National Institute of Science and Technology in Plant-Pest Interactions, Bioagro, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil.
Departament of Biochemistry and Molecular Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil.
Methods Mol Biol. 2024;2724:111-125. doi: 10.1007/978-1-0716-3485-1_9.
Protoplasts are isolated plant cells from which the cell walls have been removed by treatment with fungal cellulase and macerozyme enzymes, which degrade the primary components of the cell wall. The protoplasts are totipotent, sensitive, and versatile; thereby, they have been extensively used to study signal transduction pathways, cell-autonomous responses, and replication of plant viruses. This system has several advantages over the use of whole plants for viral replication, including a high percentage of infected cells and uncoupling virus movement from replication assays. Here, we describe a simple and efficient protocol for begomovirus transfection and replication in Arabidopsis protoplasts. The method consists of four steps, (i) protoplast isolation, (ii) PEG-calcium transfection of begomovirus infectious clones, (iii) elimination of input plasmid DNA by DpnI digestion, and (iv) quantification of the viral newly synthesized DNA by qPCR. The protoplasts can be transformed efficiently with begomovirus infectious clones, and virus replication can be monitored by the accumulation of nascent viral DNA in the infected protoplasts.
原生质体是通过用真菌纤维素酶和离析酶处理去除细胞壁而分离得到的植物细胞，这些酶可以降解细胞壁的主要成分。原生质体具有全能性、敏感性和多功能性；因此，它们已被广泛用于研究信号转导途径、细胞自主反应和植物病毒的复制。与使用整个植物进行病毒复制相比，该系统具有几个优点，包括高比例的感染细胞和将病毒运动与复制测定分离。在这里，我们描述了一种在拟南芥原生质体中进行双生病毒转染和复制的简单有效的方法。该方法包括四个步骤：（i）原生质体分离，（ii）PEG-钙转染双生病毒感染性克隆，（iii）用 DpnI 消化去除输入质粒 DNA，（iv）通过 qPCR 定量新合成的病毒 DNA。可以有效地用双生病毒感染性克隆转化原生质体，并且可以通过感染的原生质体中新生病毒 DNA 的积累来监测病毒复制。
