Phosphorylation of Plant Ferredoxin-like Protein Is Required for Intensifying PAMP-Triggered Immunity in Arabidopsis thaliana.

The immune response triggered when plant cell surface receptors recognize pathogen-associated molecular patterns (PAMPs) is known as PAMP-triggered immunity (PTI). Several studies have demonstrated that extracellular plant ferredoxin-like protein (PFLP) can enhance PTI signaling, thereby conferring resistance to bacterial diseases in various plants. The C-terminal casein kinase II (CK2) phosphorylation region of PFLP is essential for strengthening PTI. However, whether phosphorylation at this site directly enhances PTI signaling and consequently increases plant disease resistance remains unclear. To investigate this, site-directed mutagenesis was used to generate PFLPT90A, a non-phosphorylatable mutant, and PFLPT90D, a phospho-mimetic mutant, for functional analysis. Based on the experimental results, none of the recombinant proteins were able to enhance the hypersensitive response induced by the HrpN protein or increase resistance to the soft rot pathogen Pectobacterium carotovorum subsp. carotovorum ECC17. These findings suggest that phosphorylation at the T90 residue might be essential for PFLP-mediated enhancement of plant immune responses, implying that this post-translational modification is likely required for its disease resistance function in planta. To further explore the relationship between PFLP phosphorylation and endogenous CK2, the Arabidopsis insertion mutant cka2 and the complemented line CKA2R were analyzed under treatment with flg22_Pst from Pseudomonas syringae pv. tomato. The effects of PFLP on the hypersensitive response, rapid oxidative burst, callose deposition, and susceptibility to soft rot confirmed that CK2 is required for these immune responses. Furthermore, expression analysis of PTI-related genes FRK1 and WRKY22/29 in the mitogen-activated protein kinase (MAPK) signaling pathway demonstrated that CK2 is necessary for PFLP to enhance flg22_Pst-induced immune signaling. Taken together, these findings suggest that PFLP enhances A. thaliana resistance to bacterial soft rot primarily by promoting the MAPK signaling pathway triggered by PAMP recognition, with CK2-mediated phosphorylation being essential for its function.