-Pro was not detected in the total proteins of chloroplasts isolated from SCMV-infected maize leaves by western blotting.Y2H and Co-IP assays demonstrated that ZmVDE-HC-Pro interaction was virus-and host-specific.In addition,the glutamine(Q)at position 292 of ZmVDEm and the amino acids 101-460 in HC-Pro were the crucial amino acid and the crucial domain,respectively,essential for their interaction.The subcellular localization assay in Nicotiana benthamiana leaves showed that ZmVDEp,ZmVDEm(ZmVDE lacking the transit peptide)and its mutant ZmVDEmQ292A all localized in the cytoplasm,and ZmVDEp was processed to distribute mainly in the chloroplasts and slightly in the cytoplasm and chloroplast stromules.However,when these different forms of ZmVDE and the mutant ZmVDEmQ292A were co-expressed with SCMV HC-Pro in N.benthamiana leaves,these different forms of ZmVDE and the mutant ZmVDEmQ292A re-distributed to HC-Pro-containing aggregate bodies,but their expression had no obvious effect on the quantity of HC-Pro-containing aggregate bodies.In addition,ZmVDE re-localized to aggregate bodies in the presence of SCMV infection.The N.benthamiana leaves transiently co-expressing GFP,SCMV HC-Pro and GUS exhibited high green fluorescence under UV light due to the RNA silencing suppression activity of SCMV HC-Pro.But when different forms of ZmVDE(3Myc-ZmVDEm?ZmVDEp-3Myc and 3Myc-ZmVDEp)was co-expressed with GFP and SCMV HC-Pro,respectively,the green fluorescence intensity was evidently decreased,and consistent with the fluorescence intensity,the accumulations of GFP protein and mRNA were also decreased to varying extent.These results suggested that ZmVDE could attenuate the RNA silencing suppression activity of SCMV HC-Pro,and further analysis results showed that this capacity of ZmVDE depended on the interaction between these two proteins.In this study,ZmVDE,a host factor of maize,is reported to be likely involved in secondary defense responses against viral RNA slilencing suppressor in monocot plants,revealing an additional role of a chloroplast protein which responds to biotic stresses in addition to its well-known function in responding to abiotic stresses.