Investigating Potential Regulators of Cell Morphology in Aureobasidium pullulans Using Targeted Gene Deletions

Abstract

Aureobasidium pullulans is a polyextremotolerant, ubiquitous fungus with pronounced morphological plasticity, enabling transitions between yeast-like and filamentous growth forms, and displays stress-induced melanisation. Although these morphological shifts are thought to contribute to ecological fitness and industrial performance, the genetic regulatory mechanisms underlying morphological plasticity in A. pullulans remain insufficiently studied and understood.In this study, a reverse genetic approach was used to investigate regulators of morphological plasticity in A. pullulans. Candidate genes were selected based on homology to well-characterised morphological regulators in other fungi, particularly Candida albicans and Saccharomyces cerevisiae. The selected genes represent different regulatory levels, including signal perception, intracellular signalling and transcription factors. Targeted gene deletions were generated by protoplast-mediated transformation combined with CRISPR/Cas9-assisted genome editing and the resulting strains were analysed under defined cultivation conditions to assess effects on morphology.Phenotypic characterisation under cultivation conditions designed to activate nutrient-responsive, stress-associated and morphogenetic signalling pathways revealed that several gene deletions led to pronounced changes in growth morphology and pigmentation. In particular, deletions of a putative Wor1-like transcription factor and a Hog1-like mitogen-activated protein kinase were associated with increased filamentous growth and/or melanisation. In contrast, deletion of several upstream sensors and transcriptional regulators resulted in only mild or condition-dependent phenotypic effects.Overall, this work provides first functional insights into genetic factors influencing morphology in A. pullulans. The results contribute to understanding a central trait of this extremotolerant fungus and establish a foundation for future mechanistic studies of morphogenetic regulation.