Abstract
Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae) is an economically important virus infecting wheat in the Great Plains region of the USA. Previously, we reported that the P1 protein of WSMV acts as a viral suppressor of RNA silencing. In this study, we delineated the minimal region of WSMV P1 and examined its mechanisms in suppression of RNA silencing. We found that the 25 N-terminal amino acids are dispensable, while deletion of a single amino acid at the C-terminal region completely abolished the RNA silencing suppression activity of P1. Electrophoretic mobility shift assays with in vitro expressed P1 revealed that the P1 protein formed complexes with green fluorescent protein-derived 180-nt dsRNA and 21 and 24-nt ds-siRNAs, and WSMV coat protein-specific 600-nt dsRNA. These data suggest that the P1 protein of WSMV binds to dsRNAs in a size- and sequence-independent manner. Additionally, in vitro dicing assay with human Dicer revealed that the P1 protein efficiently protects dsRNAs from processing by Dicer into siRNAs, by forming complexes with dsRNA. Sequence comparison of P1-like proteins from select potyvirid species revealed that WSMV P1 harbors a glycine-tryptophan (GW) motif at the C-terminal region. Disruption of GW motif in WSMV P1 through W303A mutation resulted in loss of silencing suppression function and pathogenicity enhancement, and abolished WSMV viability. These data suggest that the mechanisms of suppression of RNA silencing of P1 proteins of potyvirid species appear to be broadly conserved in the family Potyviridae.
Highlights
RNA silencing or post-transcriptional gene silencing (PTGS) is one of the most conserved eukaryotic regulatory pathways of many biological processes such as development, genome stability, and responses to abiotic and antiviral defenses [1,2,3]
These data suggest that the mechanisms of suppression of RNA silencing of P1 proteins of potyvirid species appear to be broadly conserved in the family Potyviridae
Despite the complexity and diversity of RNA silencing, the basic components are highly conserved among many eukaryotes [7]. These components include RNase III DCL (Dicer-like), which is involved in the dicing of dsRNAs into siRNAs [8], Piwi containing Argonaute (AGO-PIWI), which is the effector protein of siRNA-induced silencing complex that mediates target hydrolysis [9], and RNA-dependent RNA polymerase (RDR), which amplifies RNA silencing into transitivity [10]
Summary
RNA silencing or post-transcriptional gene silencing (PTGS) is one of the most conserved eukaryotic regulatory pathways of many biological processes such as development, genome stability, and responses to abiotic and antiviral defenses [1,2,3]. Despite the complexity and diversity of RNA silencing, the basic components are highly conserved among many eukaryotes [7]. These components include RNase III DCL (Dicer-like), which is involved in the dicing of dsRNAs into siRNAs [8], Piwi containing Argonaute (AGO-PIWI), which is the effector protein of siRNA-induced silencing complex (siRISC) that mediates target hydrolysis [9], and RNA-dependent RNA polymerase (RDR), which amplifies RNA silencing into transitivity [10]. Despite strong and specific RNA silencing employed by hosts, viruses gain access to and establish stable and systemic infection. One of the strategies viruses employ to overcome the host RNA silencing is through encoding viral suppressors of RNA silencing (VSR) [11]
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