RESUMO
The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Heterotriméricas de Ligação ao GTP , Proteínas RGS , Arabidopsis/metabolismo , Fosforilação , Proteínas de Arabidopsis/metabolismo , Proteoma/metabolismo , Proteínas RGS/química , Proteínas RGS/genética , Proteínas RGS/metabolismo , Transdução de Sinais , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Flagelina/farmacologia , Flagelina/metabolismo , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
Coupling assay for transposase-accessible chromatin sequencing (ATAC-seq) with microfluidic separation and cellular barcoding has emerged as a powerful approach to investigate chromatin accessibility of individual cells. Here, we define a protocol for constructing single-cell ATAC-seq libraries from maize seedling nuclei and the preliminary computational steps for assessing data quality. This protocol can be readily adapted to other plant species or tissues with minor changes to reveal chromatin accessibility variation among individual cells. For complete details on the use and execution of this protocol, please refer to Marand et al. (2021).