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

Watkins, J. M.; Jones, A. M.; Walley, J.; Clark, N. M.; Urano, D.; Mishra, B.; Mukhtar, M. S.; Oliveira, C.; Song, G.; Brachova, L.; Seifert, C.; Mitchell, M.; Reis, P.

doi:10.1101/2021.12.06.471472

bioRxiv · 10.1101/2021.12.06.471472

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

Watkins, J. M.·Jones, A. M.·Walley, J.·Clark, N. M.·Urano, D.·Mishra, B.·Mukhtar, M. S.·Oliveira, C.·Song, G.·Brachova, L.·Seifert, C.·Mitchell, M.·Reis, P.

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Abstract

flg22 is a 22-amino peptide released from bacterial flagellin, a Microbe-Associated Molecular Pattern (MAMP) 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 (MAPK) 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 ATB, a substrate-recognition sub-unit of the PP2A Ser/Thr phosphatase and an interactor to Arabidopsis thaliana REGULATOR OF G SIGNALING 1 protein (AtRGS1), 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 confers high basal endocytosis of AtRGS1, suggesting sustained phosphorylated status. Loss of ATB confers traits associated with loss of AtRGS1. Because the basal level of AtRGS1 is lower in the atb 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 G protein complex. Thus, the role of ATB is now established as a central component of phosphorylation-dependent regulation of system dynamics in innate immunity.

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Watkins, J. M., Jones, A. M., Walley, J., Clark, N. M., Urano, D., Mishra, B., Mukhtar, M. S., Oliveira, C., Song, G., Brachova, L., Seifert, C., Mitchell, M., Reis, P.. 2021-12-07. Phosphorylation dynamics in a flg22 induced, heterotrimeric G protein dependent signaling network in Arabidopsis thaliana reveals a candidate PP2A phosphatase involved in AtRGS1 trafficking. https://doi.org/10.1101/2021.12.06.471472

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