A PAMP‐triggered MAPK cascade inhibits phosphatidylinositol 4,5‐bisphosphate production by PIP5K6 in Arabidopsis thaliana

Abstract

Summary The phosphoinositide kinase PIP5K6 has recently been identified as a target for the mitogen‐activated protein kinase (MAPK) MPK6. Phosphorylation of PIP5K6 inhibited the production of phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2), impacting membrane trafficking and cell expansion in pollen tubes. Here, we analyzed whether MPK6 regulated PIP5K6 in vegetative Arabidopsis cells in response to the pathogen‐associated molecular pattern (PAMP) flg22. Promoter‐β‐glucuronidase analyses and quantitative real‐time reverse transcription polymerase chain reaction data show PIP5K6 expressed throughout Arabidopsis tissues. Upon flg22 treatment of transgenic protoplasts, the PIP5K6 protein was phosphorylated, and this modification was reduced for a PIP5K6 variant lacking MPK6‐targeted residues, or in protoplasts from mpk6 mutants. Upon flg22 treatment of Arabidopsis plants, phosphoinositide levels mildly decreased and a fluorescent reporter for PtdIns(4,5)P2 displayed reduced plasma membrane association, contrasting with phosphoinositide increases reported for abiotic stress responses. Flg22 treatment and chemical induction of the upstream MAPK kinase, MKK5, decreased phosphatidylinositol 4‐phosphate 5‐kinase activity in mesophyll protoplasts, indicating that the flg22‐activated MAPK cascade limited PtdIns(4,5)P2 production. PIP5K6 expression or PIP5K6 protein abundance changed only marginally upon flg22 treatment, consistent with post‐translational control of PIP5K6 activity. PtdIns(4,5)P2‐dependent endocytosis of FM 4‐64, PIN2 and the NADPH‐oxidase RbohD were reduced upon flg22 treatment or MKK5 induction. Reduced RbohD‐endocytosis was correlated with enhanced ROS production. We conclude that MPK6‐mediated phosphorylation of PIP5K6 limits the production of a functional PtdIns(4,5)P2 pool upon PAMP perception.

Functions of Anionic Lipids in Plants

“…The role of post-translational modifications (e.g. phosphorylation) has been spear headed by Ingo Heilmann's group and this is an area of research that is likely to expand in the future (33,80). 3.…”

Coordinated Localization and Antagonistic Function of NtPLC3 and PI4P 5-Kinases in the Subapical Plasma Membrane of Tobacco Pollen Tubes

“…Cells were ruptured by ultrasound on ice, using five 60 s bursts in a Sonifier Cell Disruptor B15 (Branson Dietzenbach, Germany) at 50% power and 50% impulse settings. The protein extracts were cleared by centrifugation and stored at −20 • C. Proteins were analyzed by immunodetection as previously described [42,43].…”

Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes

“…Besides a distinction between PtdIns(4,5)P 2 pools based on spatial separation, which may or may not be detectable by microscopy, it is possible that diffusely localizing PI4P 5-kinases may display a patchy pattern of activated and inactivated states, resulting in islands of active PI4P 5-kinases within a population of possibly not activated proteins that is diffusely distributed. This hypothesis is based on recent reports that the type B PI4P 5-kinase AtPIP5K6 is regulated in its activity by the mitogen-activated protein kinase (MAPK) MPK6, and that MPK6mediated phosphorylation does not impair membrane association of PIP5K6 (Hempel et al, 2017;Menzel et al, 2019).…”