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.
Highlights
The best characterized plant pathogen-associated molecular pattern (PAMP) perception system is the recognition of bacterial flagellin and its elicitor-active epitope, flg22, by the Arabidopsis pattern recognition receptors (PRRs) FLS2
plasma membrane (PM) fractions of pooled 15N- or 14N-labeled treatment and control samples were extracted by two-phase partitioning and subsequently detergent-resistant membranes (DRMs) were isolated by Triton X-100 treatment and sucrose gradient centrifugation
Functional category (FC); Arabidopsis Genome Initiative code (AGI code); average fold-change; number of TM domains based on the consensus predicted by ARAMEMNON (TM, [17]); experimental evidence for PM association (PM, [17,18,19,20]); transcriptionally co-expressed with FLS2 [46], number indicates rank of co-expressed gene according to ATTED (ATTED); elevated transcript levels in response to flg22 treatment (flg22 up, [14, 21]); phosphorylated after flg22 treatment (P flg22, [15, 22]); mutants of according genes were analyzed for flg22 responsiveness in this study (MA); flg22-induced reactive oxygen species (ROS) (ROS). enriched, dephosphorylated, phosphorylation below the significance threshold (ߛ), not germinated, no ROS ϭ 1, weak ROS ϭ 2, wild-type ROS ϭ 3, ROS higher than wild-type ϭ 4
Summary
Metabolic Labeling of Suspension Cell Cultures—Full metabolic 15N/14N-labeling of Arabidopsis thaliana (Col-0) suspension cell cultures was carried out as described [10]. Protein abundance ratios were converted into log values and were normalized to the median log ratio of all proteins identified in the non-treated sample (0 min) Those proteins for which intensity ratios were obtained in both of the paired reciprocal experimental sets were considered for further analysis. I.e. with high ratios in one of the reciprocal experiments and low ratios in the other, lie away from this diagonal Using this information, for each data point the p value was determined by a 2-tailed t-distribution [11], and a multiple testing correction was applied to the whole data set using the false discovery rate (FDR) method introduced by Benjamini and Hochberg [13]. Immunoblot Analysis and Bioassays—Immunoblot analysis of FLS2 in PM-derived DRMs and mutant seedlings, the oxidative burst assay, mitogen-activated protein kinase (MAPK) activity assay, measurement of stomatal aperture, callose (aniline blue) and cell death (trypan blue) staining as well as the bacterial infection assays are described in detail in the supplemental Methods
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