Study Of The Interaction Between The Coat Protein Of Cucumber Mosaic Virus And Host Factors

Cucumber mosaic virus(CMV)is the type member of the genus Cucumovirus(family Bromoviridae).It consists three single-stranded RNAs(RNA1-3),encoding five proteins(1a,2a,2b,MP and CP).CMV usually induces very severe symptoms,such as mosaic,stunting and leaf disformation.But it is still unclear how the virus could induce such various symptoms on host,especially the mechnism of mosaic symptoms,which is always the research hotspot for plant virology.Here,the M strain of CMV(CMV-M)can induced extremely yellow-white symptoms on tobacco and is the good choice to study the mechanism of mosaic development.As the cDNA of CMV-M RNA1 is not stable and toxic to most clone strains,the sequencing clone method is used to construct the infectious cDNA clone of the gene,showing that this method can effectively overcome the toxic problem of the cDNA of RNA1.The yellow-white symptom induced by CMV-M is mapped to viral RNA3 with pseudo-recombinant method.Further investigations find that the virus CP is directly related with the yellow-white symptom.Site-directed mutation is also used to find the critical amino acid of CP which is responsible for yellow-white symptom.Except that,we construct two different 2b mutants of CMV-M and also explore the role of 2b for the develpoment of the mosaic symptoms,finding that CMV-M 2b is not the direct determinant for the yellow-white symptom.To explain how the CMV-M CP could induce yellow-white symptoms on tobacco,the subcellular localization of CP is firstly studied by using green fluorescent protein(GFP)combined with transient expression system,showing that the CP mainly localizes in cytoplasm and nucleus.Secondly,the cDNA library of tobacco is constructed and yeast two hybrid system(YTH)is performed with the bait of M-CP.One host interacting protein is picked and found to be Ferredoxin I(FdI),which is encoded by nuclear genome and function in chloroplast.Bimolecular Fluorescence Complementation(BiFC)is also used to confirm that the interaction occurs in plant cytoplasm.The transient peptide of FdI is predicted by online software and further verified with transient expression systerm.It is found that the transient peptide influences the interaction between FdI and CP.The CMV-Fny could induce green mosaic symptoms on tobacco,while CMV-Q could not.Reuslts show that the FdI could interact with Fny-CP,but not with Q-CP,showing than the FdI is related with mosaic symptom developing.The accumulation of FdI is down-regulated after virual infection,especially in the yellow-white leaf.When the mRNA of FdI is partly silenced with VIGS(virus induced gene silencing)systerm,the host also shows mosaic symptom,further revealing that the down-regulation of FdI is directly related with the develpoment of mosaic symptom.FdI is encoded by a small nuclear gene,synthesized in the cytoplasm and then imported into chloroplast with its N-terminal transient peptide,so we speculate that the CP could interfer the process of Fdl imported into chloroplast by interacting with FdI,resulting the repression of FdI in chloroplast and further leading the development of mosaic symptoms.Lastly,Arabidopsis mutants library is used to identify mutans which is senstive to viral infection.One mutant,named 78,could affect the viral RNA accumulation.Further work will be done to identify the mutant gene and its role during viral infection.