CCR4, a RNA decay factor, is hijacked by a plant cytorhabdovirus phosphoprotein to facilitate virus replication
- State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, China
- College of Plant Protection, China Agricultural University, China
- Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Shandong Province Key Laboratory of Agricultural Microbiology, China
Figures
Figure 1 with 7 supplements
The barley HvCCR4 protein interacts with the BYSMV P protein in vitro and in vivo.
(A) Modular organization of BYSMV P, HvCCR4 and schematic presentation of deletion mutants. The BYSMV P protein consists of three structural domains, including an N terminal domain (NTD), a central …
Figure 1—figure supplement 1
Phylogenetic tree of plant and animal CCR4 orthologues.
Plant CCR4 orthologues are from rice (Os), wheat (Ta), barley (Hv), N. benthamiana (Nb) and Arabidopsis (At). Animal CCR4 orthologues are from human (Hs), fruit fly (Dm), and planthopper (Ls). The …
Figure 1—figure supplement 2
Sequence alignments of the zf-MYND-like, LRR, and EEP domains of various CCR4 protein.
(A) Sequence alignments of the zf-MYND-like domains of rice, wheat, barley, N. benthamiana and Arabidopsis CCR4 proteins (B) Sequence alignments of human, fruit fly, and planthopper CCR4 protein LRR …
Figure 1—figure supplement 3
qRT-PCR analysis of the expression of CCR4 in BYSMV-infected and mock-treated barley at 15 dpi or planthoppers at seven dpi.
The values of CCR4 accumulation in mock-treated barley and planthopper were set to 1. Error bars indicate standard errors of three independent experiments. Letters above the bars indicate …
Figure 1—figure supplement 4
BiFC assays showing negative Rub controls with HvCCR4 and LsCCR4.
Leaves were infiltrated with Agrobacterium strains harboring pairs of YN and YC tagged proteins. Images were taken at three dpi with a confocal scanning laser microscope. Bar = 50 μm.
Figure 1—figure supplement 5
Time-lapse confocal images of YN-P and YC-HvCCR4 in N.
benthamiana leaves. YN-P and YC-HvCCR4 were co-expressed in N. benthamiana leaves following treatment with DMSO or latrunculin B (LatB) at two dpi to disrupt actin assembly. Images were taken 3 hr …
Figure 1—figure supplement 6
CCR4 does not affect self-interaction of BYSMV P in vitro and in vivo.
(A) Competitive GST pull-down in vitro. GST-P and P-his were pulled down by glutathione sepharose beads incubated with increasing concentrations of HvCCR4-His. Input and GST pull down proteins were …
Figure 1—figure supplement 7
BiFC assays of NCMV-P with HvCCR4 or LsCCR4.
N. benthamiana leaves were infiltrated with Agrobacterium strains expressing YC-NCMV-P and YN-HvCCR4, YN-N-HvCCR4, YN-C-HvCCR4 or YN-NCMV-P and YC-LsCCR4. YFP fluorescence was photographed by …
Figure 2 with 2 supplements
BYSMV P sequestration of HvCCR4 from the PBs.
(A–C) Confocal micrographs showing subcellular co-localization of HvCCR4-GFP and DCP1-mCherry (A), GFP-P and HvCCR4-mCherry (B), GFP-P and DCP1-mCherry (C) co-expressed in N. benthamiana epidermal …
Figure 2—figure supplement 1
Co-localization of GFP-P or GFP-P1-207 with DCP1-mCherry.
N. benthamiana leaves were infiltrated with Agrobacterium strains expressing DCP1-mCherry with GFP-P or GFP-P1-207 were photographed by microscope at two dpi. Bar = 50 µm.
Figure 2—figure supplement 2
Quantitative data showing localization of CCR4-GFP and DCP1-mCherry bodies in the absence and presence of P protein.
Figure 3
BYSMV P recruitment of HvCCR4 into viroplasm-like bodies.
(A) Confocal micrographs showing subcellular localization of the YN-P and YC-L BiFC combination and CFP-N together with HvCCR4-mCherry or free mCherry in N. benthamiana leaves. Bar = 20 μm. (B) …
Figure 4
HvCCR4 enhancement of BYSMV minigenome replication.
(A) Schematic diagrams of pBY-agMR, pGD-NLP, pGD-VSRs, pMD-HvCCR4, and derivative plasmids. Mixtures of these plasmids transiently express anti-genomic minireplicon RNA, BYSMV N, P, and L proteins, …
Figure 5 with 4 supplements
HvCAF1 negatively regulates minigenome RNA replication.
(A) Schematic representation of CAF1 linking CCR4 and the Not1 scaffold in the CCR4-NOT complex. (B) RFP foci in N. benthamiana leaves agroinfiltrated with Agrobacterium mixture harboring BYSMV-agMR …
Figure 5—figure supplement 1
Phylogenetic tree (A) and sequence alignments (B) of plant CAF1 orthologues.
Plant CCR4 orthologues from sorghum (Sb), rice (Os), maize (Zm), barley (Hv), N. benthamiana (Nb) and Arabidopsis (At). The phylogenetic tree was constructed by mega 5.0. Accession numbers of genes …
Figure 5—figure supplement 2
BiFC assays examining interactions between the P, HvCCR4 and HvCAF1 proteins.
(A) BiFC analysis of interactions between HvCAF1-YC and HvCCR4N-YN or CCR4C-YN in coinfiltrated N.benthamiana leaves. Bar = 50 μm. (B) BiFC analysis of the interactions between YN-P and HvCCR4-YC, …
Figure 5—figure supplement 3
Western blotting analysis of accumulation of expressed proteins in competitive BiFC assays.
The accumulation of YN-P and YC-HvCCR4 with anti-GFP, HvCAF1-Flag with anti-Flag, CBB represents the quantify of protein.
Figure 5—figure supplement 4
HvCAF1 negatively affect viroplasm-like structure formation.
(A) Co-localization of YN-P and YC-CCR4 with L-mCherry. N. benthamiana leaves were infiltrated with Agrobacterium strains expressing YN-P, YC-CCR4 and L-mCherry with empty vector (EV) or HvCAF1. YFP …
Figure 6 with 1 supplement
BYSMV-mediated HvCCR4 overexpression enhances virus pathogenesis in barley plants.
(A) Schematic diagrams of BYSMV pBYR-GUS and pBYR-HvCCR4 derivatives harboring GUS and HvCCR4 ORFs. (B) RFP foci in N. benthamiana leaves infiltrated with Agrobacterium strains containing pGD-NLP, …
Figure 6—figure supplement 1
HvCCR4mEEP overexpression did not enhances virus pathogenesis in N. benthamiana leaves.
(A) Schematic diagrams of BYSMV pBYR-GUS and pBYR-HvCCR4 derivatives harboring GUS and HvCCR4 ORFs. (B) RFP foci in N. benthamiana leaves infiltrated with Agrobacterium strains containing pGD-NPL, …
Figure 7 with 1 supplement
Requirement of the planthopper LsCCR4 protein for efficient BYSMV infection in SBPHs.
(A) Schematic diagrams of the BYSMV pBY-RFP derivative containing an RFP gene between the N and P genes of the antigenome cDNA. (B) GST pull-down analysis of interactions between BYSMV P and LsCCR4. …
Figure 7—figure supplement 1
BiFC assays examining interactions between BYSMV P and LsCCR4.
(A) Schematic diagrams of LsCCR4 and different domains for BiFC assays. (B) BiFC analysis of the interactions of YN-P with LsCCR4-YC, LsCCR4N-YC, LsCCR4C-YC, or Rub-YC in coinfiltrated N. benthamiana…
Figure 8 with 1 supplement
CCR4 facilitates binding of BYSMV N with viral genome RNAs by triggering N-bound cellular mRNA decay.
(A) Northwestern blotting assays detecting non-specific RNA-binding abilities of HvCCR4, BYSMV N, P protein by digoxigenin-labeled Luciferase mRNA (See Materials and methods for details). (B) BYSMV …
Figure 8—figure supplement 1
HvCCR4 did not exhibit obvious activities involved in RNAi.
(A) Confocal micrographs showing subcellular co-localization of GFP-P and SGS3-RFP. (B) Transgene GFP-induced silencing co-expressed with HvCCR4OE and HvCCR4RNAi, TBSV P19 suppressor, and empty …
Videos
Video 1
Motile cytoplasmic BiFC bodies of YN-P and YC-CCR4 in the epidermal cells of agroinfiltrated N. benthamiana leaves.
Images were taken at three dpi with a Leica laser scanning microscope. Bar = 50 μm.
Video 2
Intracellular movement of YN-P and YC-CCR4 inclusion bodies in epidermal cells of agroinfiltrated N. benthamiana leaves expressing mCherry-HDEL after DMSO treatment.
Scale bar = 2 μm.
Video 3
Intracellular movement of YN-P and YC-CCR4 inclusion bodies in epidermal cells of agroinfiltrated N. benthamiana leaves expressing mCherry-HDEL with LatB (10 μM) treatment.
Scale bar = 2 μm.
Video 4
High mobility of GFP-P and CCR4-mCherry colocalized bodies in epidermal cells of agroinfiltrated N. benthamiana leaves.
Scale bar = 20 μm.
Video 5
High mobility of GFP-P bodies and DCP-mCherry bodies in epidermal cells of agroinfiltrated N. benthamiana leaves.
Scale bar = 2 μm.
Video 6
A representative movie showing CFP-N, L-mCherry, and CCR4-GFP in epidermal cells of agroinfiltrated N. benthamiana leaves expressing BYSMV P.
Scale bar = 20 μm.
Additional files
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Supplementary file 1
List of the BYSMV P protein interacting barley proteins obtained in IP-MS assays.
- https://cdn.elifesciences.org/articles/53753/elife-53753-supp1-v2.docx
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Supplementary file 2
Primers used in this study.
- https://cdn.elifesciences.org/articles/53753/elife-53753-supp2-v2.docx
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Supplementary file 3
Key Resources Table.
- https://cdn.elifesciences.org/articles/53753/elife-53753-supp3-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53753/elife-53753-transrepform-v2.pdf
