Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins

Abstract

We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement

“…All mutated sequences were verified by sequencing. Abaxial infiltrations of N. benthamiana leaves with Agrobacterium tumefaciens were performed essentially as described previously (Lim et al, 2009), applying a final concentration of 0.6 OD 600 , with constructs mixed in equal proportions where appropriate.…”

“…Subcellular localization of several class I (hordeivirus-like) and class II (potexvirus-like) TGB proteins has been investigated by using fluorescent gene fusions (Haupt et al, 2005;Ju et al, 2005;Lim et al, 2009;Lough et al, 1998;Solovyev et al, 2000). To assess localization of AltMV TGB3, we expressed TGB3 from pGDR and pGDG (Goodin et al, 2002) or pHVLR and pHVLG vectors for Agrobacteriummediated transient expression of DsRed or green fluorescent protein (GFP) fusions (Fig.…”

Tyrosine Phosphorylation of the Triple Gene Block Protein 3 Regulates Cell-to-Cell Movement and Protein Interactions of Potato Mop-Top Virus

“…These roles of TGBp3 and TGBp2 are consistent with their ability to increase the size exclusion limit of plasmodesmata (23). Yeast two-hybrid assays have shown that TGBp3 and TGBp2 self-interact and interact with each other (24,25), which is important for TGB functions, because disruption of the TGBp2-TGBp3 interaction inhibits the movement of Barley stripe mosaic virus (BSMV) (genus Hordeivirus) (26,27). Previous studies suggested that TGB proteins must be expressed at suitable relative molarities for viral movement to occur.…”

“…Previous studies suggested that TGB proteins must be expressed at suitable relative molarities for viral movement to occur. For example, the optimal relative expression ratio of TGBp2 and TGBp3 is 10:1 in BSMV and PMTV (25,28), and TGBp2 and TGBp3 should be expressed at 10-and 100-fold-lower levels, respectively, than TGBp1 (27,29,30). Previous studies on PMTV TGBp3 have been done by producing the protein from a plant expression vector or heterologous virus (e.g., see references [23][24][25].…”

Mechanical vs Agroinfiltration: Efficient mechanism of plant RNA virus infection

“…It also serves as a more economically feasible approach compared to instances where in vitro RNA transcription is required for the infection of cells for mechanical inoculation [25]. Even a robust approach like agrobacterium mediated gene transfer doesn't come without limitations, as it is limited to dicotyledonous plants and is not amenable for monocots [26]. Moreover, there is difference in the virulence of strains for Agrobacterium tumefaciens, and requires extensive optimization of technique as a protocol for transformation differs from one species to another [25,27].…”