RIN4 functions with plasma membrane H+-ATPases to regulate stomatal apertures during pathogen attack

Abstract

Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4-mediated immune signal transduction, we purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H(+)-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines exhibit differential PM H(+)-ATPase activity. PM H(+)-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H(+)-ATPase activity and, importantly, its stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H(+)-ATPase to regulate stomatal apertures, inhibiting the entry of bacterial pathogens into the plant leaf during infection.

Figure 1. RIN4 and AHA interact in planta and in yeast.

(A) RIN4 interacts with the C terminus of both AHA1 and AHA2 using the Matchmaker yeast two-hybrid system (Clontech). BD, binding domain vector (pGBKT7); AD, activation domain vector (pGADT7); SD-3, synthetic dextrose media lacking leucine, tryptophan, and histidine; YPDA, yeast potato dextrose agar. AHA1 and AHA2 were cloned into the AD vector. (B) AHA1 and AHA2 associate with RIN4 in vivo. We were able to detect a specific interaction between AHA1 and AHA2 with RIN4 by BiFC across three replications. The experiments to detect BiFC fluorescence with AHA1 and AHA2 were conducted independently. N. benthamiana leaves co-expressing either AHA1 or AHA2 and RIN4 results in detectable GFP fluorescence on the membrane (upper panel, a–b). No interaction with RPS2 (lower panel, d–e) or AUX1 (c, f) could be detected. YFP fluorescence was excited at 488 and imaged at 518–540 nm by confocal microscopy, except for b and e (used excitation 512 and emission 525–540 nm). Chlorophyll emission was detected at 618 nm.

Figure 2. Plasma membrane H⁺-ATPase enzymatic activity is altered in RIN4 overexpression and knockout lines.

Plant leaf plasma membranes were purified by an aqueous polymer two-phase system. The H⁺-pumping activity assay was conducted on inside-out plasma membrane vesicles as described in the Materials and Methods. In this assay, the plasma membrane H⁺-ATPase hydrolyzes ATP and pumps H⁺ into vesicles, which creates the pH gradient across the membrane. The pumping activity was measured by the pH probe acridine orange quenching at an absorbance of 495 nm. (A) and (C) H⁺-pumping activity decreased in the RIN4 mutant line rpm1/rps2/rin4, but not in rpm1/rps2 plants. (B) and (D) RIN4 overexpression results in an increase in H⁺-pumping activity in comparison to Col 0. (C) and (D) The initial slope of acridine orange absorbance quenching was graphed from (A) and (B) respectively. H⁺-pumping activity is reported as ΔA_495nm/mg protein/min. Dexamethasone (Dex) inducible RIN4 lines and Col 0 were sprayed with water and 0.025% silwett or 20 µM Dex in 0.025% silwett. Leaf tissue was harvested after 48 h, and plasma membranes were immediately purified. (E) RIN4 immunoblot showing RIN4 expression levels in Col 0 (1), Dex:RIN4 (2), and rpm1/rps2/rin4 mutant lines (3) 48 h after Dex treatment. Each experiment was repeated two times with independent plasma membrane isolations. Statistical differences were detected by Fisher's LSD alpha = 0.05 for (C) and a two-tailed t-test for (D).

Figure 3. RIN4 positively regulates plasma membrane H⁺-ATPase enzymatic activity in vitro.

Purified recombinant RIN4 protein (3 µg) or elution buffer (EB) were added in the assay medium and pre-incubated at 25°C for 10 min. The same activity assay is performed as in Figure 2. (A) RIN4 recombinant protein enhanced H⁺-pumping activity in the rpm1/rps2/rin4 mutant in vitro, but not in wild-type Col 0. The assays with EB served as the control. (B) The initial slope of acridine orange absorbance quenching was graphed from (A). H⁺-pumping activity is reported as ΔA_495nm/mg protein/min. (C) An SDS-PAGE gel stained with coomassie blue demonstrating the purity of the recombinant RIN4 protein. Each experiment was repeated two times with independent plasma membrane isolations. Results are shown as the mean (n = 3), including standard deviation from plasma membrane vesicles isolated at one time point. Statistical differences were detected with a two-tailed t-test.

Figure 4. Constitutively active AHA1 lines display enhanced susceptibility to spray but not syringe inoculation with P. syringae pv. tomato strain DC3000.

(A) 4-wk-old Arabidopsis plants were inoculated by syringe infiltration or spray inoculation with Pst DC3000 and subjected to growth curve analysis 3 d post-inoculation. (B) Arabidopsis plants were inoculated by syringe infiltration or spray inoculation with Pst DC3000 (AvrRpt2) and subjected to growth curve analysis 3 d post-inoculation. (C) The 35S:AHA2_(1–837) overexpression line displays enhanced susceptibility to spray inoculation with Pst DC3000. (D) Disease symptoms in ost2-2D, ost2-1D, Col 0, and Landsberg (Ler) 3 d after spray inoculation with Pst DC3000 (bottom panel). Experimental control plants were sprayed with water (top panel). All syringe inoculations were performed at a concentration of 0.5×10⁵ CFU/ml; spray inoculations were performed at a concentration of 1×10⁹ CFU/ml. Results are shown as the mean (n = 6), including standard deviation.

Figure 5. Constitutively active AHA1 lines do not exhibit enhanced susceptibility to nonmotile P. syringae pv. tomato.

(A) Col 0 plants were inoculated by syringe infiltration with 0.5×10⁵ CFU/ml Pst DC3000 and the nonmotile flagellin mutant Pst DC3000 flaA ⁻. Bacterial growth was measured 3 d post-inoculation. (B) Constitutively active AHA1 mutants and corresponding wild-type Arabidopsis ecotypes were inoculated by syringe infiltration and spray inoculation with Pst DC3000 flaA ⁻ and subjected to growth curve analysis 3 d post-inoculation. All syringe inoculations were performed at a concentration of 0.5×10⁵ CFU/ml; spray inoculations were performed at a concentration of 1×10⁹ CFU/ml. Results are shown as the mean (n = 6), including standard deviation.

Figure 6. rin4 knockout lines do not re-open their stomata after exposure to virulent Pst DC3000.

Pst DC3000 induces stomatal closure at 1 h (A) and re-opening after 3 h (B) on Col 0 but not rin4 knockout lines. (C) The rpm1/rps2/rin4 mutant displays enhanced resistance to spray inoculation with Pst DC3000. 4-wk-old rpm1/rps2 and rpm1/rps2/rin4 plants were syringe infiltrated or spray inoculated with Pst DC3000 and leaves were subjected to growth curve analyses 4 d post-inoculation. Bacterial growth curve results are shown as the mean (n = 6), including standard deviation. These experiments are representative of at least three independent replicates. In this and all other figures, results are shown as the mean (n = 50–80) stomata ±SEM and statistical differences were detected with a two-tailed t-test.

Figure 7. RIN4 is expressed in guard cells.

Guard cell protoplasts (GCPs) were purified from Col 0 leaves and visually inspected for purity by light microscopy. Half of the GCP sample was used for RNA extraction and half for total protein extraction. (A) RNA was isolated from entire Arabidopsis leaves or GCPs and subjected to RT-PCR. RIN4 mRNA is highly expressed in guard cells. The expression of phosphoenolpyruvate carboxylase 2 (ATPPC2, At2g42600), which has low-level expression in guard cells and high-level expression in mesophyll cells served a control for guard cell protoplast purity . Actin (AtACT2) served as a loading control. (B) Anti-RIN4 immunoblots detected RIN4 protein expression in both Col 0 leaf tissue and GCPs. Thirty µg of total protein extract was loaded per lane. (C) RNA samples from (A) were subjected to RT-PCR to detect the expression of additional innate immune signaling components. EDS1, PAD4, RPS2, NDR1, EFR, and CERK1 transcript levels were detected in GCPs and leaf tissue after a 28-cycle amplification. +, RT-PCR, −, no-RT control.

Figure 8. AHA1 constitutively active mutant lines are insensitive to PTI-mediated stomatal closure.

Stomatal apertures were measured in epidermal peels of wild-type Col 0 and Ler, as well as the AHA1 mutants ost2-2D and ost2-1D after incubation with water or 1×10⁸ CFU/ml Pst DC3000 for 1 h (A) and 3 h (B). (C) Arabidopsis epidermal peels were floated on MES buffer containing the flagellin peptide Flg22 (5 µmol/l) and LPS 100 ng/µl). Stomatal apertures were measured after 3h. MES served as a control.

Figure 9. Model of PAMP-induced stomatal closure.

RIN4 acts in concert with AHA1 and/or AHA2 to regulate stomatal apertures in response to pathogen attack during PTI. (A) Virulent pathogens are able to overcome PTI and induce stomata to re-open 3 h after pathogen perception. Activation of the PM H⁺-ATPase can lead to hyperpolarization of the plasma membrane and subsequent induction of inward K⁺ channels. These events lead to an increase in guard cell turgor and stomatal opening. (B) RIN4 is a negative regulator of plant innate immunity. In resistant genotypes, pathogens are not able to overcome PTI and stomata remain closed after pathogen perception. Pathogen PAMPs are detected by pattern recognition receptors (PRRs) and the induction of PTI induces stomatal closure. Posttranslational modification of RIN4 (elimination or possibly phosphorylation) inhibits the association between RIN4 and AHA1/AHA2, resulting in inactivation of the PM H⁺-ATPase.