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Filamentous fungi are ubiquitous in nature and serve as important biological models in various scientific fields including genetics, cell biology, ecology, evolution, and chemistry. A significant obstacle in studying filamentous fungi is the lack of tools for characterizing their growth and morphology in an efficient and quantitative manner. Consequently, assessments of the growth of filamentous fungi are often subjective and imprecise. In order to remedy this problem, we developed Fungal Feature Tracker (FFT), a user-friendly software comprised of different image analysis tools to automatically quantify different fungal characteristics, such as spore number, spore morphology, and measurements of total length, number of hyphal tips and the area covered by the mycelium. In addition, FFT can recognize and quantify specialized structures such as the traps generated by nematode-trapping fungi, which could be tuned to quantify other distinctive fungal structures in different fungi. We present a detailed characterization and comparison of a few fungal species as a case study to demonstrate the capabilities and potential of our software. Using FFT, we were able to quantify various features at strain and species level, such as mycelial growth over time and the length and width of spores, which would be difficult to track using classical approaches. In summary, FFT is a powerful tool that enables quantitative measurements of fungal features and growth, allowing objective and precise characterization of fungal phenotypes., Author summary One of the main obstacles to study filamentous fungi is the lack of tools for characterizing fungal phenotypes in an efficient and quantitative manner. Assessment of cell growth and numbers rely on tedious manual techniques that often result in subjective and imprecise measurements. In response to those limitations, we developed Fungal Feature Tracker (FFT), a user-friendly software that allows researchers to characterize different phenotypic features of filamentous fungi such as sporulation, spore morphology and mycelial growth. In addition, FFT can recognize and quantify other fungal structures including the fungal traps developed by nematode-trapping fungi. In order to show the capabilities and potential of our software, we conducted a detailed characterization and comparison of different fungal species. Our comparison relies on a series of experimental set-ups using standard and easily accessible equipment to ensure reproducibility in other laboratories. In summary, FFT is an easy to use and powerful tool that can quantitatively characterize fungal morphology, cell number and quantitatively measures the filamentous growth, which will advance our understanding of the growth and biology of filamentous fungi.

Fatty acid synthase 1 (FAS I) from Mycobacterium. tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4’-Phosphopantetheine (4’-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4’-PP transferase, termed acyl carrier protein synthase (AcpS). In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid.We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structurefunction analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

Coprinopsis cinerea is a model mushroom particularly suited to study fungal fruiting body development and the evolution of multicellularity in fungi. While microRNAs (miRNAs) are extensively studied in animals and plants for their essential roles in post-transcriptional regulation of gene expression, miRNAs in fungi are less well characterized and their potential roles in controlling mushroom development remain unknown. To identify miRNA-like RNAs (milRNAs) in C. cinerea and explore their expression patterns during the early developmental transition of mushroom development, small RNA libraries of vegetative mycelium and primordium were generated and putative milRNA candidates were identified following the standards of miRNA prediction in animals and plants. Two out of 22 novel predicted milRNAs, cci-milR-12c and cci-milR-13e-5p, were validated by northern blot and stem-loop reverse transcription real-time PCR. Cci-milR-12c was differentially expressed whereas the expression levels of cci-milR-13e-5p were similar in the two developmental stages. Target prediction of the validated milRNAs resulted in genes associated with fruiting body development, including pheromone, hydrophobin, cytochrome P450, and protein kinase. Besides, essential genes for miRNA biogenesis, including three coding for Dicer-like (DCL), two for Argonaute-like (AGO-like) and one for quelling deficient-2 (QDE-2) proteins, were identified in the C. cinerea genome. Phylogenetic analysis showed that the DCL and AGO-like proteins of C. cinerea were more closely related to those in other basidiomycetes and ascomycetes than to animals and plants. Taken together, our findings provided the first evidence of milRNAs in the model mushroom and their potential roles in regulating fruiting body development. Information on the evolutionary relationship of milRNA biogenesis proteins across kingdoms has also provided new insights into further functional and evolutionary studies of miRNAs.