Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus

We have recently used a green fluorescent protein (GFP) fusion to the gamma b protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The gammab protein is involved in expression of th e triple gene block (TGB) proteins encoded by RNA beta but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replic ation or movement substantially, and mutagenesis experiments demonstrated t hat the three most abundant TGB-encoded proteins, betab (TGB1), betac (TGB3 ), and betad (TGB2), are each required for cell-to-cell movement (D. M. Law rence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001). We have now ext ended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivati ves containing the TGB1 fusion were able to move from cell to cell and esta blish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion prot ein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localizatio n of the GFP-TGB1 protein indicated an association with the endoplasmic ret iculum and with plasmodesmata. The subcellular localization of the TGB1 pro tein was altered in infections in which site-specific mutations were introd uced into the six conserved regions of the helicase domain and in mutants u nable to express the TGB2 and/or TGB3 proteins. These results are compatibl e with a model suggesting that movement requires associations of the TGB1 p rotein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.