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Characterization and Functional Analysis of a New Calcium/Calmodulin-Dependent Protein Kinase (CaMK1) in the Citrus Pathogenic Fungus Penicillium italicum

ABSTRACT: Calcium (Ca2+)/calmodulin-dependent protein kinases (CaMKs) act as a class of crucial elements in Ca2+-signal transduction pathways that regulate fungal growth, sporulation, virulence, and environmental stress tolerance. However, little is known about the function of such protein kinase in phytopathogenic Penicillium species. In the present study, a new CaMK gene from the citrus pathogenic fungus P. italicum, designated PiCaMK1, was cloned and functionally characterized by gene knockout and transcriptome analysis. The open reading frame of PiCaMK1 is 1209 bp in full length, which encodes 402 amino acid residues (putative molecular weight ~45.2 KD) with the highest homologous (~96.3%) to the P. expansum CaMK. The knockout mutant ΔPiCaMK1 showed a significant reduction in vegetative growth, conidiation, and virulence (i.e., to induce blue mold decay on citrus fruit). ΔPiCaMK1 was less sensitive to NaCl- or KCl-induced salinity stress and less resistant to mannitol-induced osmotic stress, indicating the functional involvement of PiCaMK1 in such environmental stress tolerance. In contrast, the PiCaMK1-complemented strain ΔPiCaMK1COM can restore all the defective phenotypes. Transcriptome analysis revealed that knockout of PiCaMK1 down-regulated expression of the genes involved in DNA replication and repair, cell cycle, meiosis, pyrimidine and purine metabolisms, and MAPK signaling pathway. Our results suggested the critical role of PiCaMK1 in regulating multiple physical and cellular processes of citrus postharvest pathogen P. italicum, including growth, conidiation, virulence, and environmental stress tolerance.

SUBMITTER: Li G

PROVIDER: S-EPMC9323541 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The calcium calmodulin-dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium-calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as the first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich the discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

Dataset Information

Characterization and Functional Analysis of a New Calcium/Calmodulin-Dependent Protein Kinase (CaMK1) in the Citrus Pathogenic Fungus Penicillium italicum

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