Plant Protein-Mediated Inhibition of Virus Cell-to-Cell Movement: Far-Western Screening and Biological Analysis of a Plant Protein Interacting with a Viral Movement Protein

Cell-to-cell movement via plasmodesmata is a crucial step for plant RNA viruses to determine their host ranges. Many viruses including Tomato mosaic virus (ToMV) encode one or more movement proteins (MPs) that are indispensable for cell-to-cell movement. During movement processes, MPs are thought to interact directly with many plant proteins that may be involved in supporting or inhibiting cell-to-cell movement of viruses. In order to understand the molecular mechanisms that regulate viral spread positively or negatively, it is important to discover such MP-interacting plant proteins and analyze their functions in viral cell-to-cell movement in efficient ways. In this chapter, we provide protocols of a radioisotope-based far-western screening strategy to construct a λ phage cDNA library from a nonhost Brassica campestris (syn. rapa) for ToMV and identify plant proteins that bind directly to the ³²P-labeled probe of ToMV MP, and subsequently a biolistic bombardment method to examine whether a plant protein selected have a function as an inhibitory factor that can interfere with virus cell-to-cell movement.