W342F Mutation in CCaMK Enhances Its Affinity to Calmodulin But Compromises Its Role in Supporting Root Nodule Symbiosis in Medicago truncatula.

The calcium/calmodulin-dependent protein kinase (CCaMK) is regulated by free Ca2+ and Ca2+-loaded calmodulin. This dual binding is believed to be involved in its regulation and associated physiological functions, although direct experimental evidence for this is lacking. Here we document that site-directed mutations in the calmodulin-binding domain of CCaMK alters its binding capacity to calmodulin, providing an effective approach to study how calmodulin regulates CCaMK in terms of kinase activity and regulation of rhizobial symbiosis in Medicago truncatula. We observed that mutating the tryptophan at position 342 to phenylalanine (W342F) markedly increased the calmodulin-binding capability of the mutant. The mutant CCaMK underwent autophosphorylation and catalyzed substrate phosphorylation in the absence of calcium and calmodulin. When the mutant W342F was expressed in ccamk-1 roots, the transgenic roots exhibited an altered nodulation phenotype. These results indicate that altering the calmodulin-binding domain of CCaMK could generate a constitutively activated kinase with a negative role in the physiological function of CCaMK.

Calcium signatures and signaling events orchestrate plant-microbe interactions.

Calcium (Ca2+) acts as an essential second messenger connecting the perception of microbe signals to the establishment of appropriate immune and symbiotic responses in plants. Accumulating evidence suggests that plants distinguish different microorganisms through plasma membrane-localized pattern recognition receptors. The particular recognition events are encoded into Ca2+ signatures, which are sensed by diverse intracellular Ca2+ binding proteins. The Ca2+ signatures are eventually decoded to distinct downstream responses through transcriptional reprogramming of the defense or symbiosis-related genes. Recent observations further reveal that Ca2+-mediated signaling is also involved in negative regulation of plant immunity. This review is intended as an overview of Ca2+ signaling during immunity and symbiosis, including Ca2+ responses in the nucleus and cytosol.

Autophosphorylation of calcium/calmodulin-dependent protein kinase (CCaMK) at S343 or S344 generates an intramolecular interaction blocking the CaM-binding.

The Ca2+ and Ca2+/calmodulin-dependent protein kinase (CCaMK) is an important effector protein of Ca2+/calmodulin-mediated signaling, and in legumes, it is a critical regulator of plant-rhizobia and mycorrhizal symbioses. CCaMK contains a kinase domain, a calmodulin-binding/autoinhibitory domain and a visinin-like domain. Previous studies revealed the presence of 2 phosphorylation sites, S343 and S344, in the calmodulin-binding domain. Mutations at these sites affected the kinase activity and downstream rhizobium and mycorrhizal symbioses, which highlighted the importance of these residues in regulating protein activity. This addendum further clarifies the regulation of CCaMK by identifying an intramolecular interaction between residue(s) in the kinase domain and phosphorylation sites S343 and S344. This interaction turns off the substrate phosphorylation capacity of CCaMK.