植物叶肉原生质体的分离和转染在病毒学研究中的应用。

Isolation and Transfection of Plant Mesophyll Protoplasts for Virology Research.

机构信息

London Research and Development Centre, Agriculture and Agri-Food Canada, London, ON, Canada.

Department of Biology, University of Western Ontario, London, ON, Canada.

出版信息

Methods Mol Biol. 2022;2400:43-53. doi: 10.1007/978-1-0716-1835-6_5.

DOI:10.1007/978-1-0716-1835-6_5

PMID:34905189

Abstract

Protoplasts are the naked plant cells lacking the rigid cell wall and have been broadly utilized as an excellent tool to study the molecular virus-plant interactions, particularly at the early stages of the infection process, such as virion disassembly, viral genome translation, intracellular trafficking, and virus replication. Compared to the use of whole plants, the protoplast system has several major advantages in plant virology research, including homogeneous cell populations, high percentage of infected cells, synchronous infection, effects free from other cells/tissues, and ease of extraction of the viral RNA. This chapter describes a simple, streamlined, and efficient protocol for isolation and purification of mesophyll protoplasts from the model plants Arabidopsis thaliana and Nicotiana benthamiana, and subsequent transfection of the isolated protoplasts with a potyvirus infectious clone.

摘要

原生质体是缺乏刚性细胞壁的裸露植物细胞，已广泛用作研究分子病毒-植物相互作用的优秀工具，特别是在感染过程的早期阶段，如病毒体解体、病毒基因组翻译、细胞内运输和病毒复制。与使用整个植物相比，原生质体系统在植物病毒学研究中有几个主要优势，包括同质细胞群体、高比例的感染细胞、同步感染、不受其他细胞/组织影响以及易于提取病毒 RNA。本章描述了一种简单、精简和高效的方案，用于从模式植物拟南芥和本氏烟中分离和纯化叶肉原生质体，然后用马铃薯 Y 病毒感染性克隆转染分离的原生质体。