Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production

Abstract

The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition of several microbe-associated molecular patterns (MAMPs) in plants. The Arabidopsis (Arabidopsis thaliana) PI-PLC gene family is composed of nine members. Previous studies suggested a role for PLC2 in MAMP-triggered immunity, as it is rapidly phosphorylated in vivo upon treatment with the bacterial MAMP flg22. Here, we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silence PLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficient bacterial strain Pseudomonas syringae pv tomato (Pst) DC3000 hrcC^- and to the nonadapted pea (Pisum sativum) powdery mildew Erysiphe pisi However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent (Pst DC3000 AvrRPM1) P. syringae strains, conserving typical hypersensitive response features. In response to flg22, PLC2-silenced plants maintain wild-type mitogen-activated protein kinase activation and PHI1, WRKY33, and FRK1 immune marker gene expression but have reduced reactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation of ROS upon flg22 treatment is compromised in the PLC2-defficient plants, suggesting an effect of PLC2 in a branch of MAMP-triggered immunity and nonhost resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidase RBOHD, suggesting its potential regulation by PLC2.

Figure 1.

PLC2 silencing by artificial microRNAs in Arabidopsis. A, Total RNA was isolated from leaves of 4- to 5-week-old Columbia-0 (Col-0) or PLC2-silenced plants (T4 homozygous lines amiR-PLC2-4, amiR-PLC2-7, and amiR-PLC2-11). Relative transcript levels of PLC2 were determined by RT-qPCR. Transcript levels were normalized to ACT2. Error bars represent sd of three to nine individual plants. Different letters indicate significant differences (ANOVA for unbalanced samples, posthoc Tukey-Kramer test at P < 0.001). B, PLC2 protein levels were analyzed by western blot using anti-AtPLC2 antibody in leaves of 4- to 5-week-old Col-0, empty vector (EV), and amiR-PLC2-11 and amiR-PLC2-4 independent silenced lines. Ponceau S staining of Rubisco subunit L was included as a loading control.

Figure 2.

Growth of P. syringae and E. pisi in Arabidopsis PLC2-silenced plants. Wild-type (Col-0), empty vector (EV), and PLC2-silenced lines (amiR-PLC2-11 and amiR-PLC2-4) were used. A, PLC2-silenced plants are more susceptible to the Pst DC3000 hrcC⁻ mutant. Bacteria were inoculated by spray at OD₆₀₀ = 0.1, and the number of colony-forming units (CFU) per cm² of leaf extracts was determined. Data from three biological replicates each with three technical replicates were averaged (n = 9), and ANOVA was performed considering each replicate as a factor. Error bars represents se. Different letters indicate significant differences between genotypes (ANOVA, P < 0.001, posthoc Tukey’s test). dpi, Days postinoculation. B, PLC2-silenced plants do not show increased susceptibility to the Pst DC3000 hrcC⁻ mutant when the bacteria are syringe inoculated into the leaf apoplast. Bacterial suspension was inoculated at OD₆₀₀ = 0.0001, and the number of CFU per cm² of leaf extracts was determined. Data from three biological replicates each with three technical replicates were averaged (n = 9), and ANOVA was performed considering each replicate as a factor. No significant differences were observed between genotypes. Error bars represent se. C and D, PLC2-silenced lines showed no differences in susceptibility to virulent (C) and avirulent (D) Pst DC3000 infections. Pst DC3000 (virulent) and Pst DC3000:AvrRpm1 (avirulent) were inoculated by infiltration at OD₆₀₀ = 0.0002, and CFU per cm² of leaf was calculated. A representative experiment of four biological replicates is depicted. No significant differences were observed regarding the EV control according to Student’s t test (P < 0.05). E, PLC2-silenced plants are more susceptible to the nonadapted pea powdery mildew E. pisi. The penetration rate at 3 d after inoculation was calculated as the percentage of successful penetration of at least 50 germinated spores on three independent leaves. Error bars represent se. Different letters indicate significant differences (multiple comparison using one-way ANOVA, posthoc Tukey’s test at P < 0.05). One representative experiment of four biologically independent replicates is depicted.

Figure 3.

PLC2-silenced plants exhibit impaired flg22-induced callose deposition. Leaves from 4- to 5-week-old Col-0 or amiR-PLC2 plants were infiltrated with 1 µm flg22 or water as a control and incubated for 18 h, and callose deposition was measured as dots per area. Six different microscopic areas (1 mm²) were taken per leaf. Two different leaves per individual were analyzed. Three independent plants were analyzed per line per experiment. Three independent experiments were performed. Error bars represent se. Different letters indicate significant differences (ANOVA for unbalanced samples, posthoc Tukey-Kramer test at P < 0.001). EV, Empty vector.

Figure 4.

PLC2-silenced plants exhibit impaired flg22-induced stomatal closure. Epidermal peels from Col-0 and PLC2-silenced plants were incubated in opening buffer under light for 3 h. The peels were treated with water, 1 µm flg22, 50 µm ABA, or 50 µm ABA + 1 µm flg22 for 1 h. The results show means of 90 to 120 stomata measured from three independent experiments. Error bars represent se. Different letters denote statistical differences (ANOVA for unbalanced samples, posthoc Tukey-Kramer test at P < 0.05). EV, Empty vector.

Figure 5.

PLC2-silenced plants exhibit impaired flg22-induced oxidative burst. The production of ROS was measured with a luminol-based assay in Col-0 or amiR-PLC2 plants. A, Leaf discs from 4- to 5-week-old plants were incubated with 100 nm flg22, and the luminescence was measured every 1 min for 30 min and expressed as relative light units (RLU). A representative experiment is shown using wild-type (Col-0) and a PLC2-silenced line (amiR-PLC2-11) plants. B, Total ROS production was calculated integrating the areas under the curves and referring to the Col-0 wild type treated with flg22 as 100%. Averages of four independent experiments are shown. Error bars represent se. Asterisks indicate statistically significant differences compared with the flg22-treated Col-0 plant (ANOVA for unbalanced samples, multiple comparisons versus control group posthoc Dunnett’s method at P < 0.05). EV, Empty vector.

Figure 6.

PLC2 associates with RBOHD. Coimmunoprecipitation of PLC2 and ROBHD was performed in stable transgenic Arabidopsis seedlings (T3) expressing FLAG-RBOHD (pRBOHD:FLAG-RBOHD) treated (+) or not (−) with 1 µm flg22 for 15 min. Total protein extracts (Input) were subjected to immunoprecipitation (IP) with anti-FLAG beads followed by immunoblot analysis with anti-PLC2 (α-PLC2) and anti-FLAG (α-FLAG) antibodies as indicated. Protein extracts of Col-0 plants were used as negative controls. Anit-BRI1 (α-BRI1) antibodies were used as plasma membrane protein not associated with RBOHD. CBB, Coomassie Brilliant Blue. These experiments were performed three times with similar results.