title: Lipid flippases promote antiviral silencing and the biogenesis of viral and host siRNAs in Arabidopsis.
abstract: Dicer-mediated processing of virus-specific dsRNA into short interfering RNAs (siRNAs) in plants and animals initiates a specific antiviral defense by RNA interference (RNAi). In this study, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in Arabidopsis thaliana Using whole-genome sequencing and a computational pipeline, we identified aminophospholipid transporting ATPase 2 (ALA2) and the related ALA1 in the type IV subfamily of P-type ATPases as key components of antiviral RNAi. ALA1 and ALA2 are flippases, which are transmembrane lipid transporter proteins that transport phospholipids across cellular membranes. We found that the ala1/ala2 single- and double-mutant plants exhibited enhanced disease susceptibility to cucumber mosaic virus when the virus-encoded function to suppress RNAi was disrupted. Notably, the antiviral activity of both ALA1 and ALA2 was abolished by a single amino acid substitution known to inactivate the flippase activity. Genetic analysis revealed that ALA1 and ALA2 acted to enhance the amplification of the viral siRNAs by RNA-dependent RNA polymerase (RdRP) 1 (RDR1) and RDR6 and of the endogenous virus-activated siRNAs by RDR1. RNA virus replication by plant viral RdRPs occurs inside vesicle-like membrane invaginations induced by the recruitment of the viral RdRP and host factors to subcellular membrane microdomains enriched with specific phospholipids. Our results suggest that the phospholipid transporter activity of ALA1/ALA2 may be necessary for the formation of similar invaginations for the synthesis of dsRNA precursors of highly abundant viral and host siRNAs by the cellular RdRPs.
authors.affiliation: Center for Plant Cell Biology, Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521.
authors.forename: Zhongxin
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Center for Plant Cell Biology, Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521.
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Lu
authors.affiliation: Center for Plant Cell Biology, Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521.
authors.forename: Xianbing
authors.initials: X
authors.lastname: Wang
authors.affiliation: Center for Plant Cell Biology, Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521.
authors.forename: Binhui
authors.initials: B
authors.lastname: Zhan
authors.affiliation: Center for Plant Cell Biology, Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521.
authors.forename: Wanxiang
authors.initials: W
authors.lastname: Li
authors.affiliation: Center for Plant Cell Biology, Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521 shou-wei.ding@ucr.edu.
authors.forename: Shou-Wei
authors.initials: SW
authors.lastname: Ding
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000821:virology; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D017799:Cucumovirus / Q000235:genetics* / Q000502:physiology; D054884:Host-Pathogen Interactions / Q000235:genetics; D009154:Mutation; D049294:Phospholipid Transfer Proteins / Q000235:genetics* / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010935:Plant Diseases / Q000235:genetics / Q000821:virology; D030821:Plants, Genetically Modified; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D034741:RNA, Small Interfering; C000631376:AT1G14790 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase
keywords: RNA interference; cucumber mosaic virus; lipid flippase; mutCAN; viral siRNAs