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, 27 (7), 2042-56

Phosphorylation of the Plant Immune Regulator RPM1-INTERACTING PROTEIN4 Enhances Plant Plasma Membrane H⁺-ATPase Activity and Inhibits Flagellin-Triggered Immune Responses in Arabidopsis

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Phosphorylation of the Plant Immune Regulator RPM1-INTERACTING PROTEIN4 Enhances Plant Plasma Membrane H⁺-ATPase Activity and Inhibits Flagellin-Triggered Immune Responses in Arabidopsis

DongHyuk Lee et al. Plant Cell.

Abstract

The Pseudomonas syringae effector AvrB targets multiple host proteins during infection, including the plant immune regulator RPM1-INTERACTING PROTEIN4 (RIN4) and RPM1-INDUCED PROTEIN KINASE (RIPK). In the presence of AvrB, RIPK phosphorylates RIN4 at Thr-21, Ser-160, and Thr-166, leading to activation of the immune receptor RPM1. Here, we investigated the role of RIN4 phosphorylation in susceptible Arabidopsis thaliana genotypes. Using circular dichroism spectroscopy, we show that RIN4 is a disordered protein and phosphorylation affects protein flexibility. RIN4 T21D/S160D/T166D phosphomimetic mutants exhibited enhanced disease susceptibility upon surface inoculation with P. syringae, wider stomatal apertures, and enhanced plasma membrane H(+)-ATPase activity. The plasma membrane H(+)-ATPase AHA1 is highly expressed in guard cells, and its activation can induce stomatal opening. The ripk knockout also exhibited a strong defect in pathogen-induced stomatal opening. The basal level of RIN4 Thr-166 phosphorylation decreased in response to immune perception of bacterial flagellin. RIN4 Thr166D lines exhibited reduced flagellin-triggered immune responses. Flagellin perception did not lower RIN4 Thr-166 phosphorylation in the presence of strong ectopic expression of AvrB. Taken together, these results indicate that the AvrB effector targets RIN4 in order to enhance pathogen entry on the leaf surface as well as dampen responses to conserved microbial features.

Figures

Figure 1.
Figure 1.
RIN4 Is an Intrinsically Disordered Protein, and Phosphorylation Affects Flexibility. (A) PrDOS prediction of disorder in RIN4. Red and black amino acid sequences indicate predicted disordered and ordered regions, respectively. Asterisks indicate phosphorylated residues on RIN4 by RIPK (Thr-21, Ser-160 and Thr-166). (B) PrDOS plot of disorder probability of each residue along the sequence of RIN4. Residues above the red threshold line in this plot are predicted to be disordered. (C) SDS-PAGE gel stained with Coomassie blue demonstrating the purity of recombinant wild-type RIN4 (WT RIN4), phosphorylation mimic (RIN4 3D), or phosphorylation null (RIN4 3A). The residues Thr-21, Ser-160, and Thr-166 were mutated to D or A in the RIN4 phosphorylation mimic and phosphorylation null, respectively. (D) In vitro phosphorylation of RIN4 by RIPK. Recombinant RIPK (MBP-RIPK) was incubated with recombinant wild type RIN4 (WT RIN4) in the presence and absence of ATP. RIPK was subsequently removed based on its molecular mass using a centrifugal filter unit. Top panel: RIN4 pThr166 (pRIN4) proteins detected by anti-pRIN4 Thr-166 antibody. Bottom panel: SDS-PAGE gel stained with Coomassie blue confirmed equal amounts of RIN4. (E) Far-UV CD spectroscopy of purified RIN4 proteins from (C). Wild-type RIN4’s trace is shown in black, the RIN4 phosphorylation mimic (3D) trace is shown in red, and the RIN4 phosphorylation null (3A) trace is shown in blue. (F) Far-UV CD spectroscopy of phosphorylated RIN4 proteins from (D). Unphosphorylated RIN4’s trace is shown in black and phosphorylated RIN4’s trace is shown in red.
Figure 2.
Figure 2.
Transgenic Lines Expressing the RIN4 Phosphorylation Mimic Exhibit Enhanced Disease Susceptibility to P. syringae DC3000 in a Susceptible Genetic Background. The Arabidopsis rpm1/rps2/rin4 mutant was complemented with T7-tagged with genomic RIN4 (WT), gRIN4 3D (3D), or gRIN4 3A (3A) under the control of RIN4's native promoter. RIN4 Thr-21/Ser-160/Thr-166 residues are mutated to D or A in 3D or 3A lines, respectively. (A) Anti-RIN4 protein gel blot illustrating RIN4 expression in T4 homozygous lines expressing wild type (WT 5-1 and WT 7-4), phosphorylation mimics (3D 1-7, 3D 2-8, and 3D 9-10), and phosphor-null mutants (3A 4-1 and 3A 10-7) in the rpm1/rps2/rin4 genetic background. Total proteins were extracted from 10-d-old seedlings and subjected to immunoblot with anti-RIN4. SDS-PAGE gel stained with Coomassie blue (CBB) was used as a loading control. r1r2r4 = rpm1/rps2/rin4; r1r2 =rpm1/rps2. The white line indicates that samples were run on two separate protein gel blots but extracted and processed at the same time. (B) Complementation analysis with npro:T7-gRIN4 in the rpm1/rps2/rin4 mutant. Four-week-old r1r2, r1r2r4, or wild-type RIN4 complementation lines (WT RIN4 5-1 and 7-4) were spray-inoculated with 1 × 108 cfu/mL of Pto DC3000. Bacterial population sizes were quantified 4 d postinoculation. Results represent means ± se, n = 6. (C) RIN4 phosphorylation mimic lines (RIN4 3D) exhibit enhanced disease susceptibility to Pto DC3000. Wild-type RIN4 (WT 5-1), RIN4 phosphorylation mimic (3D 1-7 and 9-10), or phosphor-null (3A 4-1 and 10-7) lines were inoculated and bacterial growth was determined as described in (B). Results represent means ± se, n = 6. Different letters above bars indicate statistical differences in means, detected by a Fisher’s LSD (α = 0.05).
Figure 3.
Figure 3.
Purified Recombinant RIN4 Phosphorylation Mimics Enhance H+-ATPase Activity. Inside-out plasma membrane vesicles from the Arabidopsis rpm1/rps2/rin4 mutant were incubated with different recombinant RIN4 proteins (RIN4 WT, 3D, 3A, T21D, S160D, or T166D) in the assay medium to measure H+-ATPase activity. In this assay, the plasma membrane H+-ATPase hydrolyzes ATP and pumps H+ into vesicles, creating a pH gradient across the membrane. The pumping activity was measured by the pH probe acridine orange (Δ495 nm/mg protein/min). (A) SDS-PAGE gel stained with Coomassie blue showing the purity of recombinant RIN4 wild type (WT), triple (3D), or single (T21D, S160D, and T166D) phosphorylation mimics and phosphorylation null (3A). Proteins were extracted at the same time but run on two separate SDS-PAGE gels. (B) The RIN4 triple phosphorylation mimic (3D) enhances H+-ATPase activity in vitro. Results represent means ± sd, n = 3. Different letters above bars indicate statistical differences in means, detected by a Fisher’s LSD (α = 0.05). (C) RIN4 single phosphorylation mimics are unable to enhance H+-ATPase activity in vitro. Results represent mean ± sd, n = 3. Statistical differences were detected as described in (B). All assays used 50 µg of plasma membrane protein and 5 µg of recombinant RIN4.
Figure 4.
Figure 4.
RIN4 Phosphorylation Mimics for All Three Residues Exhibit the Strongest Association with AHA1 in Planta. AHA1 was coexpressed with different RIN4 phosphorylation mimics in N. benthamiana using Agrobacterium tumefaciens-mediated transient expression. The luciferase complementation assay used AHA1-NLuc and CLuc-RIN4 variants. Luciferin (1 mM) was infiltrated 48 h after Agrobacterium infiltration and the luminescence signal was quantified on a luminometer. (A) The RIN4 phosphorylation mimic (RIN4 3D) exhibits the strongest enhanced association with AHA1 in N. benthamiana. (B) The RIN4 triple (RIN4 3D) phosphorylation mimic, but not single phosphorylation mimics (RIN4 T21D, S160D, or T166D), show increased association with AHA1. For (A) and (B), the bioluminescence signal in the leaf was captured using a CCD camera and the intensity was quantified by a luminometer. Results represent mean ± sd, n ≥ 5. Different letters above bars indicate statistical differences in means, detected by a Fisher’s LSD (α = 0.05). (C) Expression of CLuc-RIN4 and AHA1-NLuc in N. benthamiana. Anti-RIN4 and Anti-LUC antibody were used to detect RIN4 (top) and AHA1 (middle), respectively. An SDS-PAGE gel stained with Coomassie blue (CBB) was used to visualize protein loading (bottom). (D) Anti-RIN4 protein gel blot confirmed RIN4 expression. The membrane was stained with Coomassie blue to visualize protein loading.
Figure 5.
Figure 5.
RIN4 Phosphorylation Mimics Display Wider Basal Stomatal Apertures, and the ripk Knockout Does Not Reopen Its Stomata in Response to Virulent P. syringae. (A) The Arabidopsis ripk knockout line does not reopen stomata after treatment with virulent Pto DC3000. Stomatal apertures were measured in epidermal peels from wild-type Col-0 and the ripk knockout in the Col-0 background after incubation with either water or virulent Pto DC3000 at a concentration of 1 × 108 cfu/mL. Stomatal apertures were measured 3 h after treatment. Results represent means ± se, n = 30. Statistical differences were detected by a two-tailed t test compared with the wild type; asterisks indicate α = 0.001. (B) The ripk knockout is more resistant after spray inoculation with Pto DC3000, but not the coronatine-deficient strain Pto DC3118. Four-week-old wild-type (WT) Col-0 or the ripk knockout (ripk-1) were spray-inoculated with 1 × 108 cfu/mL of Pto DC3000 or DC3118 Cor-. Bacterial population sizes were quantified 4 d postinoculation. Results represent mean ± se, n = 6. Statistical differences were detected by a two-tailed t test compared with the wild type; asterisks indicate α = 0.01. (C) The RIN4 3D phosphorylation mimic exhibits wider basal stomatal apertures compared with controls. Basal stomatal apertures were measured 3 h after light treatment. Genotypes included rpm1/rps2 (r1r2), rpm1/rps2/rin4 complemented with T7-RIN4 (WT), and rpm1/rps2/rin4 complemented with T7-RIN4 T21D/S160D/T166D (3D). Results represent mean ± se, n ≥ 100. Different letters above bars indicate statistical differences in means, detected by a Fisher’s LSD (α = 0.05). (D) The RIN4 3D phosphorylation mimic exhibits wider basal stomatal apertures compared with RIN4 phosphorylation null (3A). Basal stomata opening was measured in WT5-1, 3D 9-10, and 3A 10-7 lines. Results represent mean ± se, n ≥ 90. Statistical differences were detected as described in (C). (E) RIN4 phosphorylation mimic (3D) lines exhibit enhanced disease susceptibility to Pto DC3118 Cor-. Pathogen inoculation and bacterial population measurement were performed as described in (B). Results represent means ± se, n = 6. Statistical differences were detected as described in (B). (F) Pto DC3118 Cor- expressing AvrB exhibited enhanced bacterial growth in planta compared empty vector (EV) alone. RIN4 complementation lines were inoculated with Pto DC3118 Cor- expressing pBRR1-MCS5 (Cor- +EV) or npro:AvrB:3xFlag (Cor- +AvrB). Pathogen inoculation and bacterial growth measurement were performed as described in (E). Results represent means ± se, n = 6. Statistical differences were detected as described in (C).
Figure 6.
Figure 6.
RIN4 Phosphorylation Mimics Exhibit Compromised PTI Responses. (A) The RIN4 phosphorylation mimic (3D line 9-10) showed enhanced bacterial growth after inoculation with Pto DC3000 ∆hrcC. Genotypes included rpm1/rps2 (r1r2), rpm1/rps2/rin4 complemented with T7-RIN4 (WT), and rpm1/rps2/rin4 complemented with T7-RIN4 T21D/S160D/T166D (3D). Four-week-old Arabidopsis plants were syringe-inoculated with 1 × 105 cfu/mL of Pto DC3000 ∆hrcC and bacterial growth was quantified zero and 3 d postinoculation. Results represent means ± se, n = 6. (B) RIN4 phosphorylation mimics suppress the ROS burst upon flg22 perception. Leaf disks were harvested from rpm1/rps2, WT5-1, and 3D9-10, treated with 100nM flg22, and the ROS burst analyzed with a luminometer. Total ROS generation was monitored over 35 min. Results represent means ± se, n = 16. (C) RIN4 Thr-166 is the critical residue required for suppression of the flg22-induced ROS burst. ROS burst was measured in leaf discs of wild-type line 5-1, 3D line 9-10, T21D line 1-5, and T166D line 6-1 after 100 nM flg22 treatment as described in (B). Total ROS generation was monitored over 35 min. Results represent means ± se, n = 16. Different letters above bars indicate statistical differences in means, detected by a Fisher’s LSD (α = 0.05) for all panels.
Figure 7.
Figure 7.
RIN4 Thr-166 Phosphorylation Is Suppressed during Flagellin Perception. (A) The phosphorylation of RIN4 Thr-166 is decreased after flg22 treatment. Proteins were extracted 10 min after vacuum infiltration with 1 µM flg22 or water. Numbers indicate three biological repeats. RIN4 and RIN4 pThr166 (pRIN4) proteins were detected by anti-RIN4 and anti-pRIN4 Thr166 antibodies, respectively. In order to visualize basal RIN4 phosphorylation, a Femto enhanced chemiluminescence substrate was used to enable detection of a weak signal. Phosphorylated MAPK3/6 were detected by anti-p44/42 ERK antibody. The membrane was stained with Coomassie blue (CBB) to detect protein loading. (B) Flg22 perception is unable to decrease pThr166 in the presence of strong ectopic AvrB expression. rpm1-3 or rpm1-3 carrying Dex-inducible GVG:AvrB-HA was treated with 30 µM Dex, then vacuum infiltrated with water or 1 μM flg22 and total proteins were extracted 10 min later. RIN4 and RIN4 pThr166 (pRIN4) proteins were detected by anti-RIN4 and anti-pRIN4 Thr-166 antibodies, respectively. Short and long indicate exposure time of the anti-pRIN4 blot. Protein samples were run on the same gel, but cropped to remove additional lanes. The membrane was stained with Coomassie blue to verify protein loading. (C) Flg22 perception can decrease pThr166 in the presence of weak ectopic AvrB expression. Plant genotypes were treated with water or 3 µM Dex prior to infiltration with water or flg22 and immunoblotting as described in (B). (D) Detecting AvrB expression by RT-PCR. Four-week-old plants were not treated or treated with 3 µM Dex and total RNA was extracted for RT-PCR 16 h later. ELONGATION FACTOR1-α was used as a control for gene expression. RT, reverse transcriptase. (E) P. fluorescens (EtHAn) expressing AvrB can induce RIN4 Thr-166 phosphorylation. rpm1-3 plants were syringe-inoculated with 5 × 107 cfu/mL of P. fluorescens expressing a broad host range vector pBRR1 MCS5 (EV) or npro:AvrB-3xFLAG. MgCl2 (10 mM) was used as the mock treatment. Total proteins were extracted 6 h postinfiltration. RIN4 and RIN4 pThr166 (pRIN4) proteins were detected by anti-RIN4 and anti-pRIN4 Thr-166 antibodies, respectively. The membrane was stained with Coomassie blue to verify protein loading. (F) AvrB is expressed in the rpm1-3 genotype after infection with P. fluorescens (EtHAn). Arabidopsis leaves were syringe-infiltrated as described in (E). Total proteins were extracted from leaves at 6 and 12 h postinfiltration and AvrB was detected by immunoblotting using anti-FLAG antibody (top). The membrane stained with Coomassie blue verified protein loading (bottom).

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