Fikry, C. | bioRxiv Science

bioRxiv2026

Regulation of polarity during morphological transitions in Aureobasidium pullulans

Many fungi are able to switch between different morphologies by regulating the behavior of the cytoskeleton. In turn, the cytoskeleton is regulated by Rho-family GTPases, whose differing spatiotemporal patterns of activity lead to different modes of growth. The polymorphic fungus Aureobasidium pullulans alternates between multiple growth modes, including multibudding, isotropic, and hyphal growth. Here, we describe hyphal growth and the yeast-to-hyphal transition in this organism, with a focus on cell polarity. Yeast cells switched between cycles of budding and isotropic expansion in a variable manner prior to initiating hyphal extension. Initial hyphal extension proceeded in a stop-and-start manner with frequent pauses, before switching to continuous growth. Hyphal branching occurred from the sides of hyphae, and septation at multiple sites occurred in more mature hyphae. Previous work showed that either of two Rho-family GTPases, Rac1 and Cdc42, were sufficient promote budding. However, while Rac1 was also able to drive hyphal extension without Cdc42, Cdc42 could not promote hyphal growth without Rac1. Our findings provide the basis for an investigation of how polarity components are re-purposed for different fungal growth modalities.

cell biology↗

bioRxiv2025

Pooled CRISPRi screening reveals fungal-specific vulnerabilities across environments and genetic backgrounds

The rising rate of drug-resistant fungal infections and the emergence of fungal pathogens with intrinsic resistance phenotypes are a growing concern. The close evolutionary distance between mammals and fungi complicates the design of new antifungals and increases the chances of toxic off-target effects. As such, antifungal drug development usually focuses on fungal-specific proteins when considering potential new targets. Ideal drug targets should mediate essential cell processes and be highly sensitive to inhibition. Targeted gene repression can serve as a model for drug-mediated inhibition and for determining the dosage-sensitivity profile of genes of interest. In the fungal pathogen Candida albicans, classical approaches for gene repression can be labour-intensive and limited to one genetic background due to low throughput. Here, we adapt pooled CRISPRi screening in C. albicans for the first time and exploit this technique for large-scale functional genomic analysis. Through pooled CRISPRi screening, we test the repression sensitivity of over a hundred essential genes conserved in fungi but absent in humans, and successfully identify highly dosage-sensitive genes across multiple cell components and pathways. By extending our analysis to ten diverse environmental conditions, we show how the environment influences dosage-sensitivity profiles. Finally, we extend our experiments to two clinical drug-resistant C. albicans strain backgrounds and demonstrate that many of the fitness defects we observed are conserved in resistant clinical isolates. Together, our results highlight a set of genes that are highly dosage-sensitive across different genetic and environmental contexts, making them attractive targets for further investigation. By facilitating rapid, efficient large-scale functional genomics assays across diverse genetic backgrounds, CRISPRi pooled screening will open new frontiers in C. albicans biology.

microbiology↗