Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity
Tuesday, 2021/01/26 | 08:27:03
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Naganand Rayapuram, Mai Jarad, Hanna M. Alhoraibi, Jean Bigeard, Aala A. Abulfaraj, Ronny Völz, Kiruthiga Gayathri Mariappan, Marilia Almeida-Trapp, Maria Schlöffel, Emmanuelle Lastrucci, Ludovic Bonhomme, Andrea A. Gust, Axel Mithöfer, Stefan T. Arold, Delphine Pflieger, and Heribert Hirt
PNAS January 19, 2021 118 (3) e2004670118.
SignificanceMitogen-activated protein kinases (MAPKs) function in all eukaryotes in signaling extracellular stimuli to intracellular responses and ultimately link them to chromatin events by targeting transcription factors and chromatin remodeling complexes. In plants, MAPKs play crucial roles in immunity, development, and stress responses, but so far no attempts have been made to identify phosphorylation of chromatin-associated proteins. By using a phosphoproteomic approach on MAPK mutants, we identified a number of chromatin-associated MAPK substrates and characterize an AT-hook motif containing nuclear localized (AHL) DNA-binding protein 13 in plant immunity and demonstrate that phosphorylation regulates AHL13 protein stability and, in turn, its function in response to pathogens. AbstractIn many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis. The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.
See: https://www.pnas.org/content/118/3/e2004670118
Figure 1: Phosphoproteomics experimental workflow and data analysis. (A) Schematic representation of the phosphoproteomics experimental workflow and data analysis for the identification of MAPK substrates. Phosphopeptides were enriched using IMAC from WT plants and mpk3, mpk4, and mpk6 mutants treated with and without flg22 and analyzed by LC-MS/MS. Database searches were carried out using the program Mascot and phosphosite localization was ascertained using FragMixer. Relative quantification was carried out using MassChroQ, followed by statistical analysis of quantitative data to highlight sequences whose abundance was significantly modulated. The list of identified phosphopeptides was subjected to motif analysis using motif-x. (B) Number of phosphorylation sites per peptide. (C) Distribution of phosphorylated amino acids. (D) Motifs enriched by motif-x. (E) Principal component analysis of the identified phosphopeptides across the eight conditions. (F) Principal component analysis of a few candidate proteins are highlighted. (G) Venn diagram showing the overlap of candidate proteins identified in this study with other relevant studies (34, 35, 37). |
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