IBR5 Modulates Temperature-Dependent, R Protein CHS3-Mediated Defense Responses in Arabidopsis

Abstract

Plant responses to low temperature are tightly associated with defense responses. We previously characterized the chilling-sensitive mutant chs3-1 resulting from the activation of the Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR)-type resistance (R) protein harboring a C-terminal LIM (Lin-11, Isl-1 and Mec-3 domains) domain. Here we report the identification of a suppressor of chs3, ibr5-7 (indole-3-butyric acid response 5), which largely suppresses chilling-activated defense responses. IBR5 encodes a putative dual-specificity protein phosphatase. The accumulation of CHS3 protein at chilling temperatures is inhibited by the IBR5 mutation. Moreover, chs3-conferred defense phenotypes were synergistically suppressed by mutations in HSP90 and IBR5. Further analysis showed that IBR5, with holdase activity, physically associates with CHS3, HSP90 and SGT1b (Suppressor of the G2 allele of skp1) to form a complex that protects CHS3. In addition to the positive role of IBR5 in regulating CHS3, IBR5 is also involved in defense responses mediated by R genes, including SNC1 (Suppressor of npr1-1, Constitutive 1), RPS4 (Resistance to P. syringae 4) and RPM1 (Resistance to Pseudomonas syringae pv. maculicola 1). Thus, the results of the present study reveal a role for IBR5 in the regulation of multiple R protein-mediated defense responses.

Fig 1. Identification of a suppressor of chs3-1, suc5.

(A) Morphology of wild-type (Col), chs3-1 and chs3 suc5 plants grown at 16°C. The photographs show 4-week-old plants grown in soil (left panel). The rosette leaves of chs3 suc5 have increased leaf serration compared with Col (right panel). (B, C) Trypan blue and DAB staining of leaves from 3-week-old wild type, chs3-1 and chs3 suc5 plants grown at 16°C. Bars: 200 μm. (D) Levels of total and free SA in 4-week-old, wild-type, chs3-1 and chs3 suc5 plants grown at 16°C. (E, F) Expression of PR1, PR2 (E) and CHS3 (F) genes in 3-week-old wild-type, chs3-1 and chs3 suc5 plants grown at 16°C. The values were normalized to the expression of ACTIN2. The error bars represent the SD of three replicates. Similar results were obtained in three independent experiments. (G) Growth of P.s.t. DC3000 on wild-type, chs3-1, and chs3 suc5 plants. Three-week-old plants were dipped with P.s.t. DC3000 (OD₆₀₀ = 0.05). Bacterial growth was monitored as described in the Materials and Methods. The error bars represent the SD of three replicates, and the asterisks indicate significant differences compared with wild-type Col (**P < 0.01, t-test).

Fig 2. Identification of SUC5.

(A) Map position and genomic structure of the SUC5 gene. Exons, introns and UTRs are presented as gray boxes, lines and white boxes, respectively. The point mutation and T-DNA insertions are shown. (B) Morphology of the wild-type, chs3-1, chs3-1 ibr5-7, complemented transgenic plants (IBR5:IBR5 chs3 ibr5-7), T-DNA insertion mutant (ibr5-3) and chs3-1 ibr5-3 double mutant. The photographs show 3-week-old plants grown in soil at 16°C. (C) Trypan blue staining of true leaves from wild-type, chs3-1, chs3-1 ibr5-7 and IBR5:IBR5 chs3-1 ibr5-7 plants. Bars: 200 μm. (D) IBR5 protein levels in wild-type, chs3-1 and ibr5 mutants. Total protein was extracted from 2-week-old seedlings. IBR5 protein was analyzed by immunoblotting using an anti-IBR5 antibody. Anti-RBCL was used as a loading control.

Fig 3. Interaction of IBR5 with CHS3.

(A) Predicted protein structure of CHS3. TIR, Toll/Interleukin–1 receptor domain (aa 1–138); NB, nucleotide binding domain (aa 139–468); LRR, leucine-rich repeat domain (aa 469–729); Unknown, domain with no known function (aa 730–1240); LIM, LIM (Lin-11, Isl-1 and Mec-3 domains)-containing domain (aa 1135–1614); mLIM, LIM-containing domain with the same mutation in chs3-1, which contains a stop codon at 1422. (B) Interaction between IBR5 and CHS3 in yeast. The different CHS3 domains shown in (A) were fused with the pGADT7 vector. IBR5 and mutated IBR5^C129S were fused with the pGBKT7 vector. The constructs were transformed into AH109 yeast cells and spotted onto SD media lacking Trp and Leu (-2SD) or lacking Trp, Leu, His and Ade (-4SD). The experiments were performed four times with similar results. (C) Co-immunoprecipitation of IBR5 and the TIR domain of CHS3. Total proteins were extracted from protoplasts expressing 35S:HA-Flag-IBR5 (35S:HF-IBR5) with Super: TIR domain of CHS3 (Super:TIR-Myc) or Super:Myc, and immunoprecipated with anti-Myc antibody. The proteins were obtained from crude lysates (Input) and the immunoprecipated proteins were detected using an anti-HA antibody. (D) Interaction of IBR5 and full-length CHS3 in vivo. Total proteins were extracted from N. benthamiana leaves transfected with 35S:HF-IBR5 and Super:CHS3-1-Myc, and were immunoprecipated with anti-Myc antibody. The proteins from crude lysates (Input) and the immunoprecipated proteins were detected using an anti-HA antibody. (E) Expression of IBR5 in 3-week-old Super:IBR5-Myc and Super:IBR5 ^C129S -Myc transgenic plants in chs3 ibr5 grown at 16°C. (F) The phenotypes of Super:IBR5-Myc and Super:IBR5 ^C129S -Myc transgenic plants in chs3 ibr5. The photographs show 3-week-old plants grown in soil at 16°C. (G) The expression of PR1 in 3-week-old Super:IBR5-Myc and Super:IBR5 ^C129S -Myc transgenic chs3 ibr5 plants grown at 16°C.

Fig 4. CHS3 is promoted by IBR5 at both mRNA and protein levels.

Genotyping of ibr5-3 chs3-2D-GFP plants. The primers used for genotyping the chs3-2D-GFP gene were GFP-P1 and GFP-P2, and the primers used for genotyping ibr5-3 were IBR5-P1, IBR5-P2 and LB1 as listed in S1 Table. The phenotype of chs3-2D-GFP transgenic plants was rescued by ibr5-3. chs3-2D-GFP and ibr5-3 chs3-2D-GFP plants were grown for 5 weeks, and representative plants are shown. Expression of CHS3-2D-GFP protein in 8-d-old chs3-2D-GFP and ibr5-3 chs3-2D-GFP plants. Bar: 100 μm. (D) Expression of CHS3-2D-GFP protein in 8-day-old chs3-2D-GFP and ibr5-3 chs3-2D-GFP plants. Bar: 100 μm. (D, E) Expression of the PR1, PR2 (D) and CHS3 (E) genes in chs3-2D-GFP and ibr5-3 chs3-2D-GFP plants. The values were normalized to the expression of ACTIN2. The error bars represent the SD of three replicates. (F) The expression of several R genes (SNC1, RPM1, RPS4 and CHS3) in 10-day-old ibr5-3 mutant grown at 22°C. The values were normalized to the expression of ACTIN2. The error bars represent the SD of three replicates. Similar results were obtained in three independent experiments. (G) The effect of IBR5 on the CHS3 protein level in Arabidopsis protoplasts. Super:CHS3-1-Myc and 35S:HF-IBR5 or 35S:HF constructs were co-expressed in Arabidopsis protoplasts. IBR5 and CHS3 proteins were detected using anti-HA and anti-Myc antibodies. The GFP-Myc construct was used as a control for transformation efficiency. The numbers show the ratios of CHS3-1-Myc to GFP-Myc protein levels. Two biological repeats are shown with similar results.

Fig 5. Genetic analysis of CHS3, IBR5 and HSP90.3.

(A) Morphology of 4-week-old wild-type Col, chs3-1, chs3 hsp90.3–1, chs3 ibr5-7 chs3 hsp90.3 ibr5 and F1 progeny of chs3 hsp90.3–1 crossed with chs3 ibr5-7 grown in soil at 16°C. (B) Trypan blue staining of the leaves from the plants described in (A). Bar: 50 μm. (C) PR1 gene expression in the plants described in (A). The values were normalized to the expression of ACTIN2. The error bars represent the SD of three replicates. The experiments were performed three times with similar results.

Fig 6. Interaction of IBR5 with HSP90 and SGT1b.

(A) Interaction of IBR5 with HSP90 as revealed via firefly luciferase complementation imaging assay in N. benthamiana leaves. nLuc+cLuc-IBR5, HSP90-nLuc+cLuc, and nLuc+cLuc were used as negative controls. (B) Interaction of IBR5 with SGT1b as revealed by a firefly luciferase complementation imaging assay in N. benthamiana leaves. nLuc+cLuc-IBR5, SGT1b-nLuc+cLuc, and nLuc+cLuc were used as negative controls. (C) Co-immunoprecipitation of IBR5 and HSP90. Total protein was extracted from 2-week-old transgenic seedlings expressing Super:IBR5-Myc or Super:Myc. Anti-Myc antibody was used for immunoprecipitation, and the immunoprecipitated proteins were analyzed by immunoblotting using an anti-HSP90 antibody. (D) Co-immunoprecipitation of IBR5 and SGT1b. Total protein was extracted from transgenic plants expressing Super:Myc/Super:SGT1b-Flag or Super:IBR5-Myc/Super:SGT1b-Flag. An anti-Myc antibody was used for immunoprecipitation, and the immunoprecipitated proteins were analyzed by immunoblotting using an anti-Flag antibody. (E) Interaction of CHS3 with IBR5 and SGT1b in plant. Total proteins were extracted from N. benthamiana leaves transfected with 35S:HF-IBR5, Super:SGT1b-Flag and Super:CHS3-1-Myc, and were immunoprecipated with anti-Myc antibody. The proteins from crude lysates (Input) and the immunoprecipated proteins were detected using an anti-Flag antibody. (F) IBR5 can stabilize CS under thermally denaturing conditions. Citrate synthase (1 μM) was incubated alone or with 5 μM BSA (1:5), 0.5 μM IBR5 (2:1), 1 μM IBR5 (1:1) or 1 μM HSP90 (1:1) at 43°C. The kinetics was determined by light scattering using a fluorescence spectrophotometer.

Fig 7. The defense phenotypes of bon1-1 ibr5-3 and bal ibr5-3.

Morphology of 3-week-old Col, ibr5-3, bon1-1, bon1-1 ibr5-3, bal, bal ibr5-3 grown at 22°C. (B) Fresh weight of plants in (A). The error bars represent the SD of three replicates, and the asterisks indicate significant differences (**P < 0.01, *P < 0.05, t-test). (C) Trypan blue staining of leaves from the plants described in (A). Bar: 0.5 mm. The expression of PR genes in plants in (A). The error bars represent the SD of three replicates, and the asterisks indicate significant differences (**P < 0.01, *P < 0.05, t-test). (E) The effect of IBR5 on pathogen resistance of bon1-1 and bal to P.s.t. DC3000. Three-week-old seedlings were dipped with P.s.t. DC3000 (OD₆₀₀ = 0.05), and leaves were taken immediately (day 0) and at day 4 after infection. The log-transformed values presented are the mean values of three replicates ± SD. The experiments were performed three times with similar results. (F) Relative bacterial growth of plants in (E) after infection with P.s.t. DC3000. The values are the ratios of bacteria number at day 4 to day 0. The error bars represent the SD of three replicates, and the asterisks indicate significant differences (**P < 0.01, *P < 0.05, t-test).

Fig 8. Interaction of IBR5 with SNC1 and RPP4.

(A) Interaction of IBR5 with TIR domains of CHS3, SNC1 and RPP4 in yeast. The experiments were performed three times with similar results. (B) Interaction of IBR5 and full-length SNC1 in vivo. Total proteins were extracted from N. benthamiana leaves transfected with 35S: HF-IBR5 and Super:SNC1-1-Myc and were immunoprecipated with an anti-Myc antibody. The proteins from crude lysates (Input) and the immunoprecipated proteins were detected using an anti-HA antibody. (C) The effect of IBR5 on SNC1 protein level in Arabidopsis protoplasts. Super:SNC1-1-Myc and 35S:HF-IBR5 or 35S:HF constructs were co-expressed in Arabidopsis protoplasts. IBR5 was detected with anti-HA antibody and SNC1-1 was detected using an anti-Myc antibody. GFP-Myc construct was used as a control for transformation efficiency.

Fig 9. Pathogen resistance analysis of ibr5 mutants.

(A) Response of ibr5 to P.s.t. DC3000. Three-week-old plants were dipped with P.s.t. DC 3000 (OD₆₀₀ = 0.05), and leaves were taken immediately (day 0) and at day 4 after infection. The log-transformed values presented are the mean values of three replicates ± SD. (B-D) Responses of ibr5 to avirulent bacteria. Three-week-old plants were dipped with P.s.t. DC3000 (avrRpt2) (B), P.s.t. DC3000 (avrRpm1) (C) and P.s.t. DC3000 (avrRps4) (D) (OD₆₀₀ = 0.2). In (A-D), the log-transformed values presented are the mean values of three replicates ± SD, and the asterisks indicate significant differences compared with wild-type Col (**P < 0.01, *P < 0.05, t-test). All experiments were repeated at least three times with similar results.