Cdc14 protein phosphatases play an important role in plant infection by several fungal pathogens. This and other properties of Cdc14 enzymes make them an intriguing target for development of new antifungal crop treatments. Active site architecture and substrate specificity of Cdc14 from the model fungus Saccharomyces cerevisiae (ScCdc14) are well-defined and unique among characterized phosphatases. Cdc14 appears absent from some model plants. However, the extent of conservation of Cdc14 sequence, structure, and specificity in fungal plant pathogens is unknown. We addressed this by performing a comprehensive phylogenetic analysis of the Cdc14 family and comparing the conservation of active site structure and specificity among a sampling of plant pathogen Cdc14 homologs. We show that Cdc14 was lost in the common ancestor of angiosperm plants but is ubiquitous in ascomycete and basidiomycete fungi. The unique substrate specificity of ScCdc14 was invariant in homologs from eight diverse species of dikarya, suggesting it is conserved across the lineage. A synthetic substrate mimetic inhibited diverse fungal Cdc14 homologs with similar low µM K i values, but had little effect on related phosphatases. Our results justify future exploration of Cdc14 as a broad spectrum antifungal target for plant protection. Plant pathogens pose a constant threat to agricultural productivity and global food security, with fungi and the fungal-like oomycetes being the most dangerous culprits 1-4. Despite the development of chemical pesticides and disease-resistant cultivars to curb crop infections over the past century, damage from fungal and other pathogens persists at nearly comparable levels 3. Estimates suggest more than 10% of the world agricultural harvest may be lost annually to fungal infections alone, equating to hundreds of billions of dollars and enough food to feed an estimated 600 million people 2-5. Post-harvest losses from fungal-induced spoilage and toxin accumulation further exacerbate the problem, especially in developing countries 6. A major challenge to effectively suppressing fungal crop diseases is the ability of fungi to rapidly develop resistance to pesticides and acquire mutations that counteract plant defenses in disease-resistant lines 2,3,7,8. Consequently, the continual battle against fungal pathogens requires a constant stream of new management strategies, including both the generation of new infection resistance mechanisms in crops along with identification of novel pesticide compounds and targets 1. The Cdc14 phosphatases, known best for roles in counteracting cyclin-dependent kinase activity during mitosis in model fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe 9,10 may be an attractive novel target for development of broad-acting antifungal agents. Deletion of the CDC14 gene in several plant pathogen species severely impairs virulence, demonstrating that Cdc14 function is important for host infection 11-13. Fusarium graminearum lacking CDC14 exhibited defective conidia and ascospore for...

The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. Here, we identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm) as the sole source of Cdc14 proliferated like the wild-type parent strain but exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a novel and conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reduced Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.

Cdc14 protein phosphatase is highly conserved across the eukaryotic kingdom, from single‐celled yeast and protozoa to multicellular organisms including mammals. In budding yeast, where it was first studied, Cdc14 is required for mitotic exit; however, this function is not widely conserved. In humans and mice, Cdc14A mutations cause deficiencies in hearing and male fertility due to defects in cilia formation and maintenance. There is evidence for Cdc14 localization to the basal bodies at the proximal end of cilia. The large number of cilia in the free‐living protozoan Tetrahymena thermophila make it an attractive model for molecular studies of cilia structure and function. Our goal is to determine the localization of Cdc14 isoforms to gain some insight into their cellular function and to test if Cdc14 cilia localization is broadly conserved. Interestingly, T. thermophilahas a larger number of Cdc14 isoforms than most organisms. Thus far, genes encoding three of the seven T. thermophilaCdc14 isoforms have been used to create C‐terminal gene fusions with YFP that are expressed in vivo. The constructs were inserted into the T. thermophilamacronuclear genome adjacent to the RPL29 gene and cell lines were selected due to the resulting conversion to cycloheximide resistance. Expression of the transgenes was regulated by a metallothionine promoter. Cells were examined via fluorescence microscopy in the presence and absence of cadmium. All three isoforms localize along ciliary rows and in the oral apparatus after induction during vegetative growth, consistent with basal body localization. No differences in localization have been noted between the three isoforms thus far. The presence of multiple isoforms with the same localization raises questions regarding the redundancy and/or functional specialization of Cdc14 in this highly ciliated organism.

The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires a small fraction of normal Cdc14 activity. It remains unclear what fungal Cdc14 functions require high Cdc14 activity. We identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm) as the sole source of Cdc14 exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation phenotypes previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reducing Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity promotes fungal cell wall integrity and, in C. albicans, is needed to orchestrate septation and hyphal differentiation, and for pathogenesis. Cdc14 may therefore be worth future exploration as an antifungal drug target.