Fungi are extremely important organisms both in nature and for the human being. In nature, they are main decomposers of organic substance and cause million dollar losses in agriculture due to their potential to colonize on food and feed products, which is frequently associated with mycotoxin contamination. Fungi are also important pathogens for plants and animals including humans. <P>On the other hand, fungi have a long history being used in food industry as producers of organic acids or for cheese production. In addition, modern biotechnology uses the enormous potential of protein secretion by filamentous fungi. In order to prevent growth and development of harmful fungi but also to use them more efficiently in industry, it is crucial to better understand their biology. One important aspect is sporulation, which is often coupled with production of mycotoxins, which cause various adverse health effects. Asexual sporulation is a common reproductive mode for a diverse group of fungi.<P> The main objective of the project is to understand the mechanisms underlying regulation of asexual sporulation (conidiation) and biosynthesis of the carcinogenic mycotoxin sterigmatocystin (ST), the penultimate precursor of aflatoxins, in Aspergillus nidulans. Previous studies identified a number of genes activating conidiation in A. nidulans. However, the detailed mechanisms for the commencement of conidiation and ST biosynthesis are unknown. <P>To address this question, suppressor mutations that bypass the need for the early developmental activator fluG in conidiation and ST production were isolated. Molecular genetic studies revealed that a critical event for developmental transition is de-repression. The hypothesis is that conidiation and ST biosynthesis in A. nidulans occurs via removal of repressive effects imposed by multiple negative regulators.<P> Two objectives of the proposal are to: 1) Identify and characterize additional suppressors of fluG 2) Investigate the molecular basis of repression of conidiation and ST production. Outcomes of the proposed research will elucidate: a) the molecular mechanisms controling the early events of conidiation and ST production, b) a group(s) of genes regulated by SfgB/C/D, and 3) a genetic cascade controlling development and toxin biosynthesis.<P> Understanding the regulatory mechanisms of sporulation and toxin production will elucidate new insights into controlling both beneficial and detrimental activities of industrially, medically, and agriculturally important fungi.
Non-Technical Summary: A) Fungal spores and mycotoxin contamination in foods and feeds occur frequently. B) Fungal spores and toxins can cause severe adverse health effect in humans and animals and have a great impact on the US economy. A) This project identifies and examines functions of one or more key genes involved in fungal spore formation and mycotoxin production. B) The purpose of this study is to learn more about the genetic regulatory mechanisms governing production of fungal spores and mycotoxins. <P> Approach: Previous studies demonstrated that two antagonistic regulatory pathways govern vegetative growth and conidiation in A. nidulans. Activation of conidiation and ST production requires the activities of two major genes called flbA and fluG. FluG is proposed to activate downstream positive regulators of conidiation as well as FlbA, an RGS (regulator of G protein signaling) protein, which in turn inactivates G protein-mediated growth signaling. Loss of fluG function results in the lack of sporulation and ST production. Although a number of genes activating conidiation have been identified, the detailed molecular mechanisms regulating entry into conidiation and ST production are largely unknown. To address this important question, 40 second-site suppressor mutations that restored conidiation and ST production in the haploid fluG deletion mutant were isolated. These (recessive) suppressors of fluG (sfg) mutations were mapped to four linkage groups, where 31 mapped to sfgA, six mapped to sfgB and one each mapped to sfgC and sfgD. We identified sfgA by transformation-based complementation and found that sfgA is predicted to encode a novel 601 aa protein with the fungal-specific Zn(II)2Cys6 binuclear cluster DNA binding motif. Deletion and 31 other sfgA mutant alleles bypassed the need for fluG in conidiation and production of ST. However, overexpression of sfgA did not completely eliminate conidiation or ST production, indicating that other sfg genes play crucial roles in negative regulation of conidiation and ST biosynthesis. The primary hypothesis of the proposed research is that developmental transition and ST production in A. nidulans occurs via removing repressive effects imposed by multiple negative regulators defined by the four sfg genes. The first approach involves the identification and characterization of SfgB/C/D. The hypothesis in Aim 1 is that SfgB/C/D constitute important negative regulatory components of conidiation functioning downstream of FluG. While we will attempt to clone sfgB/C/D, we will first focus on identification of sfgB. Once gene(s) is cloned by transformation based complementation, its function will be characterized by deletion and overexpression. Moreover, genetic interactions, expression and localization of the cloned gene product will be examined. The second approach is to understand the mechanisms underlying regulation of conidiation by SfgB via employing genomics and protein interaction studies. We will carry out genome-wide analyses of SfgB-mediated repression of conidiation using A. nidulans microarrays. Then, an SfgB-FLAG tagged strain and FLAG antibody will be used to selectively enrich for all the binding sites of SfgB in the A. nidulans genome. Samples will be collected at 6 and 12 h post induction in liquid submerged cultures and ChIP will be carried out. We will adapt and modify a well-developed yeast ChIP method. In the event of unexpected difficulties, we will obtain technical assistance from the labs of Drs. Catherine Fox or Christina Hull on campus. Outcomes of the proposed studies will provide us with the genes regulated by SfgB and additional regulatory cascades for conidiation and toxin production.