Project description:Filamentous fungi are important producers of enzymes and secondary metabolites in the biotech industry. A relatively new but highly promising fungus for industrial production is Thermothelomyces thermophilus, which is closely related to the model fungus Neurospora crassa. Nevertheless, to fully exploit the potential of T. thermophilus, a deeper understanding of its biology is necessary. A critical aspect of the filamentous fungal life cycle is the production of asexual spores (conidia), regulated by various stimuli, including nutrient availability. Under certain submerged fermentation conditions, several filamentous fungi produce conidia, which can be detrimental to product expression. In this study, we utilized RNA-seq to map transcriptomic changes during conidia production under submerged conditions in T. thermophilus. We found that the essential genes for conidiation in Aspergillus sp. and N. crassa, fluG and fluffy (fl), are not essential for conidiation in T. thermophilus. However, we identified a transcription factor, res1, whose deletion resulted in a complete loss of conidia production under fermentation conditions. This deletion also led to increased biomass production without increasing enzyme secretion. Notably, the deletion of res1 did not affect spore production on solid media. Differential gene expression analysis between the wild type and the res1 deletion mutant revealed that res1 regulates a broad range of cellular and metabolic processes, including MAPK and spore production pathways. Overexpression of res1 caused a severe growth defect and early conidia production compared to the wild type. Using ChIP-seq, we identified 35 potential target genes of res1, including known conidiation-related genes such as acon3, MAPK genes, and transcription factors like cpc1, as well as genes with unknown functions. Overall, we identified res1 to be obligatory for submerged conidia production and lay the foundation for further work on the T. thermophilus conidiation pathway and its applications.

Project description:Fungi exhibit a huge diversity in morphology and ecology, and genetic basis of morphological development in sexual reproduction of multi-cellular fungi has not been fully investigated with genome-wide approaches. Model organism Neurospora crassa is the very first sequenced genome for multi-cellular fungi, and well-annotated genome and gene markers characterized for key morphological traits make N. crassa an ideal platform to investigate transcriptome and its regulation during sexual development. RNA sequencing with different priming strategies have been efficient to provide fine-scale transcriptome landscapes for a multiple-staged development process. RNA were sampled for eight time points cross perithecial development, including two points right before and after crossing, for which mat A protoperithecia were fertilized with mat a conidia. Transcription profiles for 9717 genes were achieved for all eight time-points using multi-targeting priming and additional 14 genes for five time-points using random hexmas priming. Majority of the genome showed continually up- or down- regulated expression patterns during the later perithecial development stages after 72 or 96 h. Functionally unclassified proteins were counted for most up-regulated genes, while genes were enriched with different functions for different down-regulation patterns. We observed significant increase in transcription for mat a-1 and for mat A-1 specific pheromone precursor ccg-4 during the perithecial development, and expression of genetic markers for morphological traits in perithecium, ascus, and ascospore, and for development traits in meiosis often showed multiple peaks during the experiment. We also observed different expression patterns in transcription for other transcription factors and for genes involved in Meiotic Silencing and Repeat Induced Point Mutation, and expression of a gene encoding a protein similar to stc-1 in fission yeast, was increased after crossing and reached almost 1000 fold changes at 144h. Different expression patterns were also observed for genes involved in heterokaryotic and vegetative incompatibility and for genes functioning in the heterotrimmeric G protein signalling systems and in the MAP kinase signalling pathways. By sequencing RNAs from different development stages with two priming strategies, this study provided a large amount of data to quantifiably describe the sophisticated transcriptomic landscape in pre-mating, crossing and sexual reproduction of N. crassa. As major expression patterns for genes in different function categories were recognized during this process, our results in general supported previous observation of expression patterns for genetic markers and regulatory genes/pathway. This study provided further evidence for functional correlation among genes involved in same function or pathways, which included mating type genes and pheromones, transcription factors, pigmentation, heterokaryotic incompatibility, singaling pathways, and RNA silencing. Besides, we also disclosed different expression patterns for the regulatory genes/pathways cross the whole sexual development, and functions of some of these genes were also suggested with phenotypic studies of knockouts. Understanding how these different regulatory elements and their function networks adjust genome-wide transcriptomic and proteomic expression will provide detailed insights about morphological development of tissue-differentiated perithecia in N. crassa. RNA were sampled for eight time points cross perithecial development, including two points right before and after crossing, for which mat A protoperithecia were fertilized with mat a conidia. Two priming MTP and N6 were used separately as technique replicates for the first strain synthesis during cDNA preparation of all samples. Total 16 cDNAs were sequenced

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	20250618_N_crassa_01_05.raw	Raw

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Neurospora crassa Δccg8 compromises cell surface integrity and antifungal tolerance: Insights from in vitro and Galleria mellonella studies

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