CBP60-DB: An AlphaFold-predicted plant kingdom-wide database of the CALMODULIN-BINDING PROTEIN 60 protein family with a novel structural clustering algorithm.

Molecular genetic analyses in the model species Arabidopsis thaliana have demonstrated the major roles of different CALMODULIN-BINDING PROTEIN 60 (CBP60) proteins in growth, stress signaling, and immune responses. Prominently, CBP60g and SARD1 are paralogous CBP60 transcription factors that regulate numerous components of the immune system, such as cell surface and intracellular immune receptors, MAP kinases, WRKY transcription factors, and biosynthetic enzymes for immunity-activating metabolites salicylic acid (SA) and N-hydroxypipecolic acid (NHP). However, their function, regulation, and diversification in most species remain unclear. Here, we have created CBP60-DB (https://cbp60db.wlu.ca/), a structural and bioinformatic database that comprehensively characterized 1052 CBP60 gene homologs (encoding 2376 unique transcripts and 1996 unique proteins) across 62 phylogenetically diverse genomes in the plant kingdom. We have employed deep learning-predicted structural analyses using AlphaFold2 and then generated dedicated web pages for all plant CBP60 proteins. Importantly, we have generated a novel clustering visualization algorithm to interrogate kingdom-wide structural similarities for more efficient inference of conserved functions across various plant taxa. Because well-characterized CBP60 proteins in Arabidopsis are known to be transcription factors with putative calmodulin-binding domains, we have integrated external bioinformatic resources to analyze protein domains and motifs. Collectively, we present a plant kingdom-wide identification of this important protein family in a user-friendly AlphaFold-anchored database, representing a novel and significant resource for the broader plant biology community.

Structural diversity and stress regulation of the plant immunity-associated CALMODULIN-BINDING PROTEIN 60 (CBP60) family of transcription factors in Solanum lycopersicum (tomato).

Cellular signaling generates calcium (Ca2+) ions, which are ubiquitous secondary messengers decoded by calcium-dependent protein kinases, calcineurins, calreticulin, calmodulins (CAMs), and CAM-binding proteins. Previous studies in the model plant Arabidopsis thaliana have shown the critical roles of the CAM-BINDING PROTEIN 60 (CBP60) protein family in plant growth, stress responses, and immunity. Certain CBP60 factors can regulate plant immune responses, like pattern-triggered immunity, effector-triggered immunity, and synthesis of major plant immune-activating metabolites salicylic acid (SA) and N-hydroxypipecolic acid (NHP). Although homologous CBP60 sequences have been identified in the plant kingdom, their function and regulation in most species remain unclear. In this paper, we specifically characterized 11 members of the CBP60 family in the agriculturally important crop tomato (Solanum lycopersicum). Protein sequence analyses revealed that three CBP60 homologs have the closest amino acid identity to Arabidopsis CBP60g and SARD1, master transcription factors involved in plant immunity. Strikingly, AlphaFold deep learning-assisted prediction of protein structures highlighted close structural similarity between these tomato and Arabidopsis CBP60 homologs. Conserved domain analyses revealed that they possess CAM-binding domains and DNA-binding domains, reflecting their potential involvement in linking Ca2+ signaling and transcriptional regulation in tomato plants. In terms of their gene expression profiles under biotic (Pseudomonas syringae pv. tomato DC3000 pathogen infection) and/or abiotic stress (warming temperatures), five tomato CBP60 genes were pathogen-responsive and temperature-sensitive, reminiscent of Arabidopsis CBP60g and SARD1. Overall, we present a genome-wide identification of the CBP60 gene/protein family in tomato plants, and we provide evidence on their regulation and potential function as Ca2+-sensing transcriptional regulators.

Salicylic Acid and N-Hydroxypipecolic Acid at the Fulcrum of the Plant Immunity-Growth Equilibrium.

Salicylic acid (SA) and N-hydroxypipecolic acid (NHP) are two central plant immune signals involved in both resistance at local sites of pathogen infection (basal resistance) and at distal uninfected sites after primary infection (systemic acquired resistance). Major discoveries and advances have led to deeper understanding of their biosynthesis and signaling during plant defense responses. In addition to their well-defined roles in immunity, recent research is emerging on their direct mechanistic impacts on plant growth and development. In this review, we will first provide an overview of how SA and NHP regulate local and systemic immune responses in plants. We will emphasize how these two signals are mutually potentiated and are convergent on multiple aspects-from biosynthesis to homeostasis, and from signaling to gene expression and phenotypic responses. We will then highlight how SA and NHP are emerging to be crucial regulators of the growth-defense balance, showcasing recent multi-faceted studies on their metabolism, receptor signaling and direct growth/development-related host targets. Overall, this article reflects current advances and provides future outlooks on SA/NHP biology and their functional significance as central signals for plant immunity and growth. Because global climate change will increasingly influence plant health and resilience, it is paramount to fundamentally understand how these two tightly linked plant signals are at the nexus of the growth-defense balance.