Cla4A, a Novel Regulator of Gene Expression Networks Required for Asexual and Insect-Pathogenic Lifecycles of Beauveria bassiana.

Cla4, an orthologous p21-activated kinase crucial for non-entomopathogenic fungal lifestyles, has two paralogs (Cla4A/B) functionally unknown in hypocrealean entomopathogens. Here, we report a regulatory role of Cla4A in gene expression networks of Beauveria bassiana required for asexual and entomopathogenic lifecycles while Cla4B is functionally redundant. The deletion of cla4A resulted in severe growth defects, reduced stress tolerance, delayed conidiation, altered conidiation mode, impaired conidial quality, and abolished pathogenicity through cuticular penetration, contrasting with no phenotype affected by cla4B deletion. In ∆cla4A, 5288 dysregulated genes were associated with phenotypic defects, which were restored by targeted gene complementation. Among those, 3699 genes were downregulated, including more than 1300 abolished at the transcriptomic level. Hundreds of those downregulated genes were involved in the regulation of transcription, translation, and post-translational modifications and the organization and function of the nuclear chromosome, chromatin, and protein-DNA complex. DNA-binding elements in promoter regions of 130 dysregulated genes were predicted to be targeted by Cla4A domains. Samples of purified Cla4A extract were proven to bind promoter DNAs of 12 predicted genes involved in multiple stress-responsive pathways. Therefore, Cla4A acts as a novel regulator of genomic expression and stability and mediates gene expression networks required for insect-pathogenic fungal adaptations to the host and environment.

Characterization of BbKlf1 as a novel transcription factor vital for asexual and infection cycles of Beauveria bassiana.

The large family of C2H2-type zinc finger transcription factors (TFs) comprise the Kruppel-like factors (KLFs) that evolved relatively late in eukaryotes but remain unexplored in filamentous fungi. Here, we report that an orthologue (BbKlf1) of yeast Klf1 mediating cell wall integrity (CWI) is a wide-spectrum TF evidently localized in nucleus and cytoplasm in Beauveria bassiana. BbKlf1 features conserved domains and multiple DNA-binding motifs predicted to bind multiple promoter DNA fragments of target genes across asexual developmental and stress-responsive pathways. Despite limited impact on normal colony growth, deletion of Bbklf1 resulted in impaired CWI and hypersensitivity to Congo red-induced cell wall stress. Also, the deletion mutant was severely compromised in tolerance to oxidative and osmotic stresses, hyphal septation and differentiation, conidiation capacity (reduced by 95%), conidial quality (viability and hydrocarbon epitope pattern) and virulence. Importantly, these phenotypes correlated well with sharply repressed or nearly abolished expressions of those genes required for or involved in chitin biosynthesis, antioxidant activity, cell division and differentiation, aerial conidiation and conidial maturation. These findings indicate an essentiality of BbKlf1 for the asexual and insect-pathogenic lifecycles of B. bassiana and a novel scenario much beyond the yeast orthologue-mediated CWI, suggesting important roles of its orthologues in filamentous fungi.

Genome-Wide Insight into Profound Effect of Carbon Catabolite Repressor (Cre1) on the Insect-Pathogenic Lifecycle of Beauveriabassiana.

Carbon catabolite repression (CCR) is critical for the preferential utilization of glucose derived from environmental carbon sources and regulated by carbon catabolite repressor A (Cre1/CreA) in filamentous fungi. However, a role of Cre1-mediated CCR in insect-pathogenic fungal utilization of host nutrients during normal cuticle infection (NCI) and hemocoel colonization remains explored insufficiently. Here, we report an indispensability of Cre1 for Beauveriabassiana's utilization of nutrients in insect integument and hemocoel. Deletion of cre1 resulted in severe defects in radial growth on various media, hypersensitivity to oxidative stress, abolished pathogenicity via NCI or intrahemocoel injection (cuticle-bypassing infection) but no change in conidial hydrophobicity and adherence to insect cuticle. Markedly reduced biomass accumulation in the Δcre1 cultures was directly causative of severe defect in aerial conidiation and reduced secretion of various cuticle-degrading enzymes. The majority (1117) of 1881 dysregulated genes identified from the Δcre1 versus wild-type cultures were significantly downregulated, leading to substantial repression of many enriched function terms and pathways, particularly those involved in carbon and nitrogen metabolisms, cuticle degradation, antioxidant response, cellular transport and homeostasis, and direct/indirect gene mediation. These findings offer a novel insight into profound effect of Cre1 on the insect-pathogenic lifestyle of B. bassiana.