Date on Senior Honors Thesis

5-2022

Document Type

Senior Honors Thesis

Degree Name

B.S.

Department

Biology

Degree Program

College of Arts and Sciences

Author's Keywords

genetics; fungi; electron transport chain; aox

Abstract

Sporisorium reilianum is a pathogenic basidiomycete fungus with two formae speciales, each capable of infecting corn (SRZ) or sorghum (SRS), respectively. This fungus is also a dimorphic variety, meaning it can switch between its haploid, yeast-like sporidia and diploid teliospore stages over the course of its life cycle (Schirawski). When S. reilianum is found in a haploid state and conditions are favorable, it will mate with a compatible non-self mating type to begin filamentous growth and proliferation in the plant host (Zhao). S. relianum, like most fungi, utilizes the four classical components of the electron transport chain to produce the high energy molecule, adenosine triphosphate (ATP), via oxidative phosphorylation in the mitochondrion (Horne). Some fungal species such as Ustilago maydis, a sister species to S. reilianum, possess additional components to this process, e.g., alternative oxidase (Aox), which serves as an alternate route of electron transfer to produce basal levels of ATP if the classical electron transport chain were compromised (O. G. Juarez). Using bioinformatics, Aox was previously identified in S. reilianum by the Perlin lab (Culver). The focus of the following experiments was on the characterization of Aox and the potential effects on pathogenicity and growth it may have in SRZ. Additionally, the investigation of potential differential expression of the gene encoding Aox during two energetically distinct stages of the fungus’ life cycle was of interest. This study further confirmed the presence of an alternative oxidase as part of the ETC of SRZ. The activity of this protein renders respiration in SRZ antimycin-resistant by providing an alternate terminal point for electron transport if the classical cytochrome c pathway is compromised. The alternative oxidase of SRZ was determined to play a role in the fungus’ pathogenic stage, as its absence significantly decreases mean disease severity. Differential expression of Aox was made evident in distinct stages of the fungal life cycle and is in concordance with their specific metabolic needs.

Lay Summary

Sporisorium reilianum is a fungal species that can be infectious to corn (SRZ) or sorghum (SRS). Fungi use an organelle called the mitochondria to manufacture energy for survival. The significance of the chemical reactions within mitochondria (oxidative phosphorylation using the electron transport chain or ETC) make it a target for drug and immune responses. The focus of this study was on identifying and characterizing an alternative component, alternative oxidase (Aox), of the ETC that SRZ has evolved to contain. Components like Aox may arise to provide an alternative means to manufacture the primary energy product of the ETC, ATP, if the original components lost function. Here, we aimed to investigate if the presence of Aox has any other impacts on the growth or infection ability of SRZ. This was achieved by scoring corn plant infections. These infections were caused by wildtype SRZ strains containing Aox and mutant SRZ strains lacking Aox. Differences in growth and survival were observed between fungal cells treated with inhibitors for the classical ETC, Aox alone, and ETC and Aox in combination. Because Aox results in lower amounts of ATP, we also wanted to investigate whether SRZ might use Aox to its advantage in reducing the productivity of aerobic respiration to limit accumulation of reactive oxygen species during the teliospore stage of its lifecycle. Teliospores do not require as much energy as other cell types, so we wondered if SRZ, like another fungal species Blastocladiella emersonii, preferentially expresses the gene for Aox to this end. This would be determined by quantifying the mRNA transcripts for Aox present between haploid cells (high energy demands) and teliospores (low energy demands). We confirmed that Aox is present in SRZ and that its absence decreases the frequency of severe infection types. Additionally, we found that there is nearly a 6-fold increase in Aox expression in teliospores compared to haploid cells.

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