RNA干扰
遗传筛选
生物
突变体
正向遗传学
基因
黄瓜花叶病毒
遗传学
拟南芥
核糖核酸
位置克隆
病毒
拟南芥
反向遗传学
病毒学
作者
Zhongxin Guo,Xianbing Wang,Wanxiang Li,Shou‐Wei Ding
出处
期刊:Methods in molecular biology
日期:2019-01-01
卷期号:: 215-229
被引量:5
标识
DOI:10.1007/978-1-4939-9635-3_12
摘要
RNA interference (RNAi) acts as a natural defense mechanism against virus infection in plants and animals. Much is known about the antiviral function of the core RNAi pathway components identified mostly by genetic screens based on specific RNAi of cellular mRNAs. Here we describe a sensitized genetic screening system for the identification of novel components and regulators in the antiviral RNAi pathway established in the model plant species Arabidopsis thaliana. Our genetic screen identifies antiviral RNAi (avi)-defective Arabidopsis mutants that develop visible disease symptoms after infection with CMV-∆2b, a Cucumber mosaic virus mutant deficient in the expression of its viral suppressor of RNAi. Loss of RNAi suppression renders CMV-∆2b highly susceptible to antiviral RNAi so that it replicates to high levels and induces disease development only in avi mutants. This chapter provides the methods for the propagation of CMV-∆2b, preparation of the mutant plants for virus inoculation, identification and characterization of avi mutants, and cloning of the genes responsible for the mutant phenotype by either the genetic linkage to T-DNA insertion or a mapping-by-sequencing approach.
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