Molecular Mechanism Of 6K And P3N-PIPO Regulating The Replication And Movement Of Tobacco Vein Banding Mosaic Virus

The genus Potyvirus(family Potyviridae)is the largest genus of plant-infecting viruses and consists of more than 200 species.Viruses of Potyvirus have a wide host range and cause great economic losses to world agriculture production.Planting resistant cultivars is the most economic and effective way to control crop viral disease.However,there is a shortage of cultivars resistant to virus in the crop production.Plant virus needs to hijack host factors to establish systemic infection.The loss of the host factors necessary to virus infection will result in host resistance.Understanding the function of viral factors and the interaction between virus and host is a prerequisite to develop new resistance strategies.This research mainly revealed the interaction mechanism between Tobacco vein banding mosaic virus(TVBMV)and host in the process of virus intercellular movement and replication.The main finding is as follow:1.The mechanasim of a 6K1-6K2-PsbO1 protein complex mediating TVBMV replication: The first 6 kDa protein contained a RSD motif.Alanine scanning mutagenesis in this conserved motif decreased the replication of TVBMV.The subcellular localization showed TVBMV 6K1 distributed in the whole cell but recruited to chloroplast and co-localized with 6K2 and nuclear inclusion protein b during TVBMV infection.TVBMV 6K1 could interact with 6K2,and was recruited by 6K2 to viral replication complexes(VRCs)found to be adjacent to chloroplast.The immunoprecipitation and liquid chromatography-tandem mass spectrometry assay showed the Photosystem II oxygen evolution complex protein of Nicotiana benthamiana(NbPsb O1)was a chloroplast protein interacting with 6K2 of TVBMV and present in the VRCs.Knockdown of NbPsbO1 gene expression in N.benthamiana plants through virus-induced gene silencing significantly decreased the genomic RNA and coat protein amounts of TVBMV and another potyvirus Potato virus Y,but not Potato virus X of genus Potexvirus.NbPsbP1 and NbPsbQ1,two other components of the Photosystem II oxygen evolution complex had no interaction with 6K2 and no effect on TVBMV replication.2.The mechanism of a P3N-PIPO-CI-NbDREPP protein complex regulating TVBMV intercellular movement: We provide genetic evidence showing that mutants carrying a PIPO domain of seven,20,or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant.We also show that a developmentally regulated plasma membrane protein of N.benthamiana(referred to as NbDREPP)interacted with both P3N-PIPO and Cylindrical Inclusion(CI)of the movement-competent TVBMV.The knockdown of NbDREPP gene expression in N.benthamiana impeded the cell-to-cell movement and systemic infection of TVBMV.NbDREPP was shown to localize at the cell wall and colocalize with plasmodesmata(PD)-resident plasmodesmata-localized protein 1.And NbDREPP could also colocalize with TVBMV P3N-PIPO and CI at PD.Brefeldin A treatment and transient overexpression of ADP-ribosylation factor1-T31 N or Secretion-associated and ras superfamily-related gene1b-H74 L disrupted the PD targeting by NbDREPP.Latrunculin B treatment and transient overexpression of the dominant-negative mutant myosin XI-2 disrupted the PD targeting by NbDREPP.3.We also reveal the mechanism of symptom development by comparing transcriptomic changes in Nicotiana benthamiana plants infected with the wild-type or an attenuated mutant of TVBMV.The helper component proteinase(HCpro)of TVBMV is an RNA silencing suppressor protein.Substitution mutations introduced into the HCpro open reading frame(ORF)in a TVBMV infectious clone resulted in changes of Asp189 to Lys or Ile250-Gln251 to Asp-Glu.These amino acid changes eliminated the RNA silencing suppression activity of the mutant HCpro(HCm)and attenuated the disease symptoms caused by the mutant TVBMV(T-HCm)in Nicotiana benthamiana plants.Here,we used RNA-seq technology to compare gene expressions in the plants inoculated with the wild type TVBMV(T-WT)with that in the plants inoculated with T-HCm at 1,2 and 10 days post agroinfiltration(dpai).At 1 and 2 dpai,N.benthamiana genes related to translation machinery were up-regulated,whereas genes related to lipid biosynthesis and metabolism or related to responses to extracellular/external stimuli were down-regulated in the leaves inoculated with T-WT or T-HCm.At 10 dpai,T-WT infection repressed photosynthesis-related genes.T-WT and T-HCm infections differentially perturbed genes involved in RNA silencing pathway.The salicylic acid and ethylene signaling pathways were induced but the jasmonic acid signaling pathway was repressed after T-WT infection.Infections of TWT and T-HCm also differentially regulated the genes involved in the auxin signaling transduction,which is known to associate with the stunting phenotypes caused by TVBMV.In conclusion,TVBMV hijacks NbDREPP for intercellular movement and NbPsbO1 for replication.DREPP and PsbO1 are potential antiviral novel targets.The infection of TVBMV interferes the Calvin–Benson cycle,chlorophyll metabolism and auxin signalling transduction pathways,which associate with the chlorosis or stunting symptoms caused by TVBMV.