The calcium-dependent protein kinase CPK28 is targeted by the ubiquitin ligases ATL31 and ATL6 for proteasome-mediated degradation to fine-tune immune signaling in Arabidopsis

Abstract

Immune responses are triggered when pattern recognition receptors (PRRs) recognize microbial molecular patterns. The Arabidopsis (Arabidopsis thaliana) receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE1 (BIK1) acts as a signaling hub of plant immunity. BIK1 homeostasis is maintained by a regulatory module in which CALCIUM-DEPENDENT PROTEIN KINASE28 (CPK28) regulates BIK1 turnover via the activities of two E3 ligases. Immune-induced alternative splicing of CPK28 attenuates CPK28 function. However, it remained unknown whether CPK28 is under proteasomal control. Here, we demonstrate that CPK28 undergoes ubiquitination and 26S proteasome-mediated degradation, which is enhanced by flagellin treatment. Two closely related ubiquitin ligases, ARABIDOPSIS TÓXICOS EN LEVADURA31 (ATL31) and ATL6, specifically interact with CPK28 at the plasma membrane; this association is enhanced by flagellin elicitation. ATL31/6 directly ubiquitinate CPK28, resulting in its proteasomal degradation. Furthermore, ATL31/6 promote the stability of BIK1 by mediating CPK28 degradation. Consequently, ATL31/6 positively regulate BIK1-mediated immunity. Our findings reveal another mechanism for attenuating CPK28 function to maintain BIK1 homeostasis and enhance immune responses.

Phosphorylation status of CPK28 affects its ubiquitination and protein stability

“…Recombinant GST‐CPK28/CPK28 D188A /BIK1/BIK1 K105/K106A and HIS‐CPK28/CPK28 D188A /CPK28 S318A /CPK28 T76A /CPK28 D188/S318A /CPK28 D188/T76A /PUB25/PBL1/PBL11 fusion proteins were purified from Escherichia coli as described previously (Liu et al ., 2022). For in vitro kinase assay, kinase reactions were performed in 30 μl kinase buffer (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 0.2 mM ATP, 100 μM CaCl 2 (when CPK28 was the kinase), or 5 mM EDTA (when BIK1/PBL1/PBL11 were the kinases)).…”

“…Reactive oxygen species production was measured as described previously (Li et al ., 2014; Liu et al ., 2022). Leaf disks were collected from the leaves of 30‐d‐old cpk28 , atl31 atl6 , atl31 atl6 cpk28 plants.…”

Shotgun Proteomics as a Powerful Tool for the Study of the Proteomes of Plants, Their Pathogens, and Plant–Pathogen Interactions

“…In addition to phosphorylation, large-scale profiling of other PTMs such as acetylation and ubiquitination during plant–pathogen interactions is becoming more common. In rice [ 123 , 124 ] and Arabidopsis [ 125 , 126 , 127 ], hundreds of ubiquitin-modified proteins were identified during the plant immune responses. The acetylome profiling of maize in response to Cochliobolus carbonum infection confirmed the hyperacetylation of several proteins, including chromatin remodeling enzymes and transcription factors [ 128 ].…”

Ubiquitination of Receptorsomes, Frontline of Plant Immunity

“…Interestingly, flg22 treatment enhanced another pair of closely related ubiquitin ligases, ARABIDOPSIS TOXICOS EN LEVADURA 6 (ATL6) and ATL31 associated with CPK28 in the PM, further mediating CPK28 degradation ( Figure 1 ). ATL31/6 positively regulates immune responses through the ubiquitination of CPK28 and relieving the CPK28-mediated negative regulation of BIK1 ( Figure 1 ) [ 94 ]. Additionally, CPK28 undergoes intermolecular autophosphorylation on Ser318, which is required for the activation of CPK28 under low intracellular [Ca 2+ ] to prevent the initiation of an immune response in the absence of infection [ 95 ].…”