The Role Of Heat Shock Protein Hsp70 Involved In CMV Infection Process

Cucumber mosaic virus(CMV) is a member of genus Cucumovirus family. CMV has extremely broad host type, infecting more than 1000 host species within 365 genera 85 families, including single cotyledon and dicotyledonous and is one of the most widespread type of virus in the world. From the yeast two hybrid libraries,we obtained a host factor Hsp70(Heat shock protein 70 k Da) which interacted with CMV replicase 1a. In this study, we performed a series of works to prove that Hsp70 involved in CMV infection process. The main results are as follows:CMV replicase 1a interacted with protein Hsp70 in the host cellWe constructed two fusion protein vectors p CB301-LS1a-n YFP and p CB301-Nb Hsp70-c YFP, in which CMV 1a replicase was fused with N-terminal of YFP and Nb Hsp70 fused with C-terminal of YFP, respectively. Agrobacterium mixtures containing p CB301-LS1a-n YFP and p CB301-Nb Hsp70-c YFP were co-infiltrated on the leaves of Nicotiana benthamiana plants. At 3 days post-infiltration, yellow fluorescence appeared in the host cells and colocalized with red fluorescence from tonoplast marker(p CB301-TIP2-m Cherry) under laser confocal microscope. The results suggested that protein Hsp70 interacted with CMV replicase 1a in the host cells.CMV replicase 1a colocalized with Hsp70 on the tonoplasts of host plantsBy labeling Hsp70 with fluorescent protein to study the colocalization of replicase 1a and Hsp70. Results from co-infiltration with bacterial suspension carrying p CB301-Egfp and p CB301-1a showed that the Hsp70 was primarily cytoplasmic localization. In presence of 1a, Hsp70 protein was recruited to the vacuole membrane.Overexpression of Hsp70 promoted CMV infection in the seedlings of host plantsIn order to study the function of Hsp70 involved in CMV infection, p CMABIAT-Nb Hsp70 vector was engineered to over-express the level of Hsp70 in the tissues of N.benthamiana. Northern blot results indicated that the level of CMV genomic RNAs on the leaves infiltrated with pCMABIAT-NbHsp70 increased obviously, compared to the control empty vector pCAMBIA.Knock-down of Hsp70 resulted in the decrease of CMV genomic RNA on the host plantsWe used both TRV-induce gene silencing and chemical inhibitor of Hsp70, quercetin, to silence the expression of Hsp70. Then inoculated CMV by GV3101 agrobacterium infiltration inoculation and detect CMV accumulation. The northern blot and western blot results suggest that CMV accumulation decreased significantly compared with the control group.