RESUMO
Cells have developed mechanisms for cytoplasmic RNA transport and localization that participate in the regulation and subcellular localization of protein synthesis. In addition, plants can exchange RNA molecules between cells through plasmodesmata and to distant tissues in the phloem. These mechanisms are hijacked by RNA viruses to establish their replication complexes and to disseminate their genomes throughout the plant organism with the help of virus-encoded movement proteins (MP). Live imaging of RNA molecules is a fundamental approach to understand the regulation and molecular basis of these processes. The most widely used experimental systems for the in vivo visualization of genetically encoded RNA molecules are based on fluorescently tagged RNA binding proteins that bind to specific motifs inserted into the RNA, thus allowing the tracking of the specific RNA molecule by fluorescent microscopy. Recently, we developed the use of the E. coli RNA binding protein BglG for the imaging of RNAs tagged with BglG-binding sites in planta. We describe here the detailed method by which we use this in vivo RNA tagging system for the real-time imaging of Tobacco mosaic virus (TMV) MP mRNA.
Assuntos
Escherichia coli , Proteínas do Movimento Viral em Plantas , Escherichia coli/genética , Proteínas do Movimento Viral em Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Nicotiana/metabolismoRESUMO
Citrus psorosis virus and Mirafiori lettuce big-vein virus are two members of the genus Ophiovirus, family Ophioviridae. So far, how these viruses can interfere in the antiviral RNA silencing pathway is not known. In this study, using a local GFP silencing assay on Nicotiana benthamiana, the 24K-25K and the movement protein (MP) of both viruses were identified as RNA silencing suppressor proteins. Upon their co-expression with GFP in N. benthamiana 16c plants, the proteins also showed to suppress systemic RNA (GFP) silencing. The MPCPsV and 24KCPsV proteins bind long (114 nucleotides) but not short-interfering (21 nt) dsRNA, and upon transgenic expression, plants showed developmental abnormalities that coincided with an altered miRNA accumulation pattern. Furthermore, both proteins were able to suppress miRNA-induced silencing of a GFP-sensor construct and the co-expression of MPCPsV and 24KCPsV exhibited a stronger effect, suggesting they act at different stages of the RNAi pathway.