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The overall goal of this proposal is to delineate the sexual reproductive transcriptome of An and to expand our understanding of the regulatory network controlling expression of this transcriptome. The specific aims of this proposal are: <P>Specific Aim 1. We will use synchronously-induced cultures of wild type An to isolate mRNAs from undifferentiated hyphae and early, mid and late developmental stages of the sexual reproductive cycle. This will provide the baseline transcriptome throughout organogenesis, meiosis and sporulation.<P> Specific Aim 2. We will isolate mRNA from tissue of synchronously induced cultures of strains with deletions in one of five transcriptional factors that we have shown to be key regulators of the An sexual cycle. These are stuA (stunted), medA (medusa), steA (homolog of yeast STERILE 12), matA (mating type-HMG box) and matB (mating type-alpha box). Expression data from mutants and wild type will be compared at similar developmental time points. <P>Specific Aim 3. We will use strains overexpressing each of the five transcription factors described in Specific Aim 2. We have used ectopic gene expression in undifferentiated hyphae to identify a limited number of direct target genes for 4 of the 5 transcription factors. This analysis will now be expanded to the whole genome level using microarrays. Comparison of expression data from Specific Aims 1-3 should allow the assembly of an initial regulatory network for sexual reproduction in the filamentous fungi.
Non-Technical Summary: An understanding of the sexual reproductive transcriptome and its regulation in A. nidulans should provide important insights into the reproductive biology of filamentous fungi and ultimately, the regulatory relationship between homothallic (self fertile) and heterothallic (self sterile, cross fertile) fungal species. Of interest is the possibility of identifying ways to manipulate the potential cryptic sexual cycle of the asexual human pathogen A. fumigatus. This could provide provide valuable genetic tools that can be used to identify and manipulate genes encoding A.fumigatus virulence factors. The purpose of this project is to generate a detailed, genome wide picture of gene expression controlling sexual reproduction in fungi. <P> Approach: Specific Aim 1. Stage-specific, synchronously induced developmental tissue will be obtained using our standard laboratory methods. Analyzed tissue will include undifferentiated vegetative hyphae, conidiating cultures, early sexual tissues and mature sexual tissues. Total RNA and mRNA will be isolated from tissues using our standard laboratory methods. Microarrays are available to the PI from the Pathogen Functional Genomics Resource Center (http://pfgrc.tigr.org/resources.shtml). RNAs will be used to generate probes labeled with Cy3 or Cy5 using the standard operating procedures described by PFGRC. Hybridization of labeled probes to microarrays will be performed using standard PFGRC operating procedures. Undifferentiated, vegetative hyphae (0 hr) will be used as reference relative to selected developmental time points. Replica data sets are expected to provide a 95% confidence level for differentially expressed genes. The PI has access to the Genomics and Bioinformatics Core Facilities at Washington State University. Specific Aim 2. We will link genome wide transcriptional profiles with the regulatory network controlling the An sexual cycle. Five transcriptional regulators that control key developmental landmarks in the sexual cycle will be analyzed. Undifferentiated, vegetative hyphae from the stuA-, medA-, steA-, matA- and matB- deletion strains will be induced to undergo synchronous development as described in Specific Aim 1. Developmental RNAs from deletion strains will be compared to wild type RNAs at equivalent time Specific Aim 3. The data from Specific Aims 2 & 3 will provide gene-dependent transcriptional profiles for the 5 transcriptional regulators. However, many of the differentially genes are likely to be targets of unknown intermediary transcription factors located downstream in the regulatory pathway, rather than direct-target genes of the 5 transcription factors of this study. The objective of Specific Aim 3 is to identify direct targets genes. An strains expressing a transcriptional regulator (e.g. stuA) under control of the glucose-repressible, ethanol inducible alcohol dehydrogenase promoter [alcA(p)]. Developmentally competent, vegetative hyphae from shake cultures grown in glucose (repressive) or acetate (neutral) media are collected, washed with ethanol (inducing) media and then transferred to shake cultures containing ethanol media. RNA will be isolated from induced issue and used in transcriptional profile analysis with 0 hr time point as the reference RNA. A comparison of data sets generated in Specific Aim 3 with those of Specific Aims 1&2 will provide the basis for the transcriptional profile and regulatory network for the An sexual cycle. This information can provide the basis for understanding how the sexual cycle is regulated in the filamentous fungi and should provide the groundwork for understanding why species, such as A.fumigatus, are sterile, or are recalcitrant to laboratory manipulation of a sexual genetic cycle. I estimate the need of 16 microarrays for each transcription factor for a total of 80 microarrays for Aim 3.