Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana

Marco Incarbone,Patrice Dunoyer,Christophe Ritzenthaler,Denise Altenbach,Mathieu Erhardt,Hélene Scheer,Lauriane Kuhn,Jean-Michel Hily

doi:10.3390/v12101121

Abstract

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant–microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant–virus interactions in complement to Saccharomyces cerevisiae.

Highlights

Tomato bushy stunt virus (TBSV) is the type member of the genus Tombusvirus in the familyTombusviridae
The highly an extremely valuable asset in the genetic investigation of host–Tombusvirus interactions, which for ineffective dicing by DCL2 upon DCL4 knock-out suggests that DCL2 could be inactive during TBSV
RT-qPCR quantification of mDCL2 gene expression was performed the Arabidopsis peroxisomes upon suggests that their andwild-type function may follow similar in different genetic backgrounds infection

Summary

Introduction

Tomato bushy stunt virus (TBSV) is the type member of the genus Tombusvirus in the familyTombusviridae. TBSV has a host range limited in nature mostly to dicotyledonous species including agricultural crops, ornamentals and vegetables such as Viruses 2020, 12, 1121; doi:10.3390/v12101121 www.mdpi.com/journal/viruses. Viruses 2020, 12, 1121 tomato, pepper, lettuce, tulip, cherry, apple and grapevine [2]. Despite this wide distribution, TBSV is not considered an economically significant plant pathogen. The experimental TBSV host range covers over 120 plant species in more than 20 different families including N. benthamiana, the widely used model plant in virology, RNA interference (RNAi) and vaccine production [3,4], on which TBSV infection rapidly leads to total necrotic collapse. Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant–microbe interactions [5] is absent from the list of known TBSV host plants [2,6]

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