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

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.

Infection Defects of RNA and DNA Viruses Induced by Antiviral RNA Interference

“…The observed inhibition of CymRSV-Dp19 infection in mutant N. benthamiana plants defective in both RDR1 and RDR6 (114) resembles the incomplete rescue of CMV-D2b and TuMV-AS9-GFP in rdr1 rdr6 plants (104,122), providing evidence for antiviral activity of RDR-independent primary vsiRNAs in a different host species. It will be interesting to determine whether plants defective in the biogenesis of both primary and secondary vsiRNAs can support more efficient systemic infection with p19-deficient tombusviruses, including the BS3Ng isolate of TBSV recently shown to develop systemic infection in A. thaliana (151).…”

Special Issue “Function and Structure of Viral Ribonucleoproteins Complexes”

“…Finally, among plant viruses, the tomato bushy stunt virus (TBSV) is one of the best-studied plant viruses since its host range covers a wide spectrum of plants. Ritzenthaler and collaborators identified and characterized a TBSV isolate that efficiently infects Arabidopsis [14]. This isolate provides a new model for dissecting plant-virus interactions and was shown to replicate in association with clustered peroxisomes.…”