Extracellular ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal aperture

Abstract

In addition to acting as a cellular energy source, ATP can also act as a damage-associated molecular pattern in both animals and plants. Stomata are leaf pores that control gas exchange and, therefore, impact critical functions such as photosynthesis, drought tolerance, and also are the preferred entry point for pathogens. Here we show the addition of ATP leads to the rapid closure of leaf stomata and enhanced resistance to the bacterial pathogen Psuedomonas syringae. This response is mediated by ATP recognition by the receptor DORN1, followed by direct phosphorylation of the NADPH oxidase RBOHD, resulting in elevated production of reactive oxygen species and stomatal closure. Mutation of DORN1 phosphorylation sites on RBOHD eliminates the ability of ATP to induce stomatal closure. The data implicate purinergic signaling via DORN1 in the control of stomatal aperture with important implications for the control of plant photosynthesis, water homeostasis, pathogen resistance, and ultimately yield.

Fig. 1

Mapping of DORN1 autophosphorylation sites and importance to DORN1 function. a Schematic representation of DORN1 protein structure highlighting the autophosphorylation sites identified by mass spectrometry. The various domains of DORN1 are color coded. b–d Contribution of different DORN1 autophosphorylation sites to ATP-induced calcium influx. Different transgenic plants (5-day-old) expressing the aequorin reporter were treated with 100 μM ATP, and the luminescence was immediately monitored. RLU relative luminescence units; Error bars indicate ± SEM; n = 8 (biological replicates); **P < 0.01, Student’s t test. These experiments were repeated three times with similar results. e Self-association of DORN1 in Arabidopsis protoplasts. DORN1-HA and DORN1-Myc were co-expressed in WT protoplasts treated with either 200 μM ATP for 20 min (+) or H₂O as a control (−). Co-IP was performed using an anti-HA and anti-Myc antibodies. This experiment was repeated three times with similar results

Fig. 2

ATP triggers ROS response through DORN1 and RBOHD. a Identification of RBOHD tryptic peptides as a substrate of DORN1 kinase by KiC assay. GST-DORN1-KD kinase domain was incubated with a 2.1 K peptides library in the presence of ATP. The library was also incubated with the kinase-dead version of DORN1-KD-1, GST, or MBP as negative controls. Potential phosphorylation sites were predicted by phosphoRS. b Co-immunoprecipitation of DORN1 and RBOHD proteins in Arabidopsis protoplasts. The indicated constructs were transiently expressed in wild-type protoplasts treated with either 200 μM ATP for 20 min (+) or H₂O as a control (−). Full-length CERK1 was used as a negative control. Co-IP was performed using an anti-HA and anti-Myc antibodies. This experiment was repeated three times with similar results. c DORN1 and RBOHD are important for the ATP-induced ROS burst. ROS production was measured using wild-type or dorn1-3 and rbohd mutant plants treated with 250 μM ATPγS for 30 min. RLU relative luminescence units; values represent the mean ± SEM, n = 8 (biological replicates); *P < 0.05, **P < 0.01, Student’s t test. This experiment was repeated three times with similar results

Fig. 3

DORN1 directly interacts with RBOHD in vivo and in vitro. a DORN1 directly interacts with RBOHD N-terminal in vitro. Purified, recombinant proteins GST-DORN1-KD, GST-DORN1-KD-1 (kinase dead), GST-DORN1-KD-2 (kinase dead), or GST were incubated with His-RBOHD-N followed by GST-mediated pull-down. Purified His-Lyk4 kinase domain protein was used as a negative control. Lambda protein phosphatase (Lambda PP) was added to release phosphate groups from phosphorylated serine, threonine, and tyrosine residues. b DORN1 interacts with RBOHD in the protoplast plasma membrane. The indicated constructs were transiently expressed in wild-type protoplasts and the BiFC assay was performed. FM4-64 was added to stain the plant cell plasma membrane. Bar = 20 μm. c DORN1 interacts with RBOHD in Arabidopsis plants. Stable transgenic Arabidopsis plants (F1) generated from a cross between NP::DORN1-HA/dorn1-3 and 35s::RBOHD-Myc/Col-0 expressing lines were treated with or without 250 μM ATP for 30 min, and total protein extract was subjected to Co-IP. All above experiments were repeated three times with similar results

Fig. 4

DORN1 phosphorylates RBOHD-N at S22 and T24 sites in vitro and in vivo. a DORN1 phosphorylates the N-terminal domain of RBOHD. Purified His-RBOHD-N recombinant protein was incubated with GST-DORN1-KD kinase domain, GST-DORN1-KD-1 (kinase dead), GST-DORN1-KD-2 (kinase dead), or GST in an in vitro kinase assay. Autophosphorylation and trans-phosphorylation were measured by incorporation of γ-[³²P]-ATP. MBP and GST-LYK5-KD kinase domain were used as positive and negative controls, respectively. The protein loading was measured by Coomassie brilliant blue (CBB) staining. This experiment was repeated three times with similar results. b DORN1 phosphorylates RBOHD-N at S22 and T24 sites in vitro. Purified GST or GST-DORN1-KD protein was incubated with His-RBOHD-N or the respective mutant proteins, S22A (His-S22A), T24A (His-T24A), S22AT24A (His-S22AT24A), followed by an in vitro kinase assay. This experiment was repeated three times with similar results. c ATP-induced phosphorylation of RBOHD through S22 and T24 sites in vivo. The indicated constructs pGBW14-RBOHD (WT, S22A, T24A, and S22AT24A) were transiently expressed in rbohd mutant protoplasts incubated with γ-[³²P]-ATP overnight. After treating with 200 μM ATP for 30 min, total protein was extracted and subjected to immunoprecipitation. Total RBOHD-HA protein was detected by anti-HA immunoblotting. Protein loading was monitored by CBB. This experiment was repeated three times with similar results. d RBOHD phosphosites are required for ATP-triggered ROS production. The indicated constructs were transiently expressed in rbohd mutant protoplasts and treated with or without 200 μM ATPγS. ROS production was measured after 30 min. RLU relative luminescence units; values represent the mean ± SEM, n = 8 (biological replicates). Means with different letters are significantly different (P < 0.01; one-sided ANOVA). Total RBOHD-HA protein was detected by anti-HA immunoblot and CBB staining was used to monitor protein loading. This experiment was repeated three times with similar results

Fig. 5

DORN1 and RBOHD positively regulate stomatal immunity. a, b DORN1 and RBOHD are required for the ATP- and ADP-induced stomatal closure. Stomatal aperture was measured after treatment with 2 mM ATP, 250 μM ATPγS, 2 mM ADP, or 5 μM ABA. Bar = 10 μm. Values represent the mean ± SEM, n ≥ 50 (biological replicates); means with different letters are significantly different (P < 0.01; one-sided ANOVA). c DORN1-mediated RBOHD phosphosites are required for stomatal closure. The RBOHD transgenic lines in rbohd mutant background were used to measure stomatal closure after treatment with 250 μM ATPγS. Values represent the mean ± SEM, n ≥ 50 (biological replicates); means with different letters are significantly different (P < 0.01; one-sided ANOVA). d, e DORN1 and RBOHD are required for stomatal immunity. Fourteen-day-old seedlings were flood inoculated with a P. syringae DC3000 suspension (5 × 10⁶ CFU ml⁻¹) with or without the addition of ATP. Bacterial colonization was determined by plate counting 3 days post inoculation. Values represent the mean ± SEM, n ≥ 8 (biological replicates). Means with different letters are significantly different (P < 0.05; one-sided ANOVA). f DORN1-mediated RBOHD phosphosites are required for bacterial defense. Two independent T2 transgenic lines (L1 and L2) were used to measure bacterial growth after 3 days post inoculation with 200 μM ATP treatment. Values represent the mean ± SEM, n ≥ 8 (biological replicates); *P < 0.05, Student’s t test. All above experiments were repeated three times with similar results

Fig. 6

The release of ATP triggers DORN1 protein modification to regulate ROS during pathogen infection. a Bacteria induce ATP release in the guard cells. Luciferase fluorescence shows that ATP accumulates around the stomata on P. syringae DC3000, hrcC ⁻ (OD₆₀₀ = 0.2) and 10 μM flg22 infecting plant leaves. Bar = 20 μm. b Relative quantification of the eATP concentration released from stomata. RLU relative light units; values represent the mean ± SD, n = 50 (biological replicates). Means with different letters are significantly different (P < 0.01; one-sided ANOVA). This experiment was repeated three times with similar results. c DORN1 and RBOHD are responsible for bacteria-induced ROS production. ROS production was measured using leaf discs inoculated with P. syringae DC3000, hrcC ⁻ (OD₆₀₀ = 0.1) and 1 μM flg22. Values represent the mean ± SEM, n = 8 (biological replicates); means with different letters are significantly different (P < 0.01; one-sided ANOVA). This experiment was repeated three times with similar results. d Bacteria elicit DORN1 protein modification similar to ATP, likely due to protein phosphorylation. DORN1 protein modification (★-DORN1-HA) was determined in NP::DORN1-3 × HA/dorn1-3 expressing transgenic plants after treatment with 1 μM flg22, 250 μM ATP, P. syringae and hrcC ⁻ (OD₆₀₀ = 0.2) for 30 min. CBB Coomassie brilliant blue staining. This experiment was repeated three times with similar results. e The proposed model depicting the mechanism by which ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal immunity