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

Nobumitsu Sasaki, Yasuhiko Matsushita, Hiroshi Nyunoya

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages123-144
Number of pages22
DOIs
Publication statusPublished - 2019 Jan 1

Publication series

NameMethods in Molecular Biology
Volume2028
ISSN (Print)1064-3745

Keywords

  • Biolistic bombardment
  • Cell-to-cell movement
  • Far-western screening
  • Fluorescent protein
  • Inhibitory effect
  • Movement protein
  • Protein–protein interaction
  • λ Phage cDNA library

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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