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, 9 (1), 1268

Beclin1 Restricts RNA Virus Infection in Plants Through Suppression and Degradation of the Viral Polymerase

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Beclin1 Restricts RNA Virus Infection in Plants Through Suppression and Degradation of the Viral Polymerase

Fangfang Li et al. Nat Commun.

Abstract

Autophagy emerges as an essential immunity defense against intracellular pathogens. Here we report that turnip mosaic virus (TuMV) infection activates autophagy in plants and that Beclin1 (ATG6), a core component of autophagy, inhibits virus replication. Beclin1 interacts with NIb, the RNA-dependent RNA polymerase (RdRp) of TuMV, via the highly conserved GDD motif and the interaction complex is targeted for autophagic degradation likely through the adaptor protein ATG8a. Beclin1-mediated NIb degradation is inhibited by autophagy inhibitors. Deficiency of Beclin1 or ATG8a enhances NIb accumulation and promotes viral infection and vice versa. These data suggest that Beclin1 may be a selective autophagy receptor. Overexpression of a Beclin1 truncation mutant that binds to NIb but lacks the ability to mediate NIb degradation also inhibits virus replication. The Beclin1-RdRp interaction further extends to several RNA viruses. Thus Beclin1 restricts viral infection through suppression and also likely autophagic degradation of the viral RdRp.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
TuMV infection activates autophagy. a Confocal micrographs showing N. benthamiana leaf cells co-infiltrated with Agrobacterium harboring a YFP-NbATG8a expression construct and Agrobacterium carrying an empty vector (mock) or a TuMV infectious clone (TuMV) or a TuMV replication-defective mutant (TuMV-ΔGDD) at 60 h post infiltration (hpi). Bars, 50 μm. b The average number of YFP-NbATG8a spots per 10 cells. Infiltration experiments were repeated three times and 60 cells in total were counted for the punctate spots. The average number was calculated using 10 cells as a unit. Values represent the mean spots ±standard deviation (SD) per 10 cells. c, d Effects of viral infection on the expression of autophagy components at 3 and 7 days post infiltration (dpi). N. benthamiana leaves were agroinfiltrated with mock, TuMV, or TuMV-ΔGDD. Total RNAs were extracted from infiltrated zones at 3 dpi (c) or from newly emerged leaves at 7 dpi (d). Values represent the mean relative to the mock-treated plants (n = 3 biological replicates) and were normalized with NbActin as an internal reference. e, f Immunoblotting analysis of total protein isolated from upper non-inoculated leaves of plants agroinfiltrated with mock, TuMV, and TuMV-ΔGDD at 7 dpi with anti-Beclin1 (e) or anti-ATG8 (f) antibody. Coomassie Brilliant Blue R-250 (CBB)-stained Rubisco large subunit serves as a loading control. g Representative TEM images from upper non-inoculated leaves of N. benthamiana plants infected with mock, TuMV, and TuMV-ΔGDD at 7 dpi. Obvious autophagic structures (red arrows) were observed in TuMV-infected samples and the corresponding region in the white box in the left panel is magnified in the right panel. Cp chloroplast, CW cell wall, P particle, S starch, V vacuole. Bars = 1 μm. h The number of typical double-membrane autophagosomes in different samples in g. Experiments were repeated twice and typical autophagic structures were counted in 30 cells in each treatment. Values represent the mean number of autophagosomes ±SD per 10 cells. b, c, d, h Data were analyzed using Student’s t-test and asterisks denote significant differences between mock (or TuMV-ΔGDD) and TuMV-infected leaves (two-sided, *P < 0.05, **P < 0.01)
Fig. 2
Fig. 2
NbBeclin1 interacts with NIb. a Yeast-two hybrid (Y2H) assays for possible interactions between NbBeclin1 and each of the 11 TuMV proteins. NbBeclin1 and 11 viral proteins were fused with a GAL4 activation domain (AD-NbBeclin1) and a GAL4-binding domain (BD-P1, BD-HC-Pro, BD-P3, BD-P3N-PIPO, BD-6K1, BD-CI, BD-6K2, BD-NIa-VPg, BD-NIa-Pro, BD-NIb, BD-CP), respectively. Y2H Gold yeast cells co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Trp, -Leu, -His, -Ade or SD/-Trp, -Leu medium to screen for positive interactions at 3 days after transformation. Yeast co-transformed with AD-T7-T+BD-T7-53 serves as a positive control; yeast cells co-transformed with AD-NbBeclin1 and the empty BD or with the empty AD and BD-NIb are negative controls. b BiFC assays between NbBeclin1 and NIb in the leaves of H2B-RFP transgenic N. benthamiana. Confocal imaging was performed at 48 hpi. NbBeclin1 and NIb were fused to the N (YN) and C-terminal (YC) fragments of yellow fluorescent protein (YFP). The NbBeclin1-NIb interaction led to the reconstituted fluorescence-competent structure and restoration of yellow fluorescence (green). Nuclei of tobacco leaf epidermal cells are indicated by the expression of H2B-RFP transgene (red). Bars, 50 μm. c Co-localization of NIb-YFP with NbBeclin1-CFP in the leaf cells of H2B-RFP transgenic N. benthamiana by confocal microscopy at 48 hpi. Arrow indicates yellow fluorescence, which was produced from the overlapping of NIb-YFP (green) and NbBeclin1-CFP (red). Bars, 50 μm. d Co-immunoprecipitation (Co-IP) analysis of NbBeclin1-CFP and Myc-NIb in vivo. N. benthamiana leaves were co-infiltrated with A. tumefaciens cells harboring expression vectors to express NbBeclin1-CFP and Myc-NIb (Lane 1), NbBeclin1-CFP and Myc-P3N-PIPO (Lane 2), Myc-NIb and GFP (Lane 3), and GFP and Myc-P3N-PIPO (Lane 4). Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody
Fig. 3
Fig. 3
NbBeclin1 or the NbBeclin1–NIb interaction complex co-localizes with the autophagosome marker NbATG8a in N. benthamiana. a Transient co-expression of NbBeclin1-YFP and NbATG8a-CFP. Arrows indicate yellow fluorescence, which resulted from the overlapping of NbBeclin1-YFP (red) and NbATG8a-CFP (green) fluorescence. b Co-localization of YN-NbBeclin1 and YC-NIb with NbATG8a-CFP. c Co-localization of YC-NbBeclin1 and YN-NIb with NbATG8a-CFP. All confocal micrographs in this figure were taken at 48 hpi. The corresponding region in the white box in the panel I of b, c is magnified in panel II. Bars, 25 µm
Fig. 4
Fig. 4
NbBeclin1 or the NbBeclin1–NIb complex co-localizes with the TuMV viral replication complex (VRC) in autophagosomes in N. benthamiana. a NbBeclin1-YFP was transiently expressed in leaf cells infected by TuMV-6K2-mCherry-CFP-NIb. NbBeclin1-YFP co-localized with CFP-NIb and 6K2-mCherry-stained aggregations. b Transient expression of NbBeclin1-CFP and a dsRNA marker (B2-YN+VP35-YC) in N. benthamiana leaves infected by TuMV-6K2-mCherry. NbBeclin1 co-localized with dsRNA in TuMV VRCs. c Co-localization of NbBeclin1 with NbATG8a and the TuMV VRC. d, e Co-localization of the interaction complex of NbBeclin1 and NIb with NbATG8a and the TuMV VRC. All confocal micrographs shown in this figure were taken at 72 hpi. Bars, 25 µm
Fig. 5
Fig. 5
Overexpression of NbBeclin1 promotes autophagy-dependent degradation of NIb and inhibits TuMV replication. a, b Immunoblotting of total protein extracts from the N. benthamiana leaves agroinfiltrated with buffer (−) or the plasmids indicated. The membrane was probed with GFP (@GFP), or Myc antibodies (@Myc). c Immunoblotting analysis of total protein extracts from leaves infiltrated with buffer (−) or TuMV-CFP-NIb together with an empty vector (Vec) or Myc-NbBeclin1 with antibodies against GFP or Myc. d Quantification of TuMV RNA levels by qRT-PCR. RNA was extracted from leaves agroinfiltrated with TuMV-CFP-NIb together with Vec or Myc-NbBeclin1 at 60 hpi. Values represent means ± SD relative to plants infiltrated with TuMV-CFP-NIb and Vec (n = 3 biological replicates). The data were analyzed using Student’s t-test and asterisks denote significant differences between the two treatments (two-sided, **P < 0.01). e Immunoblotting analysis of total protein extracts from leaves co-infiltrated with 6K2-GFP and buffer (−), empty vector (Vec), or Myc-NbBeclin1. Antibodies against GFP or Myc were used as a primary antibody. f, g The effect of the autophagy inhibitor 3-MA on the NbBeclin1-mediated degradation of NIb-YFP or TuMV-CFP-NIb. Total proteins were isolated from plant leaves agroinfiltrated with NIb-YFP alone or with Myc-NbBeclin1 (f) or TuMV-CFP-NIb alone or with Myc-NbBeclin1 (g) followed by DMSO or 3-MA treatment. h, i The effect of silencing of NbATG8a on the NbBeclin1-mediated degradation of NIb-YFP or CFP-NIb. Plants inoculated with TRV-GUS or TRV-NbATG8a at 14 dpi were agroinfiltrated with NIb-YFP alone or with Myc-NbBeclin1 (h) or TuMV-CFP-NIb alone or with Myc-NbBeclin1 (i). Total protein was extracted from infiltrated leaves at 3 dpi. Immunoblotting was performed using GFP or Myc antibodies. All immunoblotting assays in this figure were repeated at least three times, and one representative blot was shown. CBB staining of Rubisco large subunit serves as a loading control
Fig. 6
Fig. 6
The GDD motif of NIb is required for the NbBeclin1–NIb interaction and NbBeclin1, via its AIM, interacts with NbATG8a to facilitate the formation of autophagosomes. a Y2H assays to detect possible interactions between NbBeclin1 truncated proteins (NbBeclin1-N and NbBeclin1-C) and NIb and between NbBeclin1 and NIb truncated proteins (NIb-N, NIb-M, and NIb-C). b Y2H assays to detect possible interactions between NbBeclin1 and NbATG8f or NbATG8a and between NbATG8a and NbBeclin1-N, NbBeclin1-C, or NbBeclin1-N AIM mutant (NbBeclin1- NΔAIM). c BiFC assays in H2B-RFP (red) transgenic N. benthamiana leaves at 48 hpi. Yellow fluorescence (green) was observed as a consequence of the complementation of the YN and YC tagged with NbBeclin1 and NbATG8a or NbBeclin1-N and NbATG8a. Bars, 50 μm. d Representative TEM images from N. benthamiana leaf cells agroinfiltrated with buffer (mock), NbBeclin1, NbBeclin1ΔAIM, NbBeclin1-N, NbBeclin1-NΔAIM, or NbBeclin1-C at 60 hpi. Typical autophagic structures (red arrows) were observed in NbBeclin1- or NbBeclin1-N-expressing leaves in the cytoplasm. Cp chloroplast, CW cell wall, S starch, V vacuole. Bars mean 1 μm or 2 μm as indicated. e The number of typical double-membrane autophagic structures in mock, NbBeclin1-, NbBeclin1ΔAIM-, NbBeclin1-N-, NbBeclin1-NΔAIM-, or NbBeclin1-C-infiltrated leaves. Experiments were repeated three times and typical autophagic structures were counted in 20 cells in each treatment. Values represent the mean number of autophagosomes ±SD per 10 cells. Single asterisk indicates statistically significant difference (P < 0.05) between mock and NbBeclin1-infiltrated leaves, and double asterisks indicate P < 0.01 between mock and NbBeclin1-N (Student’s t-test, two-sided). f Confocal micrographs showing N. benthamiana leaf cells co-infiltrated with Agrobacterium harboring a YFP-NbATG8a expression construct and Agrobacterium carrying NbBeclin1, NbBeclin1ΔAIM, NbBeclin1-N, NbBeclin1-NΔAIM or NbBeclin1-C at 48 hpi. Bars, 25 μm
Fig. 7
Fig. 7
NbBeclin1 via its AIM interacting with NbATG8a mediates NIb degradation to repress viral replication, and one C-terminal fragment of NbBeclin1 also inhibits viral replication by binding to NIb independent of autophagy degradation of NIb. a Co-localization of NbATG8a-CFP with NIb-YFP in the expression of empty vector (+mock), Myc-tagged NbBeclin1 (+NbBeclin1) or Myc-tagged NbBeclin1 AIM mutant (+NbBeclin1ΔAIM) in N. benthamiana leaf cells. Infiltrated leaves were treated with DMSO or concanamycin A (Con A) after 48 hpi and confocal images were taken at 10 h after treatment. Bars, 25 μm. b, c Co-IP analysis of the association of NIb with NbATG8a in the presence of Myc-NbBeclin1 or Myc-NbBeclin1ΔAIM in planta. N. benthamiana leaves were agroinfiltrated with the plasmids indicated. Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP, HA, or Myc antibody. d Total protein extracts from the N. benthamiana leaves expressing the indicated recombinant plasmids were subjected to immunoblotting analysis using GFP (@GFP) or Myc antibody (@Myc). Minus sign (−) means that NIb-YFP or NIb-ΔGDD-YFP was expressed alone. All immunoblotting assays in this figure were repeated at least three times, and one representative blot was shown. CBB staining of Rubisco large subunit serves as a loading control. Yellow asterisks indicate the expected sizes. e Quantification of TuMV RNA levels by qRT-PCR. RNA was extracted from leaves infiltrated with TuMV together with Vec, NbBeclin1, NbBeclin1-N, NbBeclin1-C, NbBeclin1-C1, NbBeclin1-C2, NbBeclin1-C3, NbBeclin1-C4, or NbBeclin1-C5 at 60 hpi. Values represent means ±SD (n = 3 biological replicates) and are presented as arbitrary units relative to Vec. According to Duncan’s multiple range test (P = 0.01), the means do not differ significantly if they are indicated with the same letter. f The C1 domain and C4 domain in the C-terminal region of NbBeclin1 interact with NIb in the Y2H assay. A series of truncated NbBeclin1 proteins from NbBeclin1-C are indicated. g The truncated proteins of NbBeclin1-C fail to degrade NIb. Immunoblotting analysis of the total protein extracts from the N. benthamiana leaves expressing the plasmids indicated. Minus sign (−) means that NIb-YFP was expressed alone. Yellow asterisks indicate the expected sizes
Fig. 8
Fig. 8
Silencing of NbBeclin1 or NbATG8a promotes TuMV infection in N. benthamiana. a GFP fluorescence and viral symptoms in plants pre-inoculated with TRV1 together with TRV2-GUS (control), TRV2-NbBeclin1, or TRV2-NbATG8a for 7 days and then infected by TuMV-GFP. Plants were photographed under UV light at 3 and 6 dpi and under regular light at 6 dpi. b Quantification of TuMV genomic RNA in the above plants. RNA was extracted from TuMV-GFP-inoculated leaves at 3 dpi or systemically infected leaves at 6 dpi and 30 dpi. The values are presented as means of fold change ±SD relative to the control plants (pretreated with TVR1 and TRV2-GUS). Error bars represent SD. Three independent experiments, each consisting of three biological replicates, were carried out. Values from one representative result were used to plot a histogram and were normalized against NbActin transcripts in the same sample. The data were analyzed using Student’s t-test (two-sided, *P < 0.05, **P < 0.01). c Protoplast transfection assay. Protoplasts isolated from N. benthamiana plants pre-inoculated with TRV1 together with TRV2-GUS, TRV2-NbBeclin1, or TRV2-NbATG8a were transfected with TuMV-GFP. RNA was extracted at 16, 24, and 48 hpt and quantified by qRT-PCR to analyze TuMV genomic RNA accumulations. Values are presented as means ± SD, and error bars represent SD (n = 3 biological replicates). The data were analyzed using Student’s t-test and asterisks denote significant differences compared to the TuMV-infected protoplasts from control plants pre-treated with TRV1 and TRV-GUS (two-sided, *P < 0.05, **P < 0.01)
Fig. 9
Fig. 9
NbBeclin1 targets RdRps of plant viruses distinct to potyviruses and inhibits viral infection. a, b Schematic representation of the full-length CGMMV RdRp (a) and PepMV RdRp (b). The positions of the first and last amino acid residues of each conserved domain are indicated. Met methyltransferase domain, Hel helicase domain, RdRp2 RdRp2 domain. c NbBeclin1 interacts with CGMMV (CG) and PepMV (PE) RdRps or their RdRp2 domains but not with their GDD mutants or other domains, i.e., Met and Hel in the Y2H assays. d Co-IP analysis of possible interactions of NbBeclin1 with different domains or the GDD mutant of CGMMV and PepMV RdRps in planta. N. benthamiana leaves were agroinfiltrated with the plasmids indicated. Leaf extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody. Yellow asterisks indicate the expected band sizes. e BiFC assays for the interaction of NbBeclin1 with different domains of CGMMV RdRp or PepMV RdRp in H2B-RFP transgenic N. benthamiana leaves at 48 hpi. Bars = 50 μm. f Quantification of CGMMV or PepMV RNA levels by qRT-PCR. The plants were pre-inoculated with buffer (mock), TRV1+TRV2-GUS, or TRV1+TRV2-NbBeclin1 for 7 days. RNA was extracted from CGMMV or PepMV–inoculated or systemically infected leaves at 3 dpi and 14 dpi, respectively. The values are presented as means of fold change ±SD relative to mock-treated plants. Error bars represent SD. Three independent experiments, each consisting of three biological replicates, were carried out. Values from one representative result were used to plot a histogram and were normalized with NbActin as the internal reference. The data were analyzed using Student’s t-test and double asterisks denote significant differences compared to the CGMMV- or PepMV-infected NbBeclin1-silenced plants from control plants pretreated with mock (two-sided, **P < 0.01). g, h Symptoms of CGMMV (g) or PepMV (h) infected plants at 21 dpi. The plants were pre-inoculated with buffer (mock), TRV-GUS, or TRV-NbBeclin1 for 7 days. Mock inoculated with buffer, CGMMV inoculated with CGMMV, PepMV inoculated with PepMV
Fig. 10
Fig. 10
Proposed model for the possible roles of autophagy in the potyviral infection. Infection by positive-sense RNA viruses activates autophagy in plant cells. Beclin1 (ATG6) binds to the GDD motif of the viral RNA-dependent RNA polymerase (RdRp) to inhibit virus replication, which is independent of the autophagy pathway. Beclin1 may also serve as a cargo receptor to interact with the viral RdRp and target the RdRP-containing virus replication complex via the interaction of Beclin1 with other autophagy proteins (e.g., ATG8a) to autophagosomes for degradation. The mechanic details leading to the autophagy-mediated degradation remain to be fully understood. Simultaneously, HC-Pro, a virulence factor and the major potyviral VSR, is hijacked by host rgs-CaM to induce its degradation by the autophagy pathway. Meanwhile, the second potyviral VSR, VPg, mediates the degradation of RNA silencing components SGS3 and RDR6 via the autophagy pathway to suppress antiviral RNA silencing

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