PAMP (pathogen-associated molecular pattern)-induced changes in plasma membrane compartmentalization reveal novel components of plant immunity

Abstract

Plasma membrane compartmentalization spatiotemporally regulates cell-autonomous immune signaling in animal cells. To elucidate immediate early protein dynamics at the plant plasma membrane in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin (flg22) we employed quantitative mass spectrometric analysis on detergent-resistant membranes (DRMs) of Arabidopsis thaliana suspension cells. This approach revealed rapid and profound changes in DRM protein composition following PAMP treatment, prominently affecting proton ATPases and receptor-like kinases, including the flagellin receptor FLS2. We employed reverse genetics to address a potential contribution of a subset of these proteins in flg22-triggered cellular responses. Mutants of three candidates (DET3, AHA1, FER) exhibited a conspicuous defect in the PAMP-triggered accumulation of reactive oxygen species. In addition, these mutants showed altered mitogen-activated protein kinase (MAPK) activation, a defect in PAMP-triggered stomatal closure as well as altered bacterial infection phenotypes, which revealed three novel players in elicitor-dependent oxidative burst control and innate immunity. Our data provide evidence for dynamic elicitor-induced changes in the membrane compartmentalization of PAMP signaling components.

FIGURE 1.

Schematic representation of the experimental setup. A and B, parental ¹⁴N and ¹⁵N cultures were split for reciprocal treatment. A, flg22 treatment was compared with flg22Δ2 treatment (in reciprocal pairs). B, flg22 as well as flg22Δ2 treatments were compared with untreated cells (including reciprocal pairs). Samples for DRM extraction and subsequent ratiometric protein quantification were taken before treatment (0 min) as well as 5 and 15 min after peptide addition. Dotted lines indicate reciprocal sample pairs that were extracted and analyzed together. C, label-free quantification.

FIGURE 2.

Classification of proteins exhibiting significant redistribution into detergent-resistant membranes after flg22 elicitation (“responding”) and proteins not responding to flg22 treatment. Functional categories were assigned according to MapMan (47) and manually advanced for some proteins as described in supplemental Methods.

FIGURE 3.

FLS2 immunoblot analysis. Immunoblot showing reduced abundance of FLS2 in DSM and increased abundance of FLS2 in DRM fractions of flg22-treated cells. Cell cultures were treated with flg22 peptide for 10 min as described (+flg22) or remained untreated (−flg22). Subsequently, cell material was homogenized and DRMs were isolated. Total protein extracts of treated and untreated cells were used as a control to demonstrate unaltered overall FLS2 abundance, and Coomassie staining was employed to demonstrate equal loading.

FIGURE 4.

fer, ost2-1D, and det3 are affected in flg22-induced ROS production and MAPK activity. A–D, oxidative burst in response to 100 nm flg22 in fer (A), ost2-1D (B), det3 (C) and respective wild types was indirectly measured as relative light units (RLU). D, oxidative burst in response to 100 nm flg22 in Col-0 seedlings treated with either 5 μm concanamycin A or with respective amounts of DMSO (Control). Note the different levels of ROS production in wild-type seedlings. Relative changes were similar in all experiments. Error bars represent standard deviation of six (A), ten (B), eleven (C), and six (D) independent samples measured in a single experiment. The experiment was performed four (A) or five (B–D) times with similar results. E, MAPK activity in seedlings in response to 100 nm flg22 was determined in a time course experiment by immunoblot analysis. Ponceau staining served as a loading control. The experiment was repeated once yielding similar results.

FIGURE 5.

fer displays aberrant cell death. fer, FER-GFP(fer), and respective wild-type rosette leaves were infiltrated with 10 mm MgCl₂ and cell death was revealed by Trypan blue staining. Representative micrographs of untreated leaves or leaves 24 h after treatment are shown. Bar, 500 μm.

FIGURE 6.

det3 is hypersusceptible to bacterial infection and fer displays reduced bacterial proliferation. Arabidopsis det3 and fls2 (A), fer and FER-GFP(fer) (B) as well as respective wild-type plants were challenged with PtoDC3000ΔavrPtoΔavrPtoB. Depicted are box-plot diagrams representing the statistical distribution of the data. Statistical analysis (ANOVA and subsequent post-hoc test by Tukey's HSD) was done using R software. Thick lines indicate the median, boxes designate the interquartile range, whiskers specify the whole data range, and dots represent outliers. A, experiment was repeated three times, the box plot summarizes three representative data sets. B, experiment was repeated twice, the box plot includes all three data sets. Letters indicate significant differences at the level of p < 0.05. C, stomatal aperture in first true leaves of 2-week-old seedlings was determined following mock or flg22 treatment (3 μm, 2 h) as the ratio of the width to length of 20–63 stomata per genotype. Asterisks indicate a statistically significant difference (p < 0.01) between mock and flg22 treatment (Student's t test). The experiment was repeated once with similar results.