Investigating Auxin Biosynthesis in <i>Saccharomyces cerevisiae</i> Through Targeted CRISPR Knockouts

Abstract

Auxin, or indole-3-acetic acid, is primarily a plant growth hormone that regulates various aspects of plant development (Woodward and Bartel, 2005). While primarily associated with plants, some fungal species can also produce auxin. It is generally believed that fungi do not require auxin for their own survival and rather produce it merely to facilitate their interactions with other organisms, whether mutualistic or pathogenic. In mutualistic relationships, fungi can synthesize auxin to stimulate a plant to grow in certain beneficial ways and to facilitate the exchange of nutrients (Vadassery et al., 2008). On the contrary, pathogenic fungi are thought to produce auxin to divert nutrients from the plant and manipulate host physiology (Nagarajan et al., 2023). Auxin can also alter the morphology of the fungi themselves, including the induction of hyphal and pseudohyphal growth forms, which can enhance the ability of fungi to penetrate and colonize plant and animal tissues (Kunkel and Johnson, 2021). Despite the recognized roles of why fungi are producing auxin, understanding of the mechanisms underlying fungal auxin biosynthesis remains incomplete. Current evidence suggests that fungi do not produce auxin through the same pathway as plants. Plants primarily produce auxin through the indolepyruvate (IPA) pathway, but use enzymes of the YUCCA family, which are unique to plants. This pathway in plants is typically referred to as the TAA/YUC pathway instead of IPA (Reviewed by Gao et al., 2024). In Saccharomyces cerevisiae, the model fungal organism used in this work, auxin biosynthesis can be reduced to three main tryptophan-dependent pathways, although crossover between the pathways may be possible. These are indole-3-pyruvate (IPA), tryptamine (TAM), and indole-3-acetamide (IAM) pathways (KEGG PATHWAY: Tryptophan Metabolism - Saccharomyces Cerevisiae (Budding Yeast), n.d.). Seven genes thought to be involved in auxin biosynthesis in S. cerevisiae have been identified. These are aldehyde dehydrogenases ALD2, ALD3, ALD5 and HFD1, aromatic aminotransferase ARO8, nitrilase NIT1, and amidase AMD2. To investigate the roles these known genes play in fungal auxin biosynthesis, they were mutated using the CRISPR/Cas9 genome editing system in combinations of three genes, with the goal of creating knockout mutants. These strains were created in a background containing a genetically encoded auxin biosensor, which measures auxin in correlation with the degradation of a fluorescent protein. All but one multi-gene mutant strain had lower levels of biosensor response compared to the wildtype biosensor strain. One triple gene mutant strain repeatedly failed to produce colonies. This transformation would have mutated ARO8, NIT1, and AMD2, targeting key steps in each of the three proposed auxin biosynthesis pathways. To explore this further, a smaller library of single and double gene knockout mutants of these three genes was created in the biosensor parent strain as well as a compatible mating type strain. This allowed the creation of heterozygous mutants via mating and measurements of auxin accumulation in these heterozygous diploid strains in comparison to the haploid knockout strains. To date, all attempted transformations and matings have produced colonies, a few of which have been tested for auxin accumulation, but a larger screening with all strains has not yet been completed. Two aro8Δnit1Δamd2Δ colonies produced with this method have contained each gene knockout confirmed with sequencing or gel electrophoresis and are now awaiting whole genome sequencing. This research project aims to inform fundamental questions surrounding fungal auxin biosynthesis, focusing on investigating the roles of known enzymes within these pathways. This initial analysis has helped uncover the contribution of each of these pathways and known genes to auxin biosynthesis and will allow future research possibly uncovering unknown genes and regulatory mechanisms within these pathways. Advancing our understanding of these processes could establish a foundation for future applications, such as the development of fungal biostimulants to enhance plant growth. Additionally, identifying key enzymatic steps for auxin biosynthesis may reveal novel targets for antifungals, particularly if these pathways are linked to fungal fitness or virulence.