A Lectin Receptor-Like Kinase Mediates Pattern-Triggered Salicylic Acid Signaling

Abstract

Plant surface-localized pathogen recognition receptors (PRRs) perceive conserved microbial features, termed pathogen-associated molecular patterns (PAMPs), resulting in disease resistance. PAMP perception leads to calcium influx, MAPK activation, a burst of reactive oxygen species (ROS) mediated by RbohD, accumulation of the defense hormone salicylic acid (SA), and callose deposition. Lectin receptor-like kinases (LecRKs) belong to a specific PRR family and are important players in plant innate immunity. Here, we report that LecRK-IX.2 is a positive regulator of PRR-triggered immunity. Pathogen infection activated the transcription of Arabidopsis (Arabidopsis thaliana) LecRK-IX.2, and the LecRK-IX.2 knockout lines exhibited enhanced susceptibility to virulent Pseudomonas syringae pv tomato DC3000. In addition, LecRK-IX.2 is capable of inducing RbohD phosphorylation, likely by recruiting calcium-dependent protein kinases to trigger ROS production in Arabidopsis. Overexpression of LecRK-IX.2 resulted in elevated ROS and SA and enhanced systemic acquired resistance to P. syringae pv tomato DC3000. Our data highlight the importance of LecRKs in plant immune signaling and SA accumulation.

Figure 1.

LecRK-IX.2 positively regulates disease resistance to Pst DC3000. A, LecRK-IX.2 transcription is induced by Pst DC3000 and Pst DC3000 ΔhrcC. Four-week-old plants were infiltrated with 5 × 10⁷ colony-forming units (cfu) mL⁻¹ Pst DC3000 and Pst DC3000 ΔhrcC, then they were sampled at the indicated times. Mock is 10 mm MgCl₂. qRT-PCR was used to determine gene expression. Values are means ± sd (n = 3 biological replicates). B, Growth curve analysis of Pst DC3000 in Columbia-0 (Col-0), lecrk-IX.2, lecrk-IX.1, and lecrk-IX.2/lecrk-IX.1 double mutants. Four-week-old plants were infiltrated with 5 × 10⁴ cfu mL⁻¹ Pst DC3000. Plants were subjected to bacterial growth curve analysis at 1 and 3 d postinoculation (dpi). Values are means ± sd (n = 6; ANOVA, P < 0.01). C, Overexpression of LecRK-IX.2 enhances disease resistance to Pst DC3000. Four-week-old homozygous transgenic plants were subjected to syringe inoculation as described in B. OE6-6 and OE14-1 are two independent 35S::LecRK-IX.2 lines. Values are means ± sd (n = 6; ANOVA, P < 0.01).

Figure 2.

lecrk-IX.2 is compromised in flg22-induced PTI responses. A, Induction of LecRK-IX.2 by flg22 treatment requires FLS2. Wild-type (WT) Col-0 and fls2 were treated with 0.1 µm flg22. Samples were collected for qRT-PCR after 6 h. Mock treatment served as a negative control. Values are means ± sd (n = 3 biological replicates; Student’s t test, **, P < 0.01). B, MAPK signaling is impaired in lecrk-IX.2 mutants upon flg22 treatment. Three-week-old Col-0 and lecrk-IX.2 seedlings were sprayed with 1 μm flg22 and sampled at 0, 5, 10, and 15 min for immunoblotting. Activated MAPKs were detected by immunoblotting with phospho-p44/42 MAPK antibody. The corresponding bands are indicated for MPK3 and MPK6. Ponceau staining of Rubisco was used to estimate equal loading in each lane. The experiment was repeated three times with similar results. C, The expression of FRK1 and WRKY22 was reduced in lecrk-IX.2 mutants after flg22 treatment. Four-week-old Col-0 and lecrk-IX.2 mutants were infiltrated with 0.1 μm flg22. Relative expression levels of FRK1 and WRKY22 were analyzed at 3 h postinoculation (hpi). Values are means ± sd (n = 3 biological replicates; Student’s t test, **, P < 0.01). D, The lecrk-IX.2 mutant is compromised in callose deposition upon flg22 treatment. Four-week-old Col-0 and lecrk-IX.2 plants were infiltrated with 0.1 μm flg22. Leaves were stained with Aniline Blue at 12 h post treatment.

Figure 3.

The lecrk-IX.2 mutant showed reduced activation of flg22-induced gene expression and SA signaling. A, The induction of PR1 upon flg22 treatment is impaired in lecrk-IX.2 mutant plants. Four-week-old Col-0 and lecrk-IX.2 mutants were infiltrated with 0.1 μm flg22. qRT-PCR was performed at 0, 12, 24, and 48 hpi. Values are means ± sd (n = 3 biological replicates). B and C, The jasmonic acid-responsive genes VSP1 and PDF1.2 constitutively expressed in lecrk-IX.2 mutant plants. Four-week-old Col-0 and lecrk-IX.2 mutants were harvested for qRT-PCR. Values are means ± sd (n = 3 biological replicates; Student’s t test, **, P < 0.01). D, lecrk-IX.2 is impaired in flg22-induced systemic acquired resistance (SAR). Local leaves of 4-week-old Col-0 and lecrk-IX.2 plants were infiltrated with 0.1 μm flg22. Systemic leaves were infiltrated with 5 × 10⁴ cfu mL⁻¹ Pst DC3000 24 h later after flg22 treatment. Plants were subjected to bacterial growth curve analysis at 3 dpi. Mock is water. Values are means ± sd (n = 6; ANOVA, P < 0.01).

Figure 4.

Overexpression of LecRK-IX.2 leads to SA accumulation and SAR. A, Mild expression of LecRK-IX.2 induces SAR. Three local leaves of Col-0 and Dex12-2 were pretreated with 0.3 μm Dex, and the leaves were inoculated with Pst DC3000 (5 × 10⁴ cfu mL⁻¹) 1 d later. The bacterial growth assay for local and systemic leaves was performed 3 d later. Values are means ± sd (n = 6; ANOVA, P < 0.01). B, Mild expression of LecRK-IX.2 induces SA accumulation. Four-week-old plant leaves were infiltrated with 0.3 μm Dex. Samples were harvested at the indicated times. The SA contents of local leaves were determined by HPLC-tandem mass spectrometry. Values are means ± sd (n = 3; Student’s t test, **, P < 0.01). C, LecRK-IX.2-induced cell death requires SID2. The Dex12-2 line was crossed with sid2 mutant plants, and the F2 homozygous lines were used for Dex treatment. The Dex12-2/sid2 plants were infiltrated with 3 μm Dex, and the photographs were taken 24 h later. The gels at bottom show equal expression of the proteins. D, LecRK-IX.2-induced cell death is mediated by SA. Wild-type (WT) and the NahG transgenic N. benthamiana plant leaves transiently expressed 35S::LecRK-IX.2 or 35S::GFP (negative control), and the plant leaves were stained by Trypan Blue at 48 hpi.

Figure 5.

LecRK-IX.2 kinase activity is required for the cell death phenotype in N. benthamiana. A, Flg22 treatment activates the phosphorylation of LecRK-IX.2. Dex::LecRK-IX.2-FLAG transgenic plants were treated with 3 μm Dex and then infiltrated with 0.1 μm flg22 5 h later. Fifteen minutes after flg22 treatment, the plant leaves were sampled for immunoprecipitation with anti-Flag agarose beads. The precipitated proteins were subjected to immunoblotting. The phosphorylation of LecRK-IX.2 was probed with anti-pThr antibody. B, LecRK-IX.2 is an active kinase. The recombinant MBP-tagged cytoplasmic domain of LecRK-IX.2 and its kinase site-mutated variants were subjected to radioactive kinase assays. Myelin basic protein (MyBP) was used as a universal substrate. Kinase activity was detected by autoradiography. The bottom gel shows the protein abundance stained by Coomassie Brilliant Blue (CBB) in SDS-PAGE. The asterisk indicates a LecRK cleavage product. C, Transient expression of LecRK-IX.2 and its variants in N. benthamiana. 35S::GFP-FLAG (negative control), 35S::LecRK-IX.2-T7, and its kinase site mutant variants were expressed transiently in N. benthamiana leaves. Plant leaves were subjected to Trypan Blue staining and photographed at 48 hpi. Similar results were obtained from three biological replicates. The gels at right show equal expression of respective proteins.

Figure 6.

LecRK-IX.2 interacts physically and genetically with RbohD but cannot directly phosphorylate RbohD. A, LecRK-IX.2 interacts with RbohD in vivo by coimmunoprecipitation assay. Dex::LecRK-IX.2-Flag and 35S::RbohD-T7 were expressed transiently in N. benthamiana leaves. Plants were infiltrated with 3 μm Dex 48 h later to induce the expression of LecRK-IX.2-Flag. The immunoprecipitation was performed 3 h after Dex treatment. The experiment was repeated three times with similar results. B, LecRK-IX.2-CD interacts with the RbohD N terminus in vitro by GST pull-down assays. The recombinant GST-LecRK-IX.2 cytosolic domain and the MBP-RbohD N terminus were subjected to GST pull-down analysis. The interacting proteins were revealed by immunoblotting. The experiment was repeated three times with similar results. C, LecRK-IX.2-induced cell death requires RbohD. A Dex12-2 plant was crossed with rbohd mutant plants. Homozygous lines of Dex12-2/rbohd were treated with 3 μm Dex. The photographs were taken 24 h later. The gels at bottom show equal expression of the proteins before leaf collapse. D, LecRK-IX.2 cannot phosphorylate RbohD. The recombinant MBP-tagged cytoplasmic domain of LecRK-IX.2 was subjected to kinase activity assays with myelin basic protein (MyBP) or the RbohD N terminus as substrates. GST-BIK1 served as a positive control. Coomassie Brilliant Blue (CBB) shows the protein abundance.

Figure 7.

LecRK-IX.2 interacts with CPKs. A, LecRK-IX.2-CD interacts with CPKs in vitro by MBP pull-down assays. The recombinant MBP-LecRK-IX.2 cytosolic domain and His-CPKs were subjected to MBP pull-down analysis. Coomassie Brilliant Blue (CBB) shows the protein abundance. B, LecRK-IX.2 interacts with CPKs in N. benthamiana by split luciferase assays. N. benthamiana leaves were coinfiltrated with 35S::LecRK-IX.2 (K379R)-Nluc and 35S::CPKs-Cluc. Luciferase complementation imaging assays were performed 2 d later. This experiment was repeated three times with similar results. RLU, Relative light units.

Figure 8.

LecRK-IX.2-mediated cell death requires Ca²⁺ influx and CPKs. A, The LecRK-IX.2-induced cell death phenotype requires Ca²⁺ influx. N. benthamiana leaves transiently expressed 35S::GFP-FLAG and 35S::LecRK-IX.2-T7 and then were infiltrated with water or 1 mm LaCl₃ 24 h later. The leaves were photographed at 2 dpi and were stained with Trypan Blue. The experiments were repeated three times with similar results. B, LaCl₃ inhibits LecRK-IX.2-induced ROS in N. benthamiana. Plant leaves transiently expressed Dex::LecRK-IX.2. Dex alone or with 1 mm LaCl₃ was applied to leaves 48 hpi. A luminol-based assay for ROS production was performed 2 h after Dex and LaCl₃ treatment. Values are means ± sd (n = 3). RLU, Relative light units. C, LecRK-IX.2-induced cell death requires CPKs in N. benthamiana. The transcription of NbCPK5 and NbCPK6 was knocked down by VIGS in N. benthamiana. The silenced plants transiently expressed 35S::GFP and 35S::LecRK-IX.2 with different concentrations of A. tumefaciens. Sites 1 and 3 were infiltrated with 35S::GFP at concentrations of 1 × 10⁸ and 2 × 10⁸ cfu mL⁻¹. Sites 2 and 4 were infiltrated with 35S::LecRK-IX.2 at concentrations of 1 × 10⁸ and 2 × 10⁸ cfu mL⁻¹. Plant leaves were subjected to Trypan Blue staining and photographed at 48 hpi. The experiments were repeated three times with similar results. D, CPK5-dependent RbohD phosphorylation is required for LecRK-IX.2-induced ROS generation. Dex::LecRK-IX.2 and different variants of 35S::RbohD-T7 were coexpressed in N. benthamiana. After 48 h, 30 µm Dex was applied to leaves. Plant leaves were subjected to a luminol-based assay to measure ROS production 2 h after Dex treatment. GFP was used as a negative control. Values are means ± sd (n = 6). Six discs were from different leaves. E and F, LecRK-IX.2-induced cell death is compromised in cpk5/6. A Dex12-2 plant was crossed with cpk5/6 double mutant plants. Homozygous lines of Dex12-2/cpk5/6 were treated with 3 μm Dex. The gels at bottom show equal expression of the proteins before leaf collapse. Leaves were photographed and stained with Trypan Blue 72 h later (F).

Figure 9.

Working model of the LecRK-IX.2-mediated immune response. When plants perceive pathogen infection, some LecRKs are activated, leading to PTI activation. In addition, the activated LecRKs recruit CPKs to phosphorylate RbohD, resulting in PTI activation, ROS production, and the subsequent ROS-triggered SA accumulation. As a result, plant disease resistance is activated.