Subcellular localization of Citrus psorosis virus-encoded proteins

GABRIEL ROBLES LUNA; EDUARDO PEÑA; MANFRED HEINLEIN; MARÍA LAURA GARCÍA

Aussois

Jornada; 13e Rencontres de Virologie Végétale; 2011

Institut national de la recherche agronomique (INRA)

Ophioviruses belong to the few plant virus groups with a negative-sensed RNA genome. Citrus psorosis virus (CPsV), the type member of the genus, causes serious disease in citrus plants grown in Argentina and Uruguay. The viral genome consists of three negative-sense ssRNAs. RNA 1 codes for 24K, a 24 kDa protein of unknown function, as well as for a protein predicted to be a RNA-dependent RNA polymerase (RdRp)(1). RNA 2 codes for a 54 kDa protein. Preliminary studies indicate that this protein participates in the suppression of post-transcriptional gene silencing (PTGS) and probably also in virus movement. RNA 3 encodes a 48 kDa coat protein (48K, CP)(2).The molecular and cellular aspects of the infection cycle of ophioviruses have thus far not been investigated. In order to study the functions of ophiovirus proteins, we investigate their subcellular distribution. Subcellular fractionation experiments combined with immunoblot assays revealed that the 48K protein of CPsV is predominantly localised in the cytoplasmic soluble fraction of infected Chenopodium quinoa extracts and upon transient expression in Nicotiana benthamiana epidermal cells. The 54K protein was detected in infected C. quinoa extracts confirming its predicted molecular weight and coding assignment from the complementary strand of RNA 2. The protein was found in the soluble, microsomal, nuclear and cell wall-enriched fractions of infected C. quinoa plants and upon transient expression also in N. benthamiana plants. Confocal laser scanning microscopy (CLSM) analyses of transiently expressed fluorescent protein-tagged CP and 54K in N. benthamiana epidermal cells confirmed the cytoplasmic localization of CP. The 54K protein was found in the nucleus, cytoplasm and in a punctuate pattern along small dots at the cell wall, suggesting localization to plasmodesmata. Current research focuses on confirming the subcellular localizations of 54K and of its potential co-localization with CP. Here, we are employing Fluorescent Resonance Energy Transfer (FRET) studies, in order to identify protein-protein interactions in vivo.