Biochemical and genetic requirements for function of the immune response regulator BOTRYTIS-INDUCED KINASE1 in plant growth, ethylene signaling, and PAMP-triggered immunity in Arabidopsis

Abstract

Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) regulates immune responses to a distinct class of pathogens. Here, mechanisms underlying BIK1 function and its interactions with other immune response regulators were determined. We describe BIK1 function as a component of ethylene (ET) signaling and PAMP-triggered immunity (PTI) to fungal pathogens. BIK1 in vivo kinase activity increases in response to flagellin peptide (flg22) and the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) but is blocked by inhibition of ET perception. BIK1 induction by flg22, ACC, and pathogens is strictly dependent on EIN3, and the bik1 mutation results in altered expression of ET-regulated genes. BIK1 site-directed mutants were used to determine residues essential for phosphorylation and biological functions in planta, including PTI, ET signaling, and plant growth. Genetic analysis revealed flg22-induced PTI to Botrytis cinerea requires BIK1, EIN2, and HUB1 but not genes involved in salicylate (SA) functions. BIK1-mediated PTI to Pseudomonas syringae is modulated by SA, ET, and jasmonate signaling. The coi1 mutation suppressed several bik1 phenotypes, suggesting that COI1 may act as a repressor of BIK1 function. Thus, common and distinct mechanisms underlying BIK1 function in mediating responses to distinct pathogens are uncovered. In sum, the critical role of BIK1 in plant immune responses hinges upon phosphorylation, its function in ET signaling, and complex interactions with other immune response regulators.

Figure 1.

BIK1 Conserved Residues and in Vitro and in Vivo Kinase Assays. (A) Structure of the BIK1 protein and comparison of residues in the ADs of BIK1 and related kinases. Gray region denotes the KD and black the AD. Residues noted in the BIK1 protein indicate those substituted for in planta assays. (B) Kinase activity of recombinant BIK1 and Ala substitution mutants produced in E. coli detected by autoradiogram. CCB, Coomassie blue staining. (C) and (D) BIK1 substitution mutants in vivo detected by a mobility shift on an HA-immunoblot or by phosphoserine/Thr-specific antibody. (E) BIK1 and MBP phosphorylation is abrogated by phosphatase treatment. Protein dephosphorylation was performed according to the manufacturer’s protocol (New England Biolabs) with ~1 to 2.5 units CIP/μg protein (left) or (~2.5 μ/μg) (right). −, Buffer; +, CIP. In (C) and (D), plants were treated with ACC (Ac) or flg22 (Fl) for 3 h and assayed for changes in BIK1 and MBP phosphorylation activity. In (C) to (E), in vivo BIK1 phosphorylation was detected by ACC or flagellin-induced mobility shifts observable by HA-immunoblot. The top band corresponds to phosphorylated BIK1, which is migrating slower than the unphosphorylated form. MBP phosphorylation by BIK1 was detected by immunoblots with a phosphoserine/Thr-specific antibody.

Figure 2.

BIK1 Is Required for PTI to B. cinerea. Disease symptoms (A) and mean lesion size (B) of water (−) and flg22-treated (+) bik1 and BIK1 substitution mutants after drop inoculation with B. cinerea (2.5 × 10⁵ spores/mL). Data in (B) represent mean ± se from a minimum of 30 disease lesions. The statistical significance of the mean lesion sizes was determined using analysis of variance and Tukey’s test. The mean values followed by different letters are significantly different from each other (P = 0.01) Experiments were repeated at least three times with similar results. Images were taken 3 d after inoculation. The BIK1 site-directed mutants and the wild-type (wt) BIK1-HA are expressed in the bik1 mutant background.

Figure 3.

BIK1 Is Required for Responses to ET and Glc. (A) and (B) Triple response phenotype (A) and hypocotyl lengths (B) of bik1 seedlings on different concentrations of ACC (μM) and unsupplemented MS media. wt, wild type. (C) and (D) Sensitive growth response (C) and root length (D) of bik1 on media containing increased levels of Glc. Data in (B) and (D) represent mean ± se from a minimum of 60 seedlings. Experiments were repeated at least three times with similar results. Statistical analysis was performed as described in the legend of Figure 2.

Figure 4.

BIK1 Functions in the ET Response Pathway. (A) to (C) Expression of BIK1, as determined by quantitative RT-PCR and normalized to actin following pathogen inoculation (A) and during the triple response (B) and flg22 (Fl) treatment (C) in ET response mutants. wt, wild type. (D) ChIP-PCR showing the association of EIN3 protein with the BIK1 promoter. Chromatin from wild-type seedlings was immunoprecipitated with an anti-EIN3 polyclonal antibody. Enrichment of BIK1 promoter region was verified by qPCR using BIK1 promoter-specific primers. BIK1 Pr region A refers to position −1221 to −774 and region B from −494 to −166 in the BIK1 promoter region. The BIK1 promoter sequence is shown in Supplemental Figure 3 online. (E) EIN3 protein levels in bik1 and wild-type seedlings in response to flg22 (Fl) and ACC (Ac) treatment. (F) BIK1 phosphorylation is inhibited by blocking ET perception with silver nitrate (AgNO₃). Changes in BIK1 phosphorylation were visualized as a mobility shift observable by HA-immunoblot. The top band corresponds to the slowly migrating band caused by BIK1 phosphorylation. MBP phosphorylation was detected on an immunoblot with a phosphoserine/Thr-specific antibody. (G) and (H) Histochemical assay showing GUS activity in transgenic BIK1pr:GUS plants (G) and seedlings (H) in response to flg22, chitin, and ACC. (I) to (L) Quantitative RT-PCR determination of pathogen-induced expression of ERF4 (I), ERF104 (J), ORA59 (K), and FRK1 (L) in wild-type and bik1 plants. Phosphorylation detection was performed as described in the legend of Figure 1. Experiments were performed as described in the Methods and repeated at least three times with similar results.

Figure 5.

Ala Substitution Mutants Reveal BIK1 Residues Required for the Arabidopsis Triple Response. Triple response phenotypes (A) and hypocotyl lengths (B) of bik1 and BIK1 substitution seedlings on 20 μM ACC (+) and unsupplemented MS media (−). Data in (B) represent mean ± se from a minimum of 60 seedlings. Experiments were repeated at least three times with similar results. Statistical analysis was performed as described in the legend of Figure 2. All BIK1 site-directed mutants are expressed in the bik1 background. wt, wild type.

Figure 6.

BIK1 Mediates Plant Responses to Pst Strains and flg22. (A) and (B) Bacterial growth ([cfu]/cm² leaf area) in water- (−) and flg22-treated (+) plants 3 d after inoculation with PstDC3000 (A) or the nonpathogenic Pst hrcC⁻ strain (B), deficient in Type III secretion. CFU, colony-forming units; wt, wild type. (C) Percentage decrease in fresh weight of seedlings after growth in 10 nM flg22. Data represent mean values ± se from three experiments and a minimum of 120 seedlings for bacterial growth and fresh weights, respectively. Experiments were repeated at least three times with similar results. Statistical analysis was performed as described in the legend of Figure 2. All BIK1 site-directed mutants are in bik1.

Figure 7.

Phosphomimic Substitution at Thr-242 in the AD of BIK1 Confers Enhanced ET Responses and PTI to B. cinerea. (A) In vivo kinase activity of the BIK1^T242D and BIK1^Y245D substitution mutants in response to ACC (Ac) and flg22 (Fl). Kinase activity of the phosphomimic mutants visualized by audioradiograms of radiolabeled proteins following incubation with [γ-³²P]ATP. (B) Constitutive activation of BIK1 phosporylation by the BIK1^T242D phosphomimic mutation. BIK1 phosphorylation was visualized as a mobility shift detected by HA-immunoblot. Coomassie blue was used as a loading control. (C) Lesion diameter in water (−) and flg22 (+) pretreated plants following drop inoculation with B. cinerea (2.5 × 10⁵ spores/mL). Wt, wild type. (D) and (E) Triple response (D) and average hypocotyl length (E) of seedlings on different concentrations of ACC (μM) (+) or unsupplemented MS media (−). In (A), protein staining with Coomassie blue was used as a loading control. Data in (C) and (E) represent mean ± se from a minimum of 30 disease lesions or 60 seedlings, respectively. Experiments were repeated at least three times with similar results. Statistical analysis was performed as described in the legend of Figure 2. [See online article for color version of this figure.]

Figure 8.

Epistasis Analysis of the Interaction between BIK1 and Other Immune Response Genes in flg22-Induced PTI to B. cinerea. Lesion diameter ([A] and [D]) and disease symptoms ([B] and [C]) in water (−) and flg22 (+) pretreated single and double mutants after drop inoculation with B. cinerea (2.5 × 10⁵ spores/mL). Data in (A) and (D) represent mean ± se from a minimum of 30 disease lesions. Experiments were repeated at least three times with similar results. Statistical analysis was performed as described in the legend of Figure 2. Images were taken 3 d after inoculation. Wt, wild type.

Figure 9.

The Role of Arabidopsis Immune Response Genes on the Functions of BIK1 in flg22-Induced PTI to Virulent and Nonpathogenic Strains of Pst. (A) and (B) Bacterial growth ([cfu]/cm² leaf area) in water (−) and flg22 (+) pretreated single and double mutants 3 d after inoculation with Pst DC3000 (A) or the nonpathogenic Pst hrcC⁻ strain (B), deficient in Type III secretion. CFU, colony-forming units; Wt, wild type. (C) Percentage decrease in fresh weight of seedlings after growth in 10 nM flg22. Data in (A) to (C) represent mean values ± se from three experiments and a minimum of 120 seedlings, respectively. Experiments were repeated at least three times with similar results. Statistical analysis was performed as described in the legend of Figure 2.

Figure 10.

Expression of Defense Marker Genes in bik1 and the Double Mutants in Response to Pathogen Infection and ACC. (A) B. cinerea and PstDC3000-induced expression of PR-1. Wt, wild type. (B) B. cinerea and ACC-induced expression of PDF1.2. Expression was determined using quantitative RT-PCR 24 h after inoculation/treatment as described in Methods. Experiments were repeated at least two times with similar results.