First evidence showing that Pepper vein yellows virus P4 protein is a movement protein
Plant viruses move through plasmodesmata (PD) to infect new cells. To overcome the PD barrier, plant viruses have developed specific protein(s) to guide their genomic RNAs or DNAs to path through the PD. Results In the present study, we analyzed the function of Pepper vein yellows virus P4 protein. Our bioinformatic analysis showed that the P4 protein contains an transmembrane domain, encompassing the amino acid residue 117-138. The P4 protein was found to target PD and form small punctates
... small punctates near walls. The P4 deletion mutant or the substitution mutant lost their function to produce punctates near the walls inside the fluorescent loci. The P4-YFP fusion was found to move from cell to cell in infiltrated leaves, and P4 could complement Cucumber mosaic virus movement protein deficiency mutant to move between cells. Conclustion Taking together, we consider that the P4 protein is a movement protein of Pepper vein yellows virus. Background Plasmodesmata-mediated macromolecular trafficking is critical for plant growth and development . Plant viruses have been shown to traffic between host cells through plasmodesmata (PD), and such trafficking is crucial for viral systemic infection . PD is considered as a bottleneck for plant virus infection in plant, due mainly to its size exclusion limit (SEL) and/or the intricate and dynamic regulations controlled by the host defense mechanism . To overcome this bottleneck, plant viruses have evolved to encode movement protein(s) (MP) to facilitate their intracellular trafficking, in a form of viral replication complexes or viral particle . The MPs produced by plant viruses are strikingly different . The pioneer report of viral MP is the 30 kDa MP of Tobacco mosaic virus (TMV), this MP was considered to guide TMV virion to move between cells , and its domain of 19 amino acids (195 to 213) is essential for localization of the MP to the cell wall fraction of plant cells . Similarly as TMV, the Ourmiaviruses also encoded a 30K movement protein to guide virus movement in plant cells . The second type of MPs have two or three specialized MPs, and are referred to as double or triple gene block proteins (DGBps and TGBps) , which form polyprotein to localize to the periphery of the plant cells . The third type of MPs are low molecule MPs, such as NSm encoded by Tomato spotted wilt tospovirus (TSWV) . The Full length PeVYV P4 gene and its alanine substitution mutant or deletion mutant aa position (117-138) were constructed individually through overlap PCR using primers listed in Table S1 . The wild type (WT) P4 and its mutants were cloned into expression vector pGWB441(YFP) or pGWB505(GFP) using the Gateway technology . Plasmid pPDLP8-YFP and pPDLP8-RFP (marker protein localized in PD) was from a previously described source and was used to localize plasmodesmata in cell walls . These plasmids were introduced individually into Agrobacterium tumefaciens strain GV3101. Agrobacterium cultures containing the corresponding plansmid were grown overnight in a YEP medium (10 g yeast extract, 10 g Bacto peptone and 5 g NaCl in one liter H 2 O, pH 7.0) supplemented with 100 mg/L kanamycin and 50 mg/L rifampicin. Agrobacterium cells were pelleted and then incubated for three hours in an infiltration buffer (10 mM MgCl 2 , 10 mM MES, pH 5.9, and 150 μM acetosyringone). The cultures were further diluted to OD600 = 0.2 and then infiltrated individually into the abaxial side of N. benthamiana leaves. The infiltrated plants were again grown at 25°C with a 16 h light/8 h dark illumination. Western blotting Western blotting was operated as previously described . Total protein was separated by electrophoresis in 10% SDS-PAGE and transferred onto a PVDF membrane. The antigens on the PVDF membrane were detected with polyantibody against PeVYV P4, then incubated by AP-coupled goat anti-mouse IgG (1:5 000 dilution; Sigma) and 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (NBT/BCIP) staining (Sangon Biotech, Shanghai, China). YFP, GFP and RFP images Agro-infiltrated leaves were detached from the plants and examined for fluorescence from different YFP, GFP or RFP fusion proteins under a Nikon TI-E+C2 confocal laser-scanning microscope (Nikon Microsystems, Watford, United Kingdom). The excitation wavelength was set at 514 nm for YFP and GFP, and 555 nm for RFP, and the emission wavelength was set at 520-550 nm for YFP and GFP, and Not applicable. ) kind gifting the cucumber mosaic virus infectious clone with its movement protein 3a replaced by GFP.