Guo Z1, Wang XB2, Li WX3, Ding SW4.A Sensitized Genetic Screen to Identify Novel Components and Regulators of the Host Antiviral RNA Interference Pathway[J] Methods in Molecular Biology

  发布时间: 2019-09-02   信息员:   

Abstract

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.

KEYWORDS:

Antiviral RNAi; Causal gene identification; Innate immunity; avi mutant; siRNA

链接:https://www.ncbi.nlm.nih.gov/pubmed/31228117


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