Phosphorylation dynamics in a flg22-induced, heterotrimeric G-protein dependent signaling network in Arabidopsis thaliana reveals a candidate PP2A phosphatase involved in AtRGS1 trafficking

Abstract

flg22 is a 22 amino peptide released from bacterial flagellin a Microbe Associated Molecular 51 Pattern ( that is recognized by the plant cell as a signal indicating that bacteria are present. On its own, flg22 initiates a rapid increase in cytoplasmic calcium, extracellular reactive oxygen species, and activation of a Mitogen Activated Protein Kinase (cascade all of which are activated within 15 minutes after the cell perceives flg22. Here we show a massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome within this 15 minute duration in wildtype and a mutant deficient in G protein coupled signaling Integration of phosphoproteome with protein protein interactome data followed by network topology analyses discovered that many of the flg22 induced phosphoproteome changes fall on proteins that comprise the G protein interactome and on the most highly populated hubs of the immunity network approximately 95% of the phosphorylation changes in the G protein interactome depend on a functional heterotrimeric G protein complex some occur on proteins that interact directly with components of G coupled signal transduction. One of these is ATBα, a substrate recognition sub-unit of the PP2A Ser/Thr phosphatase and an interactor to Arabidopsis thaliana REGULATOR OF G SIGNALING 1 protein (a 7 transmembrane spanning modulator of the nucleotide binding state of the core G protein complex. AtRGS1 is phosphorylated by BAK1, a component of the flg22 receptor, to initiate AtRGS1 endocytosis. A null mutation of ATB α confer s high 67 basal endocytosis of AtRGS1 suggesting sustained phosphorylated status. Loss of ATB α confers 68 trait s associated with loss of AtRGS1. Because the basal level of AtRGS1 is lower in the atbα null mutant in a proteasome dependent manner we propose that phosphorylation dependent endocytosis of AtRGS1 is part of a mechanism to degrade AtRGS1 which then sustains activation of the 71 G protein complex Thus, the role of ATB α is now established as a central component of phosphorylation dependent regulation of system dynamics in innate immunity

G-Protein Phosphorylation: Aspects of Binding Specificity and Function in the Plant Kingdom

“…Additionally, the inactivation of three cluster sites (AtRGS1 S428/435/436A ) completely abolishes flg22-induced internalization but only partially affects the glucose-mediated internalization of AtRGS1 [ 55 ]. Furthermore, a phosphatase is also required for AtRGS1 stability, and its presence reduces the in vitro identified phosphorylation by the WNKs [ 60 ]. These findings suggest an animal-like mechanism in which the phosphorylation patterns are the key for recruitment and posterior signal distinction and transduction.…”

“…Among these residues, Ser13 responds to osmotic stress, and Ser71/169 respond to nitrate starvation [ 45 , 46 , 48 , 49 , 50 ]. Ser13 and Ser38 display increased phosphorylation signals 15 min after flg22 exposure, while serine residues 75, 185, 190, 191, 194, and 198 show decreased signals after 3 or 15 min of exposure [ 60 ]. In addition to the four mutated N-terminal serine residues, XLG2 is differentially phosphorylated at the helical domain (Ser530) by flg22 [ 51 ].…”

“…XLG3 has nine N-terminal tissue-specific phosphoresidues under normal conditions [ 26 ]. Like XLG2-Ser530 phosphorylation, Ser506 of the XLG3 helical domain is differentially phosphorylated under ABA, sucrose, mannitol, and short cold treatments [ 42 , 51 , 52 , 53 ], and it is detected with a reduced signal in the first minutes of flg22 exposure [ 60 ].…”

“…Like in XLG2, an AGG3 phosphosite is identified at Ser37 in response to end-of-day conditions and ionizing radiation ( Table 1 ) [ 30 , 44 ]. Finally, the same site displays an enhanced phosphorylation signal after 15 min of anti-bacterial immunity elicitation [ 60 ].…”

“…β-dimerized AGG2 (purple) binds to the XLGs in order to regulate gravitropism, sugar responses, and root development [ 72 , 86 ]. Phosphorylation occurs in vivo at the N-terminal portions of AGB1, AGG2, and AGG3 [ 26 , 42 , 60 ]. Beta-gamma complex models were created using AlphaFold2, and top-ranked models were selected for analysis [ 35 ].…”