Mechanism Of Glyceraldehydes 3-phosphate Dehydrogenase Involved In CMV Infection In Nicotiana Benthamiana

Cucumber mosaic virus(CMV) is a typical member of genus Cucumovirus family. CMV can infect Over 1200 species of monocots and dicots. CMV is as a model for understanding plant-virus interactions. In order to figure CMV- host interaction mechanism, we screened host factor interacted with CMV replicase 1a by using membrane yeast two-hybrid system.The main results are as follows:CMV replicase 1a as bait protein screen host factorSplit ubiquitin yeast assay results indicated that p BT3-C-LS1 a had no self-autoactivation, and so consequently used p BT3-C-LS1 a as bait vector to screen Arabidopsis c DNA library. In the Arabidopsis c DNA library screening, we obtained five positive interactors.One of them was cytoplasmic glyceraldehyde-3-phosphate dehydrogenase(At Gap C2).When plit ubiquitin yeast assay was used to analyze interaction between 1a protein and Gap C1 and Gap C2 from Nicotinia benthamiana or Arabidopsis respectively, the data suggested that replicase 1a interacted with Nb Gap C1/2 and Arabidopsis thaliana At Gap C1/2. N-terminal methyltransferase and C-terminal helicase of 1a protein was cloned into p BT3-C vector, respectively. The results showed that Gap C2 interaction with 1a protein N-terminal methyltransferase.1a and 2a subcellular location and co-localization of 1a and Gap CWe studied subcellular location of CMV replication 1a and 2a. Results indicated that 1a located on vacuole, most of 2a located in cytoplasm and 2a was recruited onto vacuole membrane in presence of 1a and 2a exist in cell. Bimolecular Fluorescence Complementation(Bi FC) assays suggested that 1a and Gap C interacted in host tissues N.benthamiana.Co-localization results suggested Gap C2 mainly localizes in cytoplasm, little account localizes on vacuole. Gap C2 was recruited by 1a onto local region of vacuole membrane to aggregate when 1a existedEffect of knockdown-Gap C in Nicotiana benthamiana on CMV infectionKnockdown of Gap C m RNA level of the seedlings of host plants by TRV-VIGS increased CMV genomic RNAs replication and inhibited viral RNA translantion analyzed by Northern blot and Western blot. The results indicated that Gap C negatively regulated replication of CMV RNA and positively regulated translation of CMV genomic RNA4.