Divergent Roles of CPK28 in Immune Homeostasis Across Land Plants

Calcium-dependent protein kinases decode cellular calcium transients and play diverse roles in plant growth and stress responses. In Arabidopsis thaliana, AtCPK28 contributes to immune homeostasis by phosphorylating subgroup IV plant U-box proteins which target the key immune receptor-like cytoplasmic kinase AtBIK1 for turnover. Here we present functional analysis of MpCPK28 in the liverwort Marchantia polymorpha. We identify the subgroup IV plant U-box protein MpPUB20e as a substrate of MpCPK28 and we provide evidence that MpPBLa (the functional ortholog of AtBIK1) undergoes proteasomal degradation and that it can be ubiquitinated by MpPUB20e. Interestingly, while loss of CPK28 function in multiple angiosperm species results in enhanced immune signaling, we find that Marchantia Mpcpk28 mutant alleles do not display enhanced immune-triggered production of reactive oxygen species or resistance to two pathogens. However, transgenic expression of MpCPK28 is able to restore function in Arabidopsis cpk28-1 mutants, suggesting latent functional conservation of MpCPK28. Furthermore, while AtCPK28-mediated phosphorylation of Thr95/94 on AtPUB25/26 is known to contribute to their activation, we could not observe a functional role for the equivalent residue Thr122 on MpPUB20e. Taken together, our results suggest that post-translational fine-tuning by CPK28 is likely to have refined the ‘PUB-BIK1’ module in the vascular plant lineages.