Nuclear dynamics of Arabidopsis calcium-dependent protein kinases in effector-triggered immunity

Plants have evolved sophisticated innate immune systems to protect themselves from potential microbial invasions. Recognition of pathogen-derived virulence effector proteins is mediated by plant resistance (R) proteins and elicits potent defense responses, collectively termed as effector-triggered immunity (ETI). It has long been known that ETI is often accompanied with the increase of cytosolic Ca(2+) levels. We recently identified six closely related calcium-dependent protein kinases (CPKs) in Arabidopsis that orchestrate bifurcate ETI signaling via distinct substrate specificity and subcellular dynamics. In particular, the activation of CPK4, 5, 6 and 11 phosphorylates a specific subgroup of WRKY transcription factors to regulate transcriptional reprogramming crucial for restriction of pathogen growth. Upon ETI activation, a significant portion of CPK5 re-localizes to nucleus where it interacts and phosphorylates WRKY8, 28 and 48. Mass spectrometry analysis identified several conserved residues, including T247/T248 in WRKY48 and T199 in WRKY28 as the phosphorylation sites by CPKs. Here we reported that mutation of T198/T199 into alanine (TT198AA) in WRKY28 completely abolished its phosphorylation by CPK4 and 11. The importance of nuclear localization of CPK5 was further demonstrated by that CPK5 fused with nuclear export signal abolished its synergistic effect with WRKY8, 28 and 48 on the activation of defense gene. In contrast, effector AvrRpt2 likely functions in the cytoplasm to activate the transcriptional reprogramming of defense genes, consistent with the plasma membrane localization of its RPS2 receptor. Our data established WRKYs as bona fide substrates of CPKs and provided a framework for the study of CPK-WRKY cascade in diverse biological processes. Our results also demonstrated that the nuclear localization and subcellular dynamics of CPKs are essential to relay distinct ETI signaling events.