The Potyvirus Silencing Suppressor Protein VPg Mediates Degradation of SGS3 via Ubiquitination and Autophagy Pathways

Abstract

RNA silencing is an innate antiviral immunity response of plants and animals. To counteract this host immune response, viruses have evolved an effective strategy to protect themselves by the expression of viral suppressors of RNA silencing (VSRs). Most potyviruses encode two VSRs, helper component-proteinase (HC-Pro) and viral genome-linked protein (VPg). The molecular biology of the former has been well characterized, whereas how VPg exerts its function in the suppression of RNA silencing is yet to be understood. In this study, we show that infection by Turnip mosaic virus (TuMV) causes reduced levels of suppressor of gene silencing 3 (SGS3), a key component of the RNA silencing pathway that functions in double-stranded RNA synthesis for virus-derived small interfering RNA (vsiRNA) production. We also demonstrate that among 11 TuMV-encoded viral proteins, VPg is the only one that interacts with SGS3. We furthermore present evidence that the expression of VPg alone, independent of viral infection, is sufficient to induce the degradation of SGS3 and its intimate partner RNA-dependent RNA polymerase 6 (RDR6). Moreover, we discover that the VPg-mediated degradation of SGS3 occurs via both the 20S ubiquitin-proteasome and autophagy pathways. Taken together, our data suggest a role for VPg-mediated degradation of SGS3 in suppression of silencing by VPg.

Importance: Potyviruses represent the largest group of known plant viruses and cause significant losses of many agriculturally important crops in the world. In order to establish infection, potyviruses must overcome the host antiviral silencing response. A viral protein called VPg has been shown to play a role in this process, but how it works is unclear. In this paper, we found that the VPg protein of Turnip mosaic virus (TuMV), which is a potyvirus, interacts with a host protein named SGS3, a key protein in the RNA silencing pathway. Moreover, this interaction leads to the degradation of SGS3 and its interacting and functional partner RDR6, which is another essential component of the RNA silencing pathway. We also identified the cellular pathways that are recruited for the VPg-mediated degradation of SGS3. Therefore, this work reveals a possible mechanism by which VPg sabotages host antiviral RNA silencing to promote virus infection.

Keywords: RDR6; RNA silencing; SGS3; VPg; VSR; autophagy; potyvirus; ubiquitination.

FIG 1

TuMV infection induces SGS3 degradation in N. benthamiana and Arabidopsis. (A) TuMV-GFP and TuMV-GFP^ΔGDD were transiently coexpressed with FLAG-4×Myc-SGS3 in N. benthamiana leaves by Agrobacterium infiltration. The FLAG-4×Myc-SGS3 protein and GFP were detected at 60 hpi. (Top) Western blotting of total protein extracts with Myc antibodies; (middle) Western blotting of total protein extracts with GFP antibodies; (bottom) Coomassie bright blue (CBB) staining showing equal loading of total protein extracts. (B) Photograph of 3-week-old wild-type (Col-0) and SGS3-overexpressing transgenic (SGS3oe-1 and SGS3oe-3) plants. (C) Western blot detection of FLAG-4×Myc-SGS3 in eight independent transgenic Arabidopsis lines expressing FLAG-4×Myc-SGS3 with Myc antibodies. FLAG-4×Myc-SGS3 was strongly detected in lines SGS3oe-1, -3, -5, and -8. Weak expression was evident in lines SGS3oe-2 and -7. No FLAG-4×Myc-SGS3 was detectable in SGS3oe-4 and -6. (D) Phenotype of TuMV-infected transgenic Arabidopsis plants overexpressing SGS3 (SGS3oe-1 and SGS3oe-3) at 20 dpi. (E) The relative TuMV genomic RNA levels shown in panel D were determined by qRT-PCR. Mean genomic RNA levels of TuMV on Col-0 plants were normalized to 1.0. Bars represent means ± standard deviations of data from five biological replicates. (F) Western blot detection of SGS3 and GFP in SGS3oe-1 and SGS3oe-3 with Myc (top) and GFP (middle) antibodies. The bottom panel shows equal loading of total proteins by CBB staining. (G) The expression levels of SGS3 shown in panel D were determined by RT-PCR. The expression levels of SGS3 in mock-infected plants were normalized to 1.0. Bars represent means ± standard deviations of data from five biological replicates. (H) Phenotype of 3-week-old Arabidopsis sgs3-14 plants transformed with na::SGS3-FLAG. (I) Western blot detection of SGS3 and GFP in na::SGS3-FLAG plants with SGS3 (top) and GFP (middle) antibodies. The position of FLAG-SGS3 is indicated by an arrow. Stars indicate the cross-reacting bands of SGS3 antibodies. CBB staining shows equal loading of the samples (bottom).

FIG 2

VPg is the only viral protein that interacts with SGS3. (A) Yeast two-hybrid assay for protein-protein interactions between SGS3 and TuMV-encoded proteins on selective media supplied with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). The positive and negative controls are yeast cotransformants with pGAD-VPg plus pGBK-eIF4E and pGAD-T plus pGBK-Lam, respectively. AD, GAL4 activation domain; BD, GAL4 DNA binding domain. SD, synthetic defined (SD) yeast minimal medium; SD-LW, SD medium lacking Leu and Trp; SD-LWHA, SD medium lacking Leu, Trp, His, and adenine hemisulfate. (B) BiFC assay for protein-protein interactions between SGS3 and VPg encoded by TuMV, SMV, and TEV. SGS3, TuMV VPg, SMV VPg, and TEV VPg were fused to either the C-terminal or N-terminal half of YFP and transiently expressed in N. benthamiana leaves. Fluorescence was monitored by confocal microscopy at 48 hpi. Bar, 50 μm. (C) Confocal micrographs of N. benthamiana coexpressing SGS3-CFP and VPg-mRFP at 48 hpi. Bar, 50 μm. (Bottom) Closeup images showing SGS3 bodies at the perinuclear area. White arrows, nuclear localized VPg; white arrowhead, cytoplasmic SGS3-VPg interaction body. Bar, 10 μm. (D) Yeast cells cotransformed with AD-SGS3 and BD, or VPg protein encoded by TuMV, TEV (VPg^TEV), or SMV (VPg^TEV) were tested as described above for panel A. The positive control is the yeast cotransformant with pGAD-VPg and pGBK-eIF4E.

FIG 3

Subcellular localization of VPg, SGS3, and their domains in N. benthamiana determined by confocal microscopy. (A) Confocal micrographs of N. benthamiana leaf cells expressing N- or C-terminally YFP-fused VPg or SGS3 in N. benthamiana leaves at 48 hpi. Insets show the nuclear localization of VPg. Bars, 50 μm. (B) Confocal micrographs of N. benthamiana leaf cells expressing N- or C-terminally YFP-fused VPg-NTP or VPg-CTD at 48 hpi. Bars, 50 μm. (C) Confocal micrographs of N. benthamiana leaf cells expressing N- or C-terminally YFP-fused SGS3-NTD, SGS3-ZF, SGS3-XS, and SGS3-CC at 48 hpi. Bars, 50 μm.

FIG 4

Mapping of the interaction domains in VPg and SGS3. (A) Schematic representations of SGS3 and VPg proteins. NTP, VPg N-terminal NTP-binding domain; CTD, VPg C-terminal domain; NTD, SGS3 N-terminal domain; ZF, putative SGS3 zinc finger domain; XS, SGS3 XS domain; CC, SGS3 coiled-coil domain. The numbers represent amino acid positions of domain boundaries. (B) Protein-protein interaction assays of BD-SGS3 and AD-VPg-NTP or AD-VPg-CTD and BD-VPg and AD-SGS3, AD-eIF4e, and AD-SGS3-NTD with AD-SGS3-ZF, AD-SGS3-XS, or AD-SGS3-CC in yeast cells. (C) BiFC assay of possible interactions between SGS3 and each of the two VPg domains in N. benthamiana leaf cells at 48 hpi. Bars, 50 μm. (D) BiFC assay of possible interactions between VPg and each of the four SGS3 domains in N. benthamiana leaf cells at 48 hpi. Bars, 50 μm.

FIG 5

VPg-mediated degradation of SGS3. (A) FLAG-4×Myc-SGS3 was transiently coexpressed with VPg-3×HA or GFP-3×HA in N. benthamiana leaf cells. (Top and middle) Total protein was extracted at 48 hpi and detected with Myc antibodies (top) or HA antibodies (middle). The relative amount of the SGS3 protein is indicated. (Bottom) Equal protein loading by CBB staining. (B) FLAG-4×Myc-RDR6 was transiently coexpressed with VPg-3×HA and SGS3-CFP in N. benthamiana leaves. Infiltrated leaf areas were harvested for protein extraction at 60 hpi. (Top) RDR6 was purified with anti-FLAG M2 affinity agarose and detected with Myc antibodies. Numbers indicate the relative amounts of the RDR6 protein. VPg and GFP were directly detected with HA antibodies. (Bottom) Equal protein loading as determined by CBB staining. (C and D) FLAG-4×Myc-SGS3 (C) or FLAG-4×Myc-VPg (D) was transiently expressed in N. benthamiana leaves by Agrobacterium infiltration. At 48 hpi, the infiltrated leaf areas were treated with MG132, 3-MA, or MG132 and 3-MA for an additional 12 h. Total protein was extracted, separated by SDS-PAGE, and detected with Myc antibodies. The relative amounts of SGS3 or VPg are indicated. The bottom panel shows equal protein loading by CBB staining. (E) FLAG-4×Myc-VPg was transiently coexpressed with HA-Ub in N. benthamiana leaves by Agrobacterium infiltration. VPg was purified at 60 hpi with anti-FLAG M2 affinity agarose, separated by SDS-PAGE, and then detected with anti-Myc antibodies or anti-HA antibodies. (F) FLAG-4×Myc-VPg was transiently coexpressed with FLAG-4×Myc-SGS3 in N. benthamiana leaves. At 48 hpi, the infiltrated leaf areas were treated with MG132, 3-MA, or MG132 and 3-MA for an additional 12 h. Total protein was extracted, separated by SDS-PAGE, and detected with Myc antibodies. The relative amount of SGS3 or VPg is indicated. The bottom panel shows equal protein loading as determined by CBB staining. Each experiment was repeated at least three times.

FIG 6

Knockout of SGS3 promotes TuMV replication. (A) Phenotype of 3-week-old VPg transgenic Arabidopsis plants overexpressing VPg. (B) Detection of VPg transcripts in transgenic plants by RT-PCR. The full coding sequence of VPg was amplified by RT-PCR. A fragment of the Arabidopsis Actin II gene was amplified as an RNA loading control. (C) Western blotting of protein extracts from transgenic plants overexpressing VPg with Myc antibodies. The bottom panel shows equal protein loading as determined by CBB staining. (D, top) Comparison of SGS3 in sgs3-14/na::SGS3-FLAG/XVE::FLAG-VPg plants after treatment with 17-β-estradiol for 16 h with rabbit antibodies against FLAG. (Middle) Expression of VPg monitored by Western blotting with anti-FLAG antibody. (Bottom) Equal protein loading as determined by CBB staining. (E) Phenotypes of TuMV-infected wild-type and SGS3 knockout Arabidopsis plants at 20 dpi. (F) Relative viral genomic RNA levels in TuMV-infected wild-type and SGS3 knockout Arabidopsis plants at 20 dpi. Actin II was used as an internal control. The mean genomic RNA level of TuMV in Col-0 plants was normalized to 1.0. Bars represent means ± standard deviations of data from 10 biological replicates, each containing three technical repeats. ** indicates a P value of <0.01, and * indicates a P value of <0.1 (Student t test).