The BIR2/BIR3-Associated Phospholipase Dγ1 Negatively Regulates Plant Immunity

Abstract

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis (Arabidopsis thaliana) PLD family consists of 12 members, and for some of these, a specific function in resistance toward a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLDγ1, but not its close homologs PLDγ2 and PLDγ3, is specifically involved in plant immunity. Genetic inactivation of PLDγ1 resulted in increased resistance toward the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea As pldγ1 mutant plants responded with elevated levels of reactive oxygen species to MAMP treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pldγ1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are not likely the cause for the enhanced immunity in the pldγ1 line. Instead, the plasma-membrane-attached PLDγ1 protein colocalized and associated with the BAK1-INTERACTING RECEPTOR-LIKE KINASES BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLDγ1 and BIR2 was further promoted upon MAMP treatment. Hence, we propose that PLDγ1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3.

Figure 1.

Genetic inactivation of PLDγ1 results in increased resistance to bacterial infection. Wild-type (WT) plants or the indicated mutant lines for pldγ1-1, pldγ1-2, complementation lines 12-4 and 11-13 (pldγ1-1/35S:: PLDγ1-GFP, T4 generation), or bir2 and bir3 were infiltrated with 10⁴ cfu/mL of virulent Pseudomonas syringae pv. tomato DC3000. At 0 and 3 d postinoculation (dpi), bacterial growth was quantified by counting colony-forming units. Box plots show the minimum, first quartile, median, third quartile, and a maximum of log cfu/cm² leaf tissue (n = 4 for 0 dpi; n = 6 for 3 dpi). Lowercase letters indicate homogenous groups according to post hoc comparisons following one-way ANOVA (Tukey-Kramer multiple comparison analysis at a probability level of P < 0.05). The entire experiment was repeated three times with similar results.

Figure 2.

pldγ1-1 mutants are more resistant to B. cinerea infection. Leaves of 6-week-old plants were inoculated with 5 × 10⁶/mL B. cinerea spores. A, After 2 d, symptom development was documented and one representative leaf per line is shown. B, Lesion sizes were determined with a pixel-based approach and then calculated using a 1-cm² standard. Box plots show the minimum, first quartile, median, third quartile, and a maximum (n ≥ 12). Lowercase letters indicate homogenous groups according to post hoc comparisons following one-way ANOVA (Tukey-Kramer multiple comparison analysis at a probability level of P < 0.05). The experiment was performed three times with similar results. WT, Wild type.

Figure 3.

ROS levels are affected in pldγ1-1 mutants. Arabidopsis leaf pieces of the indicated mutant line were treated with 1 μm flg22 or water as a control, and ROS production was monitored over time. Shown are relative light units (RLU). Box plots show the minimum, first quartile, median, third quartile, and a maximum of peak values minus background values (n = 6). Water-treated samples had no detectable ROS production and are therefore not displayed in the figure. Lowercase letters indicate homogenous groups according to post hoc comparisons following one-way ANOVA (Dunnet’s multiple comparison analysis with the wild type [WT] as a control at a probability level of P < 0.05). The experiment was performed at least three times and one representative result is shown.

Figure 4.

PA levels are not affected by PLDγ1 depletion. Five-day-old seedlings of wild-type (WT), pldγ1-1, pldγ1-2, or bir2 lines were labeled with ³²P_i for 16 h and then treated for 15 min with either 1 μm flg22 (A) or 300 mm NaCl (B) or cell-free medium (control) as indicated. Lipids were extracted and separated by EtAc-TLC, and the radioactivity incorporated into PA was quantified by phosphoimaging (bottom). Data (top) represent the average ± sd of two to three biological replicates and are expressed in relation to the radioactivity of the total phospholipids. The experiment was repeated twice with similar results.

Figure 5.

PLDγ1 colocalizes with BIR2 and BIR3 at the PM. A, PLDγ1 was transiently expressed in N. benthamiana as GFP-fusion either alone or together with BIR2-RFP or BIR3-CFP in the presence of the p19 suppressor of silencing. Infiltration of p19 alone served as control. Fluorescence in epidermal cells was monitored at day 3 using confocal laser-scanning microscopy. B, Close-up view of the samples shown in A.

Figure 6.

PLDγ1-GFP associates with native BIR2, but not FLS2 or BAK1. Leaf material was harvested from pldγ1-1 plants, the complementation lines 12-4 and 11-13 (pldγ1-1/35S:: PLDγ1-GFP), or wild-type (WT) plants 5 min after treatment with 1 μm flg22 (+) or water (−). After protein extraction the proteins (input) were subjected to immunoprecipitation with GFP-beads (GFP-IP). Immunoprecipitated and copurified proteins were detected with GFP antibodies or specific antibodies against native proteins as indicated. Ponceau S Red-staining of the membrane served as a loading control. M indicates the marker band at 55 kD. The experiment was repeated with similar results. RBC, Ribulose-bis-phosphate-carboxylase large subunit.

Figure 7.

PLDγ1 can be found in complex with epitope-tagged BIR2 and BIR3. Western blot analysis of transiently expressed proteins in N. benthamiana 3 d after infiltration. Leaf material was harvested 5 min after treatment with 1 µm flg22 (+) or water (−). After protein extraction, the proteins were subjected to immunoprecipitation (IP) with GFP- or myc-affinity beads as indicated. For different immunoprecipitations within one experiment (A and D), the same source material was used. Immunoprecipitated and copurified proteins were detected with tag-specific antibodies as indicated. A and D, Coimmunoprecipitation of BIR2-myc (A) or BIR3-myc (D) and PLDγ1-GFP using myc-trap beads or GFP-trap beads, respectively. B and E, Copurification of BIR2-GFP (B) and BIR3-GFP (E) and PLDγ1-myc precipitated with GFP-trap beads. C and F, Copurification of BIR2-GFP (C) and BIR3-GFP (F) and PLDγ1-HA precipitated with GFP-trap beads. Numbers in the images indicate quantification of signal intensities. All experiments were repeated at least three times with similar results.