Project description:Despite their importance, plant MAP kinase targets are still poorly elucidated. Here, the specific in vivo interaction of an ethylene response factor (ERF104) with the Arabidopsis MAP kinase, MPK6, is shown by fluorescence resonance energy transfer. The interaction, which is lost within minutes after treatment with the flagellin-derived flg22 peptide, is dependent on both MPK6 kinase activity and rapid ethylene signaling initiated downstream of MPK6 activation. ERF104 is an MPK6 substrate and phosphorylation site mutations affected its stability. ERF104 activates promoters with GCC elements. This was evident from microarray data of overexpressing transgenic plants, where promoters of up regulated genes contain GCC motifs and chromatin immunoprecipitation showing ERF104 association with PDF1.2 promoter. The ERF104 overexpressor did not affect biotrophic bacteria proliferation but was more susceptible to necrotrophic Botrytis cinerea. Microarray performed with erf104 or mpk6 revealed only a limited number of flg22-induced genes that require these elements - possibly as a result of functional redundancies. Thus, ERF104 phosphorylation by MPK6, in concert with ethylene signaling induced by pathogen-derived molecules, modulates defense in Arabidopsis.
Project description:Despite their importance, plant MAP kinase targets are still poorly elucidated. Here, the specific in vivo interaction of an ethylene response factor (ERF104) with the Arabidopsis MAP kinase, MPK6, is shown by fluorescence resonance energy transfer. The interaction, which is lost within minutes after treatment with the flagellin-derived flg22 peptide, is dependent on both MPK6 kinase activity and rapid ethylene signaling initiated downstream of MPK6 activation. ERF104 is an MPK6 substrate and phosphorylation site mutations affected its stability. ERF104 activates promoters with GCC elements. This was evident from microarray data of overexpressing transgenic plants, where promoters of up regulated genes contain GCC motifs and chromatin immunoprecipitation showing ERF104 association with PDF1.2 promoter. The ERF104 overexpressor did not affect biotrophic bacteria proliferation but was more susceptible to necrotrophic Botrytis cinerea. Microarray performed with erf104 or mpk6 revealed only a limited number of flg22-induced genes that require these elements - possibly as a; result of functional redundancies. Thus, ERF104 phosphorylation by MPK6, in concert with ethylene signaling induced by pathogen-derived molecules, modulates defense in Arabidopsis. Experiment Overall Design: Leaves of six week old Col-0, erf104, mpk6 and 35S::ERF104 plants were infiltrated with 1µM Flg22 or water and harvested four hours later. Total RNA was isolated and processed according to the Affymetrix protocol for biotin-labelled cRNA and hybridized to the Affymetrix ATH1 chip. The data Experiment Overall Design: was analyzed with Genespring GX 7.3.1 software (Agilent) with the following parameters: Filter on Flags for present or marginal in 50% of all considered experiments, Filter for reliable differentially expressed genes based on volcano plot (one way ANOVA p-value <0.05 and >3-fold change in expression). For the flg22 experiments, analysis for each genotype was separately performed and a composite list of flg22-regulated genes compiled â with the aim of including genes that may be differentially regulated in the genotype. Global expression profile was visualized by k-means clustering and condition tree (Genespring).
Project description:Oligosaccharyl transferase (OST) protein complex mediates the N-linked glycosylation of substrate proteins in the endoplasmic reticulum (ER), which regulates stability, activity and localization of its substrates. Although many OST substrate proteins have been identified, the physiological role of OST complex remains incompletely understood. Here, we show that OST complex in C. elegans is crucial for ER protein homeostasis and enhances resistance to pathogenic bacteria, Pseudomonas aeruginosa (PA14), via immune-regulatory PMK-1/p38 MAP kinase.
Project description:The experiments were performed to monitor dynamics in WRKY transcription factor abundancies upon treatment with flg22, a peptide derived from the bacterial flagella. Mock-treated or WT seedlings treated for 2 h with flg22 were used to prepare crude nuclear lysates. Then pull downs were performed with an anti-all-WRKY antiserum to enrich for WRKY transcription factor proteins prior to MS.
Project description:VQ-motif containing proteins (VQs) control gene expression via interaction with transcription factors. MVQ1 and MVQ8 are members of a subgroup of VQs that are phosphorylated by stress-responsive MAPKs and might play a role in the transcriptional regulation of plant defence responses. To investigate the possible roles played by MVQ1 or MVQ8, the flg22-regulated transcriptomes of an MVQ1-overexpression line, the mvq1 mutant, an MVQ8-overexpression line and WT (Col-0) plants were analysed.
Project description:Seeds establish dormancy to delay germination until the arrival of a favorable growing season. In this study, we identify a fate switch comprised of the MKK3–MPK7 kinase cascade and the ethylene response factor ERF4 that is responsible for the seed state transition from dormancy to germination. We show that dormancy-breaking factors activate the MKK3–MPK7 module, which affects the expression of some α-EXPANSIN (EXPA) genes to control seed dormancy. Furthermore, we identify a direct downstream substrate of this module, ERF4, which suppresses the expression of these EXPAs by directly binding to the GCC boxes in their exon regions. The activated MKK3–MPK7 module phosphorylates ERF4, leading to its rapid degradation and thereby releasing its inhibitory effect on the expression of these EXPAs. Collectively, our work identifies a signaling chain consisting of protein phosphorylation, degradation, and gene transcription, by which the germination promoters within the embryo sense and are activated by germination signals from ambient conditions.
Project description:In this research a high-throughput RNA sequencing based transcriptome analysis technique (RNA-Seq) was used to evaluate differentially expressed genes (DEGs) in the wild type Arabidopsis seedling in response to flg22, a well-known microbe-associated molecular patterns (MAMP), and AtPep1, a well-known peptide representing an endogenous damage-associated molecular patterns (DAMP). The results of our study revealed that 1895 (1634 up-regulated and 261 down-regulated) and 2271 (1706 up-regulated and 565 down-regulated) significant differentially expressed genes in response to flg22 and AtPep1 treatment, respectively. Among significant DEGs, we observed that a number of hitherto overlooked genes have been found to be induced upon treatment with either flg22 or with AtPep1, indicating their possible involvement in innate immunity. Here, we characterized two of them, namely PP2-B13 and ACLP1. PP2-B13 contains an F-box domain and shows similarity to carbohydrate binding proteins. ACLP1 is a protein of unknown function with highest similarity to actin cross linking proteins and includes a fascin domain. Using qPCR, we verified that the genes encoding PP2-B13, and ACLP1 were highly induced upon treatment of leaf disks with flg22. We obtained T-DNA insertion mutants and generated homozygous mutant lines. None of the mutants showed a phenotype in the absence of infection. pp2-b13 and aclp1 mutants showed an increased susceptibility to infection by the virulent pathogen Pseudomomas syringae pv tomato mutant hrcC-, as evidenced by an increased growth of the pathogen in planta. Further we present evidence that aclp1 was deficient in ethylene production upon flg22 treatment, while pp2-b13, was deficient in ROS production. In conclusion, the products of these genes contribute to plant immunity against bacterial pathogens, although there is currently no clue for their mechanism of action. The results from this research provide new information to a better understanding of the immune system in Arabidopsis.