The Arabidopsis lectin receptor kinase LecRK-V.5 represses stomatal immunity induced by Pseudomonas syringae pv. tomato DC3000

Abstract

Stomata play an important role in plant innate immunity by limiting pathogen entry into leaves but molecular mechanisms regulating stomatal closure upon pathogen perception are not well understood. Here we show that the Arabidopsis thaliana L-type lectin receptor kinase-V.5 (LecRK-V.5) negatively regulates stomatal immunity. Loss of LecRK-V.5 function increased resistance to surface inoculation with virulent bacteria Pseudomonas syringae pv tomato DC3000. Levels of resistance were not affected after infiltration-inoculation, suggesting that LecRK-V.5 functions at an early defense stage. By contrast, lines overexpressing LecRK-V.5 were more susceptible to Pst DC3000. Enhanced resistance in lecrk-V.5 mutants was correlated with constitutive stomatal closure, while increased susceptibility phenotypes in overexpression lines were associated with early stomatal reopening. Lines overexpressing LecRK-V.5 also demonstrated a defective stomatal closure after pathogen-associated molecular pattern (PAMP) treatments. LecRK-V.5 is rapidly expressed in stomatal guard cells after bacterial inoculation or treatment with the bacterial PAMP flagellin. In addition, lecrk-V.5 mutants guard cells exhibited constitutive accumulation of reactive oxygen species (ROS) and inhibition of ROS production opened stomata of lecrk-V.5. LecRK-V.5 is also shown to interfere with abscisic acid-mediated stomatal closure signaling upstream of ROS production. These results provide genetic evidences that LecRK-V.5 negatively regulates stomatal immunity upstream of ROS biosynthesis. Our data reveal that plants have evolved mechanisms to reverse bacteria-mediated stomatal closure to prevent long-term effect on CO(2) uptake and photosynthesis.

Figure 1. LecRK-V.5 negatively regulates disease resistance to bacteria.

(A) Insertional mutation sites in two lecrk-V.5 mutant lines. Ds transposon (lecrk-V.5-1) and T-DNA (lecrk-V.5-2) insertion sites are shown. Filled box represents exon and arrows denote the different positions of the primers used in the RT-PCR experiments in (B). The relative position of lectin, transmembrane (TM) and kinase domains in LecRK-V.5 predicted protein structure is indicated. (B) RT-PCR analysis of LecRK-V.5 transcripts in WT and lecrk-V.5 mutants. EF-1 was used as a control. (C) Disease symptoms assessed 3 days after dip-inoculation with 1×10⁷ cfu.ml⁻¹ Pst DC3000 in WT (Ler), lecrk-V.5-1 and two complemented lines (CL-1 and CL-2). (D) Bacterial growth at 3 days post-inoculation (1×10⁷ cfu.ml⁻¹ Pst DC3000 (Pst)) in WT (Col-0 and Ler), lecrk-V.5 mutants and two complemented lines (CL-1 and CL-2). (E) Bacterial titers evaluated 3 days after dip-inoculation with 1×10⁷ cfu.ml⁻¹ Pst DC3000 (Pst) in lines overexpressing LecRK-V.5 (OE-1 and OE-2). For (D) and (E), data represent average ± SD. Statistical differences between WT controls and mutants or transgenics are detected with a t test (P<0.01, n = 6). All experiments were repeated at least three times with similar results.

Figure 2. Altered stomatal immunity responses in lecrk-V.5-1 and in lines over-expressing LecRK-V.5.

(A) Stomatal apertures in epidermal peels exposed to MES buffer (control) or 1×10⁸ cfu.ml⁻¹ Pst DC3000 (Pst) for 1 or 3 hrs. (B) Bacterial growth assessed 3 days after dip-inoculation with 1×10⁷ cfu.ml⁻¹ Pst DC3000 (Pst) or Pst DC3000 COR⁻ (Pst COR⁻) in WT Col-0 and overexpression lines (OE-1 and OE-2). Values are the means ± SD. Statistical differences between WT controls and OE lines are detected with a t test (P<0.01, n = 6). (C) Stomatal aperture in WT Col-0 and overexpression lines (OE-1 and OE-2) after 1 and 4 hours incubation in MES buffer (Control) or 1×10⁸ cfu.ml⁻¹ Pst DC3000 COR⁻ (Pst COR⁻). For (A) and (C), results are shown as mean of ≥60 stomata measurements ± SE. Asterisks indicate significant differences between WT and mutant/CL/OE lines based on a t test (P<0.001). hpi, hour post inoculation. All experiments were repeated at least three times with similar results.

Figure 3. Altered PAMP-induced stomatal closure in lines overexpressing LecRK-V.5.

(A) Stomatal apertures in epidermal peels of WT Col-0 and overexpression lines (OE-1 and OE-2) after 3 hrs of incubation with MES buffer (Control), 5 µM flg22, 5 µM elf26 or 100 ng.µL⁻¹ LPS. (B) Stomatal apertures were measured on epidermal peels incubated in MES buffer (Control), 1 µM flg22, 1 µM elf26, 10 ng.µL⁻¹ LPS or 1 µM flg22, 1 µM elf26 and 10 ng.µL⁻¹ LPS together. Results are shown as mean of ≥60 stomata measurements ± SE. Asterisks indicate significant differences between WT and OE lines based on a t test (P<0.001). All experiments were repeated at least three times with similar results. hpi, hour post inoculation.

Figure 4. Apoplastic PTI responses in lecrk-V.5 mutants.

(A) Production of reactive oxygen species in Arabidopsis leaves after treatment with 1 µM flg22 as relative light units (RLU). Values represent averages ± SE (n = 6). (B) Callose deposition in WT or lecrk-V.5 mutants leaves infiltrated with 10 mM MgSO4 (Mock) or Pst DC3000 hrcC (Pst hrcC). Data represent the number of callose deposits per square millimeter ± SD. Differences were not significantly different to WT based on a t test (P<0.01). (C, D) FRK1, NHL10, CYP81F2 expression levels in WT or lecrk-V.5 mutant seedlings soaked in 10 mM MgSO₄ (Mock) or 1×10⁷ cfu.ml⁻¹ Pst DC3000 hrcC (hrcC). Transcript levels were determined by qRT-PCR and normalized to both EF-1 and UBQ10. Bars indicate SD (n = 9). All experiments were repeated 3 times with similar results.

Figure 5. LecRK-V.5 is localized at the plasma membrane and is expressed at guard cells upon PTI activation.

(A, B and C) Subcellular localization of LecRK-V.5-GFP fusion protein in Arabidopsis mesophyll protoplasts. LecRK-V.5-GFP expression was driven by the cauliflower mosaic virus 35S promoter and transiently expressed in Arabidopsis mesophyll protoplasts. The bright-field image (A), images of the GFP fluorescence (green) only (B), and the overlap image of the GFP (green) and chlorophyll (red) fluorescence (C) are presented. (D) Photo of a control protoplast expressing GFP alone. Bars = 20 µm. (E) The activity of LecRK-V.5 promoter was detected by GUS staining in guard cell 15 min after inoculation with 1×10⁸ cfu.ml⁻¹ Pst DC3000 (Pst) or 1 µM flg22 treatment. Bar represents 20 µm.

Figure 6. Effects of COR on flg22-mediated stomatal closure in lecrk-V.5 mutants.

(A, B) Stomatal aperture in epidermal peels of WT (Ler and Col-0) and lecrk-V.5 mutants exposed to MES buffer (Control), 0.5 ng.µL⁻¹ COR, 5 µM flg22 or 5 µM flg22 and 0.5 ng.µL⁻¹ COR together (flg22+COR) for 3 hrs. Results are shown as mean of ≥60 stomata measurements ± SE. Asterisks indicate significant differences between WT and mutants based on a t test (P<0.001). All experiments were repeated at least three times with similar results.

Figure 7. Role of LecRK-V.5 in ROS-mediated stomatal closure.

(A) ROS detected by H₂DCFDA fluorescence in non-treated WT (Ler and Col-0), lecrk-V.5 mutants and two complemented line (CL-1 and CL-2) guard cells. A representative stoma is shown. Bars represent 7.5 µm. (B) Stomatal aperture in epidermal peels of WT (Ler and Col-0) and lecrk-V.5 mutants exposed to MES buffer (Control), 20 µM DPI or 1 mM ASC for 3 hrs. (C) ROS detected by H₂DCFDA fluorescence in guard cells of WT Col-0 and lines overexpressing LecRK-V.5 (OE-1 and OE-2) after treatments with MES buffer (Control), 5 µM flg22, 5 µM elf26 or 100 ng.µL⁻¹ LPS. (D) Stomatal aperture in epidermal peels of WT Col-0 and overexpression line OE-1 and OE-2 after 3 h incubation with H₂O₂. For all experiments, results are shown as mean ± SE. In (A), (B) and (D) asterisks indicate a significant difference to WT control based on a t test analysis (n≥60; P<0.001). In (C), different letters indicate statistically significant differences compared with the non-treated WT Col-0 (Fisher's Least Significant Difference test; n≥60; P<0.05). All experiments were repeated at least three times with similar results.

Figure 8. LecRK-V.5 role in stomatal immunity is mechanistically linked to ABA signaling.

(A) Stomatal apertures in epidermal peels of WT Col-0 or LecRK-V.5 overexpression transgenics OE-1 and OE-2 were measured after 3 hrs incubation in ABA. (B) H₂DCFDA-detected ROS production after ABA treatments. (C, D) Stomatal apertures in epidermal peels of WT (Ler and Col-0) and lecrk-V.5 mutants exposed to MES buffer (Control), 0.5 ng.µL⁻¹ COR, 10 µM ABA, or 10 µM ABA together with 0.5 ng.µL⁻¹ COR (ABA+COR) for 3 hrs. (E, F) Arabidopsis WT (Ler and Col-0) or lecrk-V.5 mutants epidermal peels were floated in MES buffer (Control) or 1 µM K252a (E) or 0.5 mM butyrate (F) for 3 hrs before stomatal aperture measurement. (G) Stomatal aperture in lecrk-V.5-1 abi1-1D and lecrk-V.5-1 abi2-1D double mutants. For B and G, different letters indicate statistically significant differences compared with WT (Fisher's Least Significant Difference test; n≥60; P<0.05). For A, C, D, E and F, results are shown as mean ± SE (n≥60) and asterisks indicates significant differences between WT and mutant/OE based on a t test (P<0.001). All experiments were repeated at least three times with similar results.

Figure 9. Model of LecRK-V.5 role in PAMP-induced stomatal closure.

This model is based on the information provided in this study and references cited in the Discussion. PAMPs are perceived by pattern recognition receptors (PRRs) in the guard cell. PAMPs perception is mechanistically linked to ABA-regulated stomatal closure via ROS production by NADPH oxidase. The virulence factor COR is secreted by Pst DC3000 to interfere with stomatal closure by reverting flg22-inhibition. LecRK-V.5 negatively regulates PAMP-mediated stomatal closure downstream of ABA but upstream of ROS production.