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

Fingerprint

Plant Proteins
Viral Proteins
Cell Movement
Mosaic Viruses
Viruses
Lycopersicon esculentum
Proteins
Plasmodesmata
Biolistics
Plant RNA
Plant Viruses
Host Specificity
RNA Viruses
Brassica
Gene Library
Radioisotopes
Bacteriophages

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

Cite this

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. / Sasaki, Nobumitsu; Matsushita, Yasuhiko; Nyunoya, Hiroshi.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 123-144 (Methods in Molecular Biology; Vol. 2028).

Research output: Chapter in Book/Report/Conference proceedingChapter

Sasaki, Nobumitsu ; Matsushita, Yasuhiko ; Nyunoya, Hiroshi. / 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. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 123-144 (Methods in Molecular Biology).
@inbook{0cf2ca073ff34f4b9970ec214559b32a,
title = "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",
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.",
keywords = "Biolistic bombardment, Cell-to-cell movement, Far-western screening, Fluorescent protein, Inhibitory effect, Movement protein, Protein–protein interaction, λ Phage cDNA library",
author = "Nobumitsu Sasaki and Yasuhiko Matsushita and Hiroshi Nyunoya",
year = "2019",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-9635-3_7",
language = "English",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "123--144",
booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Plant Protein-Mediated Inhibition of Virus Cell-to-Cell Movement

T2 - Far-Western Screening and Biological Analysis of a Plant Protein Interacting with a Viral Movement Protein

AU - Sasaki, Nobumitsu

AU - Matsushita, Yasuhiko

AU - Nyunoya, Hiroshi

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

KW - Biolistic bombardment

KW - Cell-to-cell movement

KW - Far-western screening

KW - Fluorescent protein

KW - Inhibitory effect

KW - Movement protein

KW - Protein–protein interaction

KW - λ Phage cDNA library

UR - http://www.scopus.com/inward/record.url?scp=85068123461&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85068123461&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-9635-3_7

DO - 10.1007/978-1-4939-9635-3_7

M3 - Chapter

C2 - 31228112

AN - SCOPUS:85068123461

T3 - Methods in Molecular Biology

SP - 123

EP - 144

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -