The plant-specific transcription factors CBP60g and SARD1 are targeted by a Verticillium secretory protein VdSCP41 to modulate immunity
- Institute of Microbiology, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
- National Institute of Biological Sciences, China
Figures
Figure 1 with 1 supplement
VdSCP41 contributes to V. dahliae virulence in host plants.
(A) Schematic description of the generation of the VdΔscp41 mutant. (B) Southern blot analysis of the VdSCP41 gene deletion in the VdΔscp41 mutant. Genomic DNA samples isolated from V592 and VdΔscp41 strains were digested by BamHI and subjected to Southern blot analysis. (C–D) Disease symptoms of upland cotton (C) and Arabidopsis (D) plants infected with the wildtype (V592), VdΔscp41, and VdΔscp41/VdSCP41-GFP strains. (E–F) Disease index analyses of upland cotton (E) and Arabidopsis (F) infected with the indicated strains. The plants were photographed and subjected to disease index analyses 3–4 weeks post inoculation. The disease indexes were evaluated with three replicates generated from 24 plants for each inoculum. Error bars indicate the standard deviation of three biological replicates. Student’s t-test was carried out to determine the significance of difference. *Indicates significant difference at a P-value of < 0.05. The experiments were repeated three times with similar results.
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Figure 1—source data 1
Source data for Figure 1.
- https://doi.org/10.7554/eLife.34902.005
Figure 1—figure supplement 1
Expression of VdSCP41 in V. dahliae is induced by plant roots.
VdSCP41 is induced by Arabidopsis root in V. dahliae. Verticillium dahliae conidia were co-cultured with (V. dahliae-induced) or without (V. dahliae) Arabidopsis roots for 2 days. RNA was extracted for real-time PCR analyses. The experiments were repeated three times with similar results. Error bars indicate standard deviations. ** indicates significant difference at a of < 0.01.
Figure 2 with 2 supplements
VdSCP41 functions to inhibit immunity in plants.
(A) VdSCP41 localizes to the base of the hyphopodia and forms ring signals surrounding the hyphal neck. The Vd∆scp41/VdSCP41-GFP strain and Vd∆scp41/ΔspVdSCP41-GFP was cultured on a cellophane membrane for 5 days to induce the formation of hyphopodia. Localization of VdSCP41-GFP was visualized using a Leica SP8 microscope. (B) Transiently expressed VdSCP41 preferentially localizes to the nucleus of Arabidopsis cells. Arabidopsis protoplasts were transfected with either a VdSCP41-mCherry or a ΔspVdSCP41-mCherry plasmid as indicated. mCherry fluorescence was visualized 16 hr post transfection. DAPI staining of the nucleus was visualized under UV light. (C–D) Expression of ΔspVdSCP41 in Arabidopsis inhibits nlp20Vd2-induced ICS1 (C) and FMO1 (D) expression. Wildtype (WT) and transgenic plants expressing ΔspVdSCP41 or ΔspVdSCP41-nls were treated with H2O or nlp20Vd2 as indicated. RNA was extracted for real-time PCR analyses. The experiments were repeated three times with similar results. Error bars indicate standard deviations. Student’s t-test was carried out to determine the significance of difference. * Indicates significant difference at a P-value of < 0.05, whereas ** indicates significant difference at a P-value of < 0.01.
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Figure 2—source data 1
Source data for Figure 2.
- https://doi.org/10.7554/eLife.34902.009
Figure 2—figure supplement 1
VdSCP41 delivered by V. dahliae translocates into plant cells and inhibits immunity.
(A) VdSCP41 contains a predicted signal peptide (SP) and a predicted nuclear localization signal (NLS) sequence. ASP predicted by SignalP is shown in red. A predicted NLS is shown in blue. (B) VdSCP41-GFP translocates into the nucleus of onion epidermal cells. Conidia of the V592-GFP, VdSCP41-GFP or VdSCP41-nls-GFP strain were inoculated onto onion epidermal cells. GFP and DAPI staining of nucleus (UV) fluorescence was visualized 3 days post inoculation. The experiments were repeated three times with similar results. (C) Expression of mcherry-tagged VdSCP41 and ΔspVdSCP41 in Arabidopsis protoplasts. Arabidopsis protoplasts were transfected with either VdSCP41-mCherry or ΔspVdSCP41-mCherry plasmid as indicated. Total protein was extracted for anti-mcherry immunoblot. (D) Transiently expressed VdSCP41 localizes to the nucleus of Nicotiana benthamiana (N. b.) cells. N. b. leaf cells were Agro-infiltrated with Agrobacterium strains carrying either VdSCP41-mCherry or ΔspVdSCP41-mCherry. mCherry fluorescence was visualized 48 hr post infiltration. (E) Expression of ΔspVdSCP41 in Arabidopsis inhibits flg22-induced gene expression. Wildtype (WT) and transgenic plants expressing VdSCP41 were treated with H2Oor flg22. RNA was extracted for real-time PCR analyses. The experiments were repeated three times with similar results. Error bars indicate standard deviations. Student’s t-test was carried out to determine the significance of difference between WT and transgenic plants in the same treatment. * Indicates significant difference at a P-value of < 0.05, whereas ** indicates significant difference at a P-value of < 0.01.
Figure 2—figure supplement 2
VdSCP41 inhibits pathogen-induced SA accumulation and gene expression.
(A) ΔspVdSCP41 transgenic lines accumulated less free SA than did WT in response to Pst hrcC-. The plants were treated with or without Pst hrcC– and subjected to free SA measurement. (B–C) The VdΔscp41 mutant has greater expression of ICS1 and FMO1 than the V592 WT strain. Arabidopsis plants were inoculated without (mock) or with V592, or with the VdΔscp41 mutant. RNA was extracted for real-time PCR analyses of ICS1 (B) and FMO1 (C). Different letters indicate significant difference at a P-value of < 0.05. The experiments were repeated twice with similar results.
Figure 3 with 3 supplements
VdSCP41 interacts with CBP60g in plants.
(A) VdSCP41C interacts with CBP60g. Arabidopsis protoplasts were transfected with the indicated constructs. Protein was extracted 16 hr post transfection and immunoprecipitated with anti-FLAG. The presence of VdSCP41-FLAG or CBP60g-HA in the purified complex was detected by anti-FLAG or anti-HA immunoblot as indicated. (B) VdSCP41 interacts with CBP60g in N. b.. Luciferase imaging of VdSCP41 and CBP60g interaction in N. b. leaves. N. b. leaves infiltrated with an Agrobacterium strain carrying constructs as indicated were subjected to luciferase complementation imaging assay. (C) Quantitative luminescence of VdSCP41 and CBP60g interaction. N. b. leaves infiltrated with the indicated constructs were sliced into strips, and their relative luminescence was determined using a microplate luminometer. Error bars indicate standard deviations of three technical repeats. ** Indicates significant difference at P-value < 0.01. The experiments were repeated three times with similar results. (D) VdSCP41 co-localizes with CBP60g and increases its nuclear accumulation. Representative confocal images of CBP60g-GFP subcellular accumulation were visualized using a spin-disk microscope. An Agrobacterium strain carrying CBP60g-GFP was Agro-infiltrated into N. b. leaves alone, or together with an Agrobacterium strain carrying ΔspVdSCP41-mCherry, ΔspVdSCP41-nls-mCherry. An overlay of GFP and mCherry fluorescence imaging was visualized 48 hr post Agro-infiltration in N. b. leaves. The experiments were repeated three times with similar results.
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Figure 3—source data 1
Source data for Figure 3.
- https://doi.org/10.7554/eLife.34902.014
Figure 3—figure supplement 1
VdSCP41 interacts with CBP60g.
(A) VdSCP41 is co-purified with CBP60g. Arabidopsis protoplasts were transfected with the indicated constructs. Protein was extracted 16 hr post transfection and immunoprecipitated with anti-HA or anti-FLAG. (B) Expression levels of NLuc- and CLuc-fusion proteins in N. b.. All NLuc proteins were further fused with a 3 × HA tag in the NLuc-vector. Anti-HA and anti-CLuc immuneblot were used to detect the expression levels of NLuc- and CLuc-fusion proteins respectively. (C) CBP60g-GFP localizes in the nucleus of N. b. cells. N. b. leaves were infiltrated with an Agrobacterium strain carrying CBP60g-GFP, FLS2-GFP or GFP alone as indicated. GFP fluorescence was visualized 48 hr post infiltration. (D) Expression of CBP60g-GFP, ΔspVdSCP41-mCherry or ΔspVdSCP41-nls-mCherry in N. b. cells. N. b. leaves were infiltrated with Agrobacterium strain as indicated. Total protein was extracted for anti-GFP and anti-mCherry immunoblot.
Figure 3—figure supplement 2
VdSCP41 interacts and co-localizes with SARD1.
(A) VdSCP41 is co-purified with SARD1. Arabidopsis protoplasts were transfected with VdSCP41-FLAG alone or together with SARD1-HA. Protein was extracted 16 hr post transfection and immunoprecipitated with anti-HA antibody. The presence of VdSCP41-FLAG in the purified complex was detected by anti-FLAG immunoblot. The experiments were repeated three times with similar results. (B) VdSCP41 co-localizes with SARD1. Representative confocal images of SARD1-GFP subcellular accumulation were visualized using a spin-disk microscope. An Agrobacterium strain carrying SARD1-GFP was Agro-infiltrated into N. b. leaves alone, or together with an Agrobacterium strain carrying VdSCP41-mCherry or VdSCP41-nls-mCherry. An overlay of GFP and mCherry fluorescence imaging was visualized 48 hr post Agro-infiltration in N. b. leaves.
Figure 3—figure supplement 3
Luciferase complementation assay did not reveal an interaction between CBP60g and SARD1.
(A) Luciferase imaging of VdSCP41 and CBP60g interaction in N. b. leaves. (B) Quantitative luminescence of VdSCP41 and CBP60g interaction. (C) Expression levels of NLuc- and CLuc-fusion proteins as indicated in N. b.. All NLuc proteins were further fused with a 3 × HA tag in the NLuc-vector. Anti-HA and anti-CLuc immuneblots were used to detect the expression levels of NLuc- and CLuc-fusion proteins, respectively.
Figure 4 with 1 supplement
VdSCP41 binds the C-terminal portion of CBP60g to interfere with its activity.
(A–B) Expression of VdSCP41 inhibits the CBP60g-activated ICS1::LUC (A) and FMO1::LUC (B) reporter. ICS1::LUC or FMO1::LUC were transfected alone, or together with CBP60g and VdSCP41 or its variants, as indicated. 35S::RLUC was co-transfected as internal control. Error bars indicate standard deviations of three technical repeats. ** Indicates significant difference between VdSCP41 and EV (empty vector) at a P-value of < 0.01. (C) CBP60gC interacts with VdSCP41. Arabidopsis protoplasts were transfected with VdSCP41-FLAG alone or together with CBP60gN-HA, CBP60gC-HA, or CBP60g-HA. Protein was extracted 16 hr post transfection and immunoprecipitated with anti-HA antibody. The presence of VdSCP41-FLAG in the purified complex was detected by anti-FLAG immunoblot. The experiments were repeated three times with similar results. (D) VdSCP41 inhibits the DNA-binding activity of CBP60g. GST-CBP60g was incubated with [γ−32P]ATP-labeled 60-bp double-stranded DNA probe within the ICS1 promoter, and subjected to electrophoretic mobility shift assay (EMSA). Unlabeled probe was used as competitors (150×) for binding. His-tagged VdSCP41C, VdSCP41100-163 or VDAG_01962 was preincubated with GST-CBP60g for 30 min at room temperature where needed (as indicated) before EMSA. The experiments were repeated three times with similar results.
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Figure 4—source data 1
Source data for Figure 4.
- https://doi.org/10.7554/eLife.34902.017
Figure 4—figure supplement 1
VdSCP41 does not cleave CBP60g.
(A) Co-expression of VdSCP41 in Arabidopsis protoplasts does not cleave CBP60g. CBP60g-HA was transfected alone or together with VdSCP41-FLAG. CBP60g-HA and VdSCP41-FLAG were detected by anti-HA or anti-FLAG immunoblot. (B) Recombinant His-tagged VdSCP41100-end does not cleave GST-CBP60g. Purified GST-CBP60g was incubated with or without His-tagged VdSCP41100-end for 30 min at room temperature and subjected to anti-GST or anti-His immunoblot.
Figure 5
CBP60gC harbors a transcription activation domain that is required for interaction with VdSCP41.
(A) CBP60gC is required for CBP60g-mediated ICS1 and FMO1 activation. ICS1::LUC or FMO1::LUC was transfected alone, or together with CBP60g or its variants, as indicated. ** Indicates significant difference between ΔC-CBP60g or ΔC-CBP60g/CBP60g and CBP60g at a P-value of < 0.01. (B) CBP60g211-440 harbors transcription activator activity. TPS10::LUC was transfected alone, or together with bHLHMYC2 or bHLH-CBP60g211-440, as indicated. 35S::RLUC was co-transfected as internal control. ** Indicates significant difference between bHLH-CBP60g211-440 and bHLHMYC2 at a P-value of < 0.01. LUC reporter activity was determined 16 hr post transfection. The experiments were repeated three times with similar results. (C) CBP60g211-440 is required for interacting with VdSCP41. Arabidopsis protoplasts were transfected with the indicated constructs. Protein was extracted 16 hr post transfection and immunoprecipitated with anti-HA. The presence of VdSCP41-FLAG or CBP60g variants in the purified complex was detected by anti-FLAG or anti-HA immunoblot as indicated.
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Figure 5—source data 1
Source data for Figure 5.
- https://doi.org/10.7554/eLife.34902.019
Figure 6
CBP60g and SARD1 are required for VdSCP41-mediated virulence.
Wildtype (WT) and cbp60g-1/sard1-1 double mutant plants were inoculated with V592 and VdΔscp41 mutant strains. The plants were subjected to disease index analyses 3–4 weeks post inoculation. The disease indexes were evaluated with three replicates generated from 24 plants for each inoculum. Error bars indicate standard deviations of three biological replicates. *Indicates significant difference of V592 virulence at a P-value of < 0.05 between WT and cbp60g-1/sard1-1 double mutant plants. Student’s t-test was carried out to determine the significance of difference between indexes collected from three biological replicates. Lower case letters indicate a significant difference at a P-value of < 0.05. The experiments were repeated three times with similar results.
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Figure 6—source data 1
Source data for Figure 6.
- https://doi.org/10.7554/eLife.34902.021
Figure 7
GhCBP60b is targeted by VdSCP41 and required for cotton resistance against V. dahliae.
(A) VdSCP41 is co-purified with GhCBP60b in Arabidopsis protoplasts. Arabidopsis protoplasts were transfected with VdSCP41-FLAG alone or together with GhCBP60b-HA. Protein was extracted 16 hr post transfection and immunoprecipitated with anti-HA. The presence of VdSCP41-FLAG in the purified complex was detected by anti-FLAG immunoblot. (B) VdSCP41 co-localizes with GhCBP60b. GFP-tagged GhCBP60b and mCherry-tagged VdSCP41 were co-expressed in N. b. leaves. An overlay of GFP and mCherry fluorescence imaging was visualized 48 hr post Agro-infiltration. The experiments were repeated three times with similar results. (C) Cotton VIGS-GhCBP60b plants develop symptoms more rapidly than VIGS-vector plants. The percentage of plants showing the Verticillium wilt phenotype at the indicated time after infection is shown. The disease ratio was scored using 15 plants per treatment and the assays were repeated three times with similar results. (D–E) GhCBP60b is required for full resistance against V. dahliae. Cotton seedlings were infiltrated with Agrobacterium carrying pTRV1 together with pTRV2 or pTRV2-GhCBP60b as indicated. Verticillium dahliae strain V592 was inoculated 10 days post Agrobacterium infiltration. Plants showing disease symptoms were photographed 30 days post V. dahliae inoculation (D). Disease index analyses of VIGS-vector or VIGS-GhCBP60b plants infected with V592 (E). The plants were subjected to disease index analyses 3–4 weeks post inoculation. The disease indexes were evaluated in three replicates generated from 24 cotton plants for each inoculum. The experiments were repeated three times with similar results. Error bars indicate standard deviations of three technical repeats within one biological experiment. The Student’s t-test was carried out to determine the significance of difference. *Indicates significant difference at a P-value of < 0.05. (F) Expression of GhCBP60b is reduced in plants that are infiltrated with Agrobacterium carrying pTRV1 together with pTRV2-GhCBP60b (VIGS-GhCBP60b). Total RNA from infiltrated plants was extracted for RT-PCR analyses of GhCBP60b expression 20 days post V. dahliae inoculation. Error bars indicate standard deviations. *Indicates significant difference at a P-value of < 0.05.
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Figure 7—source data 1
Source data for Figure 7.
- https://doi.org/10.7554/eLife.34902.023
Figure 8
Model for VdSCP41–mediated suppression of defense in Arabidopsis during V. dahliae infection.
(A) In the absence of VdSCP41, PAMPs derived from V. dahliae, such as NLPs and chitins, induce the expression of CBP60g, which in turn upregulates the expression of a number of immune regulators to activate defense. (B) In the presence of VdSCP41, VdSCP41 secreted from V. dahliae translocates into the nucleus of plant cells. VdSCP41 targets the transcription activation domain (TAD) of CBP60g, interrupting either the activity of this domain or the recruitment of associated co-activators via this domain, to interfere with their activity and with plant immunity against V. dahliae. VdSCP41-mediated over-accumulation could provide an additional strategy to further interfere with the transcription factor activity of CBP60g.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Verticillium dahliae) | VdSCP41 | PMID:21829347 | NCBI Gene ID:20709665 | |
| Gene (Gossypium hirsutum) | GhCBP60b | NCBI Gene ID:107899061 | ||
| Strain, strain background (Verticillium dahliae) | V592 | PMID:21151869 | ||
| Other (cotton plant) | Xinluzao No. 16 | PMID:28282450 | ||
| Antibody | Anti-FALG (mouse monoclonal) | Sigma | RRID:AB_259529 | 1:10000 dilution |
| Antibody | Anti-HA (mouse monoclonal) | Roche | RRID:AB_514506 | 1:5000 dilution |
| Antibody | Anti-GFP (mouse monoclonal) | Roche | RRID:AB_390913 | 1:3000 dilution |
| Antibody | Anti-CLuc (mouse monoclonal) | Sigma | RRID:AB_439707 | 1:3000 dilution |
| Antibody | Anti-mCherry | Easybio | 1:3000 dilution | |
| Peptide | nlp20Vd2 | PMID:28755291 | ||
| Recombinant DNA reagent | pTRV1 (VIGS vector) | PMID:12220268 | ||
| Recombinant DNA reagent | pTRV2 (VIGS vector) | PMID:12220268 |
Additional files
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Supplementary file 1
Table S1: data generated from mass spectrum experiments.
Data generated from mass spectrum experiment 1 (sheet 1) and mass spectrum experiment 2 (sheet 2). Information of protein IDs, scores, number of peptides for the identified proteins are listed. Matched peptides within CBP60g are shown in red.
- https://doi.org/10.7554/eLife.34902.025
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Supplementary file 2
Primers used in this study.
Sequences of primers used in this study.
- https://doi.org/10.7554/eLife.34902.026
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Transparent reporting form
- https://doi.org/10.7554/eLife.34902.027
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The plant-specific transcription factors CBP60g and SARD1 are targeted by a Verticillium secretory protein VdSCP41 to modulate immunity
eLife 7:e34902.
https://doi.org/10.7554/eLife.34902
