Brunetti, Craig

Crop losses due to pathogens, pests, and weeds account for 20–40% of global production, with fungal pathogens responsible for the most significant yield reductions and economic impact. The diseases caused by fungi spread through dormant spores, which protect its genetic material under adverse conditions. Dormancy is maintained until favorable germination conditions are met. Despite their importance in the fungal lifecycle, the molecular transitions from dormancy to germination remain poorly understood. The research presented uses the basidiomycete Ustilago maydis, the causal agent of Common Smut of Corn, to investigate fungal spore dormancy and germination. It aims to 1) identify the molecular transitions and stages of teliospore germination and 2) the roles of RNA helicases during teliospore germination. RNA-seq and respiration analyses were used to propose teliospore germination stages and a microdissection technique was developed for studying these stages. Transcriptomic analysis identified patterns of gene transcript level changes during germination, with GO term enrichment identifying genes involved in cell morphogenesis, metabolism, and RNA metabolism. Several RNA helicases were identified with potential roles during dormancy and germination. Previous work in the Saville Laboratory proposed that mRNAs are stored as dsRNA in dormant teliospores. I hypothesized that RNA helicases function to make these mRNAs available for translation upon germination. Forty-six RNA helicases were identified in U. maydis, and 28 RNA helicases were proposed to have roles in growth, pathogenesis, stress response, and teliospore dormancy and germination. The RNA helicases udbp3 and uded1 were selected for functional analysis by creating mutant strains. The results suggest that udbp3 negatively regulates osmotic stress response, potentially modulating stress-responsive genes during dormancy. The altered uded1 expression in mutant strains leads to slow and polarized growth and dsRNA formation. This suggests uded1 represses translation by stabilizing sense/antisense transcripts in dormant spores and then reactivates translation during germination. These findings increase our understanding of the molecular events during teliospore germination and offer insights into factors contributing to disease progression in fungal plant pathogens.

Author Keywords: gene expression, genome annotation, RNA helicases, RNA-seq, teliospore germination, Ustilago maydis

Cytokinins (CKs) are adenine derivative molecules that are present in all kingdoms of life. CKs are known to have a role in cell growth and development in plants. However, the role of CKs in vertebrate systems is not well understood. Frog virus 3 (FV3) is a type species of the Iridioviridae family, genus Ranavirus. FV3 is a major contributor to the amphibian population decline in North America. In this study, we demonstrate that concurrent and pretreatment of 20 µM of either N6-isopentyldenine (iP), N6-ispoentyladenosine (iPR), N6-furfurladenine/kinetin, and N6-furfurladenosine/kinetin riboside (KR) decreased FV3 replication. To understand the mechanism of inhibition, we assessed morphological changes in host cell nuclei to assess the effect of CKs on infected nuclei. Our results show that infection with FV3 and 20µM treatment of iP or iPR reduced nuclei size. These results are the first to reveal insight into the potential mechanism in which FV3 replication is inhibited by iP and iPR.  

Author Keywords: cytokinins, frog virus 3

Giardia intestinalis is a parasitic protozoan that possesses a flavohemoglobin (gFlHb), an enzyme that plays a role in the detoxification of reactive nitrogen species (RNS) and reactive oxygen species (ROS) via its nitric oxide dioxygenase (NOD) activity as well as its NADH-oxidase activity. This enzyme is a potential target for imidazole-based antigiardial drugs that act as ligands of the iron within its heme cofactor. In this work, two rapid and relatively inexpensive assays, the colorimetric Griess assay and a fluorescence assay, were adapted, optimized, and implemented to screen for flavohemoglobin inhibitors in parallel studies that compared the response of gFlHb to that of Hmp (Escherichia coli flavohemoglobin) when a group of six different imidazole-based compounds was tested. These assays displayed isotype selectivity, showing how the different drugs elicited different responses from the two enzymes. Comparative results for gFlHb and Hmp revealed that bulkier compounds elicited higher inhibition of Hmp, while smaller compounds resulted in better inhibition of gFlHb, which might be explained by the presence of different amino acid residues in the active sites of the enzymes, with two large amino acid sequence inserts being a unique feature of gFlHb, thus blocking the active site from being reached and blocked by larger compounds.

Author Keywords: 2.3-diaminonaphthalene, Flavohemoglobin, Giardia intestinalis, Griess Assay, imidazole-based drugs, nitric oxide detoxification

The TATA-binding protein (TBP) is a key regulator of eukaryotic transcription initiation. The TBP homolog from Giardia intestinalis (gTBP) is highly divergent among all TBPs; notably lacking three of the four phenylalanine residues to unwind double- stranded DNA. I show that gTBP preferentially binds to single-stranded DNA (ssDNA) in two modes based on sequence and protein concentration. The proposed A mode likely represents multimeric binding of gTBP to ssDNA with four or more consecutive guanine bases. The B mode involves monomeric binding utilizing the structural properties of the ssDNA. To demonstrate this, I developed a novel technique using base stacking energy potentials to approximate the per-nucleotide flexibility of ssDNA. I also attempted to create a polynomial regression model to predict binding; however, further work is required to improve accuracy. Overall, this thesis presents a new perspective on eukaryotic transcription regulation based on the discovery of unconventional binding between gTBP and ssDNA.

Author Keywords: computer modelling, DNA binding protein, DNA structure, DNA transcription, general transcription factor (GTF), parasite

Flavohemoglobins are enzymes primarily implicated in nitrosative stress due to their high nitric oxide (NO) dioxygenase activity and transcriptional upregulation in response to NO. Giardia intestinalis assemblages A, B, and E possess flavohemoglobins (gFlHb) that may function beyond their NO dioxygenase activity, potentially contributing to oxidative stress regulation, as transcriptional profiling revealed that peroxide also induces gFlHb expression. This study investigates gFlHb's NADH oxidase activity in the absence of NO, structural interactions with lipids, and response to reactive oxygen species. Minor differences in NADH oxidase activity among assemblages were observed, and their susceptibilities to inhibition were assessed to evaluate gFlHb as a potential therapeutic target against Giardia infection. Under aerobic conditions, we observed that gFlHb generates hydrogen peroxide, a surprising finding suggesting a self-regulating feedback mechanism involving reactive oxygen species and heme degradation. These findings provide new insight into the role of flavohemoglobins in microaerotolerant parasites like Giardia.

Author Keywords: flavohemoglobin, Giardia intestinalis, heme, hydrogen peroxide, NADH, oxidative stress

Giardia intestinalis is a parasitic protozoan that possesses a flavohemoglobin (gFlHb), an enzyme that plays a role in the detoxification of reactive nitrogen species (RNS) and reactive oxygen species (ROS) via its nitric oxide dioxygenase (NOD) activity as well as its NADH-oxidase activity. This enzyme is a potential target for imidazole-based antigiardial drugs that act as ligands of the iron within its heme cofactor. In this work, two rapid and relatively inexpensive assays, the colorimetric Griess assay and a fluorescence assay, were adapted, optimized, and implemented to screen for flavohemoglobin inhibitors in parallel studies that compared the response of gFlHb to that of Hmp (Escherichia coli flavohemoglobin) when a group of six different imidazole-based compounds was tested. These assays displayed isotype selectivity, showing how the different drugs elicited different responses from the two enzymes. Comparative results for gFlHb and Hmp revealed that bulkier compounds elicited higher inhibition of Hmp, while smaller compounds resulted in better inhibition of gFlHb, which might be explained by the presence of different amino acid residues in the active sites of the enzymes, with two large amino acid sequence inserts being a unique feature of gFlHb, thus blocking the active site from being reached and blocked by larger compounds.

Author Keywords: 2.3-diaminonaphthalene, Flavohemoglobin, Giardia intestinalis, Griess Assay, imidazole-based drugs, nitric oxide detoxification

Fungal pathogens adapt to environmental changes faster than their hosts, due in part to their adaptive mechanisms exhibited in response to stress. Ustilago maydis was used to investigate potential natural antisense transcript (NAT) RNA-mediated mechanisms that enhance fungal adaptation to stress. Of the 349 NATs conserved amongst U. maydis and two related smut fungi, five NATs were identified as having altered transcript levels in response to multiple stress conditions. Subsequently, antisense transcript expression vectors were created for select NATs and transformed into U. maydis haploid cells. When exposed to stress conditions, two antisense expressing mutant strains exhibited alterations in growth. RT-qPCR analysis of mRNA complementary to expressed NATs revealed no significant change in mRNA levels, which suggests NAT expression may influence stress response through dsRNA formation or other RNA mediated mechanisms. These results establish a basis for further investigations into the connection between NATs and the stress response of fungi.

Author Keywords: natural antisense transcripts, non-coding RNAs, stress response, Ustilago maydis

Giardia intestinalis possesses four isotypes of cytochrome b5 (gCYTB-I-IV) that differ from their mammalian counterparts, suggesting different functions in this protozoan parasite. Although the recently discovered Giardia flavoenzyme, GiOR-1, reduces these cytochromes, its properties have not been thoroughly studied, owing to the difficulty in its expression. Here I describe successful conditions for expression of GiOR-1 using autoinduction. GiOR-1 is obtained with flavins bound as indicated by its UV-visible spectrum. Its ability to catalyze electron transfer from donors (NADH, NADPH) to acceptors (oxygen, ferricyanide, cytochrome c, gCYTB5-III) were studied in spectrophotometric rate assays. NADPH is the preferred electron donor, while cytochromes are the preferred electron acceptors. Interestingly, the His-tag used to purify gCYTB5-III decreases its reaction rate with GiOR-1, as an untagged version has slightly faster rates. These findings establish the appropriate conditions for further studies on GiOR-1, including the identification of endogenous electron acceptors.

Author Keywords: Autoinduction, Cytochrome b5, Cytochrome P450 oxidoreductase, Giardia intestinalis, GiOR-1, Polyhistidine tag

This study investigated the role of the JNK signaling pathway in cadmium-treated mouse embryo forelimb bud cells in vitro. Primary cultures of forelimb bud cells harvested at day 11 of gestation were pre-treated with JNK inhibitor SP600125, and incubated with or without CdCl2 for 15, 30, 60, 120 minutes and 24, 48 hours or 5 days. Endpoints of toxicity were measured through cell differentiation by Alcian Blue Assay and phosphorylation of JNK proteins by Western blot. The results demonstrated that, in the cell differentiation assay, inhibiting JNK activation by 20 μM SP600125 causes an enhanced toxic effect in limb cells and inhibits cell differentiation, whereas 2 μM decreases differentiated nodule numbers under both cadmium stress and normal conditions. In conclusion, the JNK pathway has an essential role in the differentiation processes of limb bud cells in normal growth conditions.

Author Keywords: Cadmium, Cell Signaling, JNK, Limbs, Mouse Embryo, Teratology