1. Plant Biology
Download icon

The transcriptional regulator BZR1 mediates trade-off between plant innate immunity and growth

  1. Rosa Lozano-Durán
  2. Alberto P Macho
  3. Freddy Boutrot
  4. Cécile Segonzac
  5. Imre E Somssich
  6. Cyril Zipfel  Is a corresponding author
  1. The Sainsbury Laboratory, United Kingdom
  2. Max Planck Institute for Plant Breeding Research, Germany
Research Article
Cite this article as: eLife 2013;2:e00983 doi: 10.7554/eLife.00983
7 figures and 7 tables

Figures

Figure 1 with 3 supplements
Activation of BZR1 is sufficient to inhibit the PAMP-triggered ROS burst.

(A) and (B) Flg22-triggered ROS burst after LiCl (A) or bikinin (B) treatment. Leaf discs were pre-treated with a 10 mM LiCl solution for 5 hr or with a 50 μM bikinin solution for 16 hr. (C) Flg22- or chitin-triggered ROS burst in Col-0 and the triple GSK3 mutant plants. (D) Flg22- or chitin-induced ROS burst in Col-0 and BZR1Δ plants. (E) Elf18-triggered ROS burst in bri1-5 and bri1-5/BZR1Δ plants. In all cases, bars represent SE of n = 28 rosette leaf discs. Asterisks indicate a statistically significant difference compared to the corresponding control (mock treatment [A and B], Col-0 [C and D] or bri1-5 [E]), according to a Student’s t-test (p<0.05). Leaf discs of four- to five-week-old Arabidopsis plants were used in these assays. Flg22 and elf18 were used at a concentration of 50 nM; chitin was used at a concentration of 1 mg/ml. Total photon counts were integrated between minutes two and 40 after PAMP treatment. All experiments were repeated at least three times with similar results.

https://doi.org/10.7554/eLife.00983.003
Figure 1—figure supplement 1
The BR-mediated suppression of PTI can be triggered independently of a competition for BAK1.

(A) Co-IP of BAK1 and FLS2 in Col-0, 35S:BRI1-cit, 35S:BRI1sud-cit and 35S:DWF4 seedlings treated with 1 μM flg22 for 10 min. Coimmunoprecipitated proteins were analyzed by using anti-FLS2 or anti-BAK1 antibodies. (B) Chitin-triggered ROS burst in Col-0, 35S:BRI1-cit and 35S:DWF4 plants. Chitin was used at a concentration of 1 mg/ml. Total photon counts were integrated between minutes two and 40 after PAMP treatment. Bars represent SE of n = 28 rosette leaf discs. (C) Root length of seven-day-old Col-0, BAK1p:BAK1-HA (in Col-0 WT background) or bak1-3 seedlings grown on medium supplemented or not with 10 nM BL. Bars represent SE of 12 ≤ n ≤ 17. Asterisks indicate a statistically significant difference between treatments according to a Student's t-test (p<0.05). (D) Flg22-triggered ROS burst in Col-0, BAK1p:BAK1-HA (in Col-0 WT background) or bak1-3 plants. Leaf discs of four- to five-week-old Arabidopsis plants were used in these assays. Flg22 was used at a concentration of 50 nM. Total photon counts were integrated between minutes two and 40 after PAMP treatment. Bars represent SE of n = 28 rosette leaf discs. All experiments were repeated at least twice with similar results.

https://doi.org/10.7554/eLife.00983.004
Figure 1—figure supplement 2
PAMP-triggered MAPK activation is not impaired upon activation of BR signaling.

(A) MAPK activation in Col-0 seedlings upon treatment with 1 μM flg22 (F) and/or epiBL (B) for 10 min (with or without a 90-min or 5-hr BL pre-treatment). (B) Quantification of total MAPK activation in the experiment shown in (A), measured as pixel intensity using ImageJ. Results are the average of two independent blots, corresponding to two independent biological replicated. (C) MAPK activation in Col-0 and Triple GSK3 mutant seedlings upon treatment with 1 μM flg22. (D) Quantification of total MAPK activation in the experiment shown in (C), measured as pixel intensity using ImageJ. Results are the average of two independent blots, corresponding to two independent biological replicated. Proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) rabbit monoclonal antibodies. Bars represent SD.

https://doi.org/10.7554/eLife.00983.005
Figure 1—figure supplement 3
Activation of BZR1, but not BES1, is sufficient to inhibit the PAMP-triggered ROS burst.

(A) Flg22- or chitin-triggered ROS burst in BZR1S173A plants. (B) Flg22-triggered ROS burst in BES1S171A plants. (C) Flg22-triggered ROS burst in mock- or bikinin-treated Col-0 or bri1-5 plants. Leaf discs were pre-treated with a 50 μM bikinin solution for 16 hr. (D) Flg22-triggered ROS burst in mock- or BRZ-treated Col-0 or BZR1Δ plants. Leaf discs were pre-treated with a 2.5 μM BRZ solution for 16 hr. In all cases, bars represent SE of 21 ≤ n ≤ 28. Asterisks indicate a statistically significant difference compared to Col-0 (A and B) or mock-treatment (C and D) according to a Student's t-test (p<0.05). Flg22 was used at a concentration of 50 nM; chitin was used at a concentration of 1 mg/ml. Total photon counts were integrated between minutes two and 40 after PAMP treatment.

https://doi.org/10.7554/eLife.00983.006
Figure 2 with 1 supplement
Activation of BZR1 results in the suppression of specific PTI outputs.

(A) Co-immunoprecipitation (Co-IP) of BAK1 and FLS2 in Col-0 and BZR1Δ seedlings after 10 min mock (−) or 1 μM flg22 (+) treatment. Proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with anti-FLS2 or anti-BAK1 antibodies. (B) MAPK activation in Col-0 and BZR1Δ seedlings upon 1 μM flg22 treatment. Proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) rabbit monoclonal antibodies. CBB: Coomassie brilliant blue. (C) Marker gene (At2g17700 and NHL10) expression in Col-0 and BZR1Δ seedlings after 1 hr mock (−) or 1 μM flg22 (+) treatment, as determined by qPCR. Bars represent SE of n = 3. (D) and (E) Seedling growth inhibition of 10-day-old Col-0 or BZR1Δ seedlings induced by increasing concentrations of flg22, as indicated. Scale bar (D), 1 cm. Bars (E) represent SE of 8 ≤ n ≤ 16. (F) Flg22-induced resistance to P. syringae pv. tomato DC3000 in Col-0 and BZR1Δ plants. Plants were pre-treated with 1 μM flg22 or water 24 hr prior to bacterial infiltration. Bars represent SE of n = 4. This experiment was repeated seven times with similar results. (G) Susceptibility of Col-0 and BZR1Δ plants to P. syringae pv. cilantro 0788-9. Bars represent SE of n = 4. Asterisks indicate a statistically significant difference compared to Col-0 according to a Student’s t-test (p<0.05); ns = not significant. All experiments were repeated at least twice with similar results unless otherwise stated.

https://doi.org/10.7554/eLife.00983.007
Figure 2—figure supplement 1
Expression of the constitutively active BZR1S173A results in the suppression of specific PTI outputs.

(A) Co-IP of BAK1 and FLS2 in Col-0 and BZR1S173A seedlings treated with 1 μM flg22 for 10 min. Co-immunoprecipitated proteins were analyzed by using anti-FLS2 or anti-BAK1 antibodies. (B) MAPK activation in Col-0 and BZR1S173A seedlings upon treatment with 1 μM flg22. Membranes were blotted with phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) rabbit monoclonal antibodies. (C) Flg22-induced resistance to Pto DC3000 in BZR1S173A plants. Plants were pre-treated with 1 μM flg22 or water 24 hr prior to bacterial inoculation. Bars represent SE of n = 4. Asterisks indicate a statistically significant difference compared to mock-treated plants according to a Student's t-test (p<0.05); ns = not significant. All experiments were repeated at least twice with similar results.

https://doi.org/10.7554/eLife.00983.008
Figure 3 with 1 supplement
WRKY transcription factors play a dual role on the BR-mediated regulation of PTI signaling.

(A) Flg22-triggered ROS burst in mutants in each BR-induced BZR1-targeted WRKY. Leaf discs of four- to five-week-old Arabidopsis plants were used in these assays. Flg22 was used at a concentration of 50 nM. Total photon counts were integrated between minutes two and 40 after PAMP treatment. Bars represent SE of n = 28. Asterisks indicate a statistically significant difference compared to Col-0 according to a Student’s t-test (p<0.05). (B) Flg22-triggered ROS burst in epiBL (BL)- or mock- pre-treated wrky40 mutant or wild-type plants. Leaf discs of four- to five-week-old plants were pre-treated with a 1 μM BL solution or mock solution for 8 hr. Flg22 was used at a concentration of 50 nM. Total photon counts were integrated between minutes two and 40 after PAMP treatment. Bars represent SE of n = 21. Asterisks indicate a statistically significant difference compared to Col-0 according to a Student’s t-test (p<0.05). (C) Co-IP of BZR1-GFP transiently expressed in N. benthamiana, alone or together with WRKY40-HA or WRKY6-HA. BZR1-GFP was immunoprecipitated with an anti-GFP antibody. Immuniprecipitated or total proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with anti-HA or anti-GFP antibodies. CBB: Coomassie brilliant blue. (D) Co-IP of BZR1-GFP transiently expressed in Arabidopsis protoplasts, alone or together with WRKY40-HA. BZR1-GFP was immunoprecipitated with an anti-GFP antibody. Immuniprecipitated or total proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with anti-HA or anti-GFP antibodies. CBB: Coomassie brilliant blue. All experiments were repeated at least twice with similar results.

https://doi.org/10.7554/eLife.00983.014
Figure 3—figure supplement 1
Mutants in WRKY11, WRKY15, WRKY18 and WRKY40 are more resistant to Pto DC3000.

(A) and (B) Pto DC3000 infections in Col-0, wrky11, wrky15, and wrky18 (A) and in Col-0 and wrky40 (B) plants. Bars represent SE of n = 4. Asterisks indicate a statistically significant difference compared to Col-0 plants according to a Student's t-test (p<0.05); ns = not significant. All experiments were repeated at least three times with similar results.

https://doi.org/10.7554/eLife.00983.015
Figure 4 with 3 supplements
Activation of BR signaling and BZR1 prioritizes growth over immunity in the dark.

(A) and (B) Relative seedling growth inhibition of 10-day-old (A) Col-0, bri1-301 and bin2-1 or (B) Col-0 and BZR1Δ seedlings induced by increasing concentrations of flg22 in either light or dark. (C) Relative seedling growth inhibition of 10-day-old Col-0 seedlings grown on medium supplemented or not with BL (1 μM), GA (1 μM), BL+GA (1 μM + 1 μM) or mock solution in light or dark. (D) Relative seedling growth inhibition of Col-0 or wrky40 seedlings induced by increasing concentrations of flg22 in either light or dark. Bars represent SE of n = 16 (A, B and D) or n = 8 (C) Asterisks indicate a statistically significant difference compared to Col-0 in the same condition (light or dark and same concentration of flg22), according to a Student’s t-test (p<0.05); ‘a’ indicates a statistically significant difference compared to the same genotype/treatment and concentration of flg22 in light, according to a Student’s t-test (p<0.05). All experiments were repeated at least three times with similar results. Values are relative to Col-0 (A, B and D) or mock-treated seedlings (C) (set to 100). Absolute values of these experiments are shown in Figure 4—figure supplement 3. (E) Schematic model depicting the BZR1-mediated inhibition of PTI. Upon BR- and DELLA-dependent activation, BZR1 induces the expression of negative regulators of PTI, such as WRKY11, WRKY15, WRKY18, or HBI1. In addition, BZR1 also inhibits the expression of immune genes, acting cooperatively with WRKY40 and possibly other WRKYs. Ultimately, the BZR1-mediated changes in transcription would lead to the suppression of PTI signaling. The PTI signaling pathway is shadowed in violet; the BR signaling pathway is shadowed in green.

https://doi.org/10.7554/eLife.00983.018
Figure 4—figure supplement 1
The BR-mediated suppression of seedling growth inhibition in the dark requires GA synthesis.

Seedling growth inhibition of 10-day-old Col-0 seedlings grown on medium supplemented or not with (A) BL (1 μM), paclobutrazol (PAC; 1 μM), BL+PAC (1 μM + 1 μM) and PAC+GA (1 μM + 1 μM), or (B) uniconazole (Uni; 100 μM), BL (1 μM) and Uni+BL (100 μM + 1 μM) induced by increasing concentrations of flg22 in light or dark. (C) Seedling growth inhibition of 10-day-old Ler and ga1-3 seedlings grown on medium supplemented or not with BL (1 μM). Bars represent SE of n = 8. Asterisks indicate a statistically significant difference compared to Col-0 in the same condition (light or dark and same concentration of flg22), according to a Student's t-test (p<0.05); ‘a’ indicates a statistically significant difference compared to the same genotype/treatment and concentration of flg22 in light, according to a Student's t-test (p<0.05). Values are relative to mock-treated seedlings (set to 100). All experiments were repeated at least twice with similar results.

https://doi.org/10.7554/eLife.00983.019
Figure 4—figure supplement 2
Phenotype of the light- or dark-grown seedlings used in the seedling growth inhibition assays (Figure 4 and Figure 4—figure supplement 1).

Representative seedlings of the seedling growth inhibition experiments depicted in: (A) Figure 4A; (B) Figure 4—figure supplement 1A; (C) Figure 4–figure supplement 1B; (D) Figure 4D. Scale bar, 1 cm.

https://doi.org/10.7554/eLife.00983.020
Figure 4—figure supplement 3
Absolute fresh weight values of seedling growth inhibition assays.

(A) Seedling growth inhibition of 10-day-old Col-0 or BES1S171A seedlings induced by increasing concentrations of flg22. (B) Absolute fresh weight values of the seedling growth inhibition assay depicted in Figure 4B, dark. (C) Absolute fresh weight values of the seedling growth inhibition assay depicted in Figure 4A. (D) Absolute fresh weight values of the seedling growth inhibition assay depicted in Figure 4C. (E) Absolute fresh weight values of the seedling growth inhibition assay depicted in Figure 4—figure supplement 1A. (F) Absolute fresh weight values of the seedling growth inhibition assay depicted in Figure 4—figure supplement 1B. (G) Absolute fresh weight values of the seedling growth inhibition assay depicted in Figure 4D. Error bars represent SE as indicated in Figure 4, Figure 4—figure supplement 1.

https://doi.org/10.7554/eLife.00983.021
Author response image 1

BL-induced dephosphorylation of BZR1 is maintained after an 8-hour BL treatment. Ten-day-old transgenic Arabidopsis seedlings expressing BZR1-YFP were treated with 1μM BL or mock solution for the indicated time. Proteins were detected using an anti-GFP antibody conjugated to HRP.

Author response image 2

Hypocotyl length of light- or dark-grown Arabidopsis seedlings in increasing concentrations of flg22. Seedlings were grown in MS plates for four days, then transferred to liquid MS supplemented with flg22 at the indicated concentrations. Hypocotyl length was measured four days later. Bars represent SE with n=8.

Author response image 3

Flg22-induced resistance to P. syringae pv. tomato DC3000 in Col-0 and wrky40 plants. Plants were pre-treated with 1 μM flg22 or water 24 hours prior to bacterial infiltration. Results are the average of three independent biological replicates; bars represent SE.

Tables

Table 1

Defense-related Gene Ontology terms (Biological Process ontology) over-represented among all BR-regulated genes, BR-regulated BZR1 targets and BR-regulated BES1 targets

https://doi.org/10.7554/eLife.00983.009
Defense-related GO termObserved frequency (%)Expected frequency (%)p-value
BR-Regulated genes
 response to bacterium2.213.31 × 10−08
 defense response to bacterium1.90.83.31 × 10−08
 response to chitin1.40.51.78 × 10−07
 defense response4.733.32 × 10−07
 response to fungus1.50.73.4 × 10−06
 response to nematode0.70.20.000532
 defense response to fungus10.50.0035
BR-regulated BZR1 targets
 response to chitin2.60.59.13 × 10−13
 response to bacterium2.310.00112
 defense response to bacterium1.90.80.00154
 response to fungus1.60.70.00495
BR-regulated BES1 targets
 response to chitin2.40.50.00439
Table 2

Gene Ontology terms (Molecular Function ontology) over-represented among all BR-regulated BZR1 targets

https://doi.org/10.7554/eLife.00983.010
Over-represented GO termObserved frequency (%)Expected frequency (%)p value
BR-regulated BZR1 targets
 nucleic acid binding transcription factor activity14.810.20.000223
 transferase activity21.616.80.00333
 kinase activity11.68.10.00702
 transcription repressor activity1.10.30.01
Table 3

Defense-related Gene Ontology terms (Biological Process ontology) over-represented among the BZR1-target BR-regulated transcription factors

https://doi.org/10.7554/eLife.00983.011
Defense-related GO TermObserved frequency (%)Expected frequency (%)p value
BZR1-target BR-regulated TFs
 response to chitin16.60.51.36 × 10−26
 defense response to bacterium7.60.84.71 × 10−07
 response to bacterium7.614.51 × 10−06
 regulation of defense response to virus by host1.400.000964
 regulation of immune effector process1.400.00151
 regulation of defense response to virus1.400.00151
 regulation of defense response2.80.30.00484
 defense response8.330.00603
 response to fungus3.40.70.01
 defense response to fungus2.80.50.02
Table 4

Over-represented cis-acting promoter elements among the defense-related BR-regulated genes according to Athena (http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/home.pl)

https://doi.org/10.7554/eLife.00983.012
Enriched TF site% promotersp value
Defense-related BR-regulated genes
 W-box72.4<10−6
Table 5

BR-regulated BZR1-target WRKY genes

https://doi.org/10.7554/eLife.00983.013
AGI numberWRKY TF
BR-Induced BZR1 targets
 AT4G31800WRKY18
 AT4G31550WRKY11
 AT4G23810WRKY53
 AT3G56400WRKY70
 AT5G49520WRKY48
 AT5G52830WRKY27
 AT1G69310WRKY57
 AT2G23320WRKY15 (Yu et al., 2011)
BR-repressed BZR1 targets
 AT4G01250WRKY22
 AT1G80840WRKY40
 AT2G24570WRKY17
 AT2G23320WRKY15 (Sun et al., 2010)
 AT2G30590WRKY21
Table 6

Overlap between the targets of WRKY40 and BZR1

https://doi.org/10.7554/eLife.00983.016
Known targets of WRKY40 (Pandey et al., 2010)Targets of BZR1 (Sun et al., 2010)
Confirmed by ChIP
EDS1Yes
RRTF1Yes
JAZ8Yes
Putative (according to expression analyses)
LOX2Yes
AOSYes
JAZ7Yes
JAZ10Yes
Table 7

Representation of the W-box motif among the BR-regulated BZR1 targets according to Athena (http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/home.pl)

https://doi.org/10.7554/eLife.00983.017
BZR1 targets% of promoters with W-box motif(s)p value
BR-induced66<10−10
BR-repressed72<10−4

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)