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A Sensitized Genetic Screen to Identify Novel Components and Regulators of the Host Antiviral RNA Interference Pathway

  • Zhongxin Guo
  • Xian-Bing WangEmail author
  • Wan-Xiang Li
  • Shou-Wei DingEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2028)

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.

Key words

Antiviral RNAi siRNA Innate immunity avi mutant Causal gene identification 

Notes

Acknowledgments

This project was supported by grants from the US Department of Agriculture Research Service (6659-22000-025), the US-Israel Binational Agricultural Research and Development Fund, National Institutes of Health (R01AI52447 and GM94396), by the Agricultural Experimental Station of the University of California, Riverside (to S.-W.D.)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Vector-Borne Virus Research Center, Haixia Institute of Science and Technology, College of Plant ProtectionFujian Agriculture and Forestry UniversityFuzhouPeople’s Republic of China
  2. 2.State Key Laboratory of Agro-Biotechnology, College of Biological SciencesChina Agricultural UniversityBeijingPeople’s Republic of China
  3. 3.Department of Microbiology and Plant Pathology and Center for Plant Cell BiologyUniversity of CaliforniaRiversideUSA

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