Molecular characterization of Tobacco rattle virus proteins involved in pathogenicity

Abstract

Tobacco rattle virus (TRV), which belongs to the genus Tobravirus , possesses a bipartite positive-single-stranded genome (RNA-1 and -2), and is naturally transmitted by the plant ectoparasites trichdorids nematodes. The virus is infecting the cultivated potato (Solanum tuberosum ssp. tuberosum) and causing spraing symptoms in progeny tubers, severely affecting tuber quality. In this study, a reliable and fast resistance test to screen for TRV resistance in three different potato cultivars, Russet Burbank, Bintje and Saturna, by leaf-inoculation with a full-length RNA-1 cDNA clone and RNA-2 cDNA clone expressing the fluorescent marker protein DsRed of TRV isolate PpK20 was developed. Two different classical resistance reactions fitting to Hypersensitive Resistance (HR) and Extreme Resistance (ER) were identified in the potato cultivars analysed. Agrobacteruim-transient expression assay demonstrated that the 29K movement protein (MP) is the elicitor of both resistance respon ses. On the other hand, it was demonstrated for the first time, using an Agrobacterium-mediated transient assay, the silencing suppression activity of the pathogenicity factor TRV 16K cystein-rich protein (CRP) in planta. Functional mapping of 16K regions using pentapeptide insertion scanning mutagenesis (PSM) suggested a strong functional and possibly structural conservation of TRV 16K. Confocal laser scanning microscopy (CLSM) analysis of N. benthamiana epidermal cells transiently expressing DsRed C terminal fusions demonstrated that 16K possesses at least two independent bipartite nuclear localization signals (NLSs) in the C-terminal half of the protein. The full-length 16K, in addition to cytoplasmic localization, was able to traffic into the nucleus and nucleolus, indicating a nuclear role of 16K. In contrast, the N-terminal half was localized mainly in the cytoplasm and excluded from the nucleus, suggesting the presence of a nuclear export or a cytoplasmic retention signal.