Browsing by Subject "RNA silencing"

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  • Saha, Shreya; Makinen, Kristiina (2020)
    The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXL phi) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVA(VPgmut)). PVA(VPgmut) RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3'RLUC) from the 3' end of the PVA genome. VPg(mut) could not support either PVA RNA stabilization or enhanced 3'RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPg(mut) increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXL phi motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis.
  • Streng, Janne (Helsingfors universitet, 2013)
    RNA silencing is a sequence specific RNA degradation mechanism which is used by plants to regulate gene expression and to combat virus infections. However, viruses have developed so called silencing suppressors, which can prevent and interfere silencing reaction by many ways. For example, virus proteins can bind to maintaining proteins of the silencing reaction or to molecules which are responsible for signaling of the silencing reaction. This thesis focused on the study of protein-protein-interactions between known silencing suppressors of crini- and potyviruses and four maintaining plant proteins of RNA silencing. Protein-protein-interactions were studied using the yeast two-hybrid system (YTHS) and the bimolecular fluorescence complementation assay (BiFC). The latter method enables visualization of the studied protein interactions in plant cells. Protein expression of the cloned genes in yeast vectors were studied by using western blot. BiFC analysis was focused on protein interactions which were found by YTHS. This study detected three previously unknown protein interactions. Two virus proteins were found for the first time to bind directly to silencing maintaining proteins that are known to be targets of other silencing suppressors. Because the functions of these silencing maintaining proteins are known, it is possible that the three interactions described in this study interfere RNA silencing by impeding the functions of the plant proteins.