SapB and the rodlins are required for development of Streptomyces coelicolor in high osmolarity media

Streptomyces coelicolor produces spore-forming aerial hyphae after a period of vegetative growth. These aerial structures are decorated with a hydrophobic coating of rodlets consisting of chaplins and rodlins. Here, we show that rodlins and the surface-active peptide SapB are essential for development during growth in a medium with high osmolarity. To this end, both vegetative and aerial hyphae secrete SapB, whereas rodlins are only secreted by the spore-forming aerial hyphae.

Rapid turnover of hyphae of mycorrhizal fungi determined by AMS microanalysis of C-14

Processes in the soil remain among the least well-characterized components of the carbon cycle. Arbuscular mycorrhizal (AM) fungi are ubiquitous root symbionts in many terrestrial ecosystems and account for a large fraction of photosynthate in a wide range of ecosystems; they therefore play a key role in the terrestrial carbon cycle. A large part of the fungal mycelium is outside the root ( the extraradical mycelium, ERM) and, because of the dispersed growth pattern and the small diameter of the hyphae (<5 micrometers), exceptionally difficult to study quantitatively. Critically, the longevity of these. ne hyphae has never been measured, although it is assumed to be short. To quantify carbon turnover in these hyphae, we exposed mycorrhizal plants to fossil ("carbon-14 - dead") carbon dioxide and collected samples of ERM hyphae ( up to 116 micrograms) over the following 29 days. Analyses of their carbon-14 content by accelerator mass spectrometry (AMS) showed that most ERM hyphae of AM fungi live, on average, 5 to 6 days. This high turnover rate reveals a large and rapid mycorrhizal pathway of carbon in the soil carbon cycle

Preparation of Neurospora crassa mitochondria

The fungus Neurospora crassa represents a eukaryotic cell with high biosynthetic activities. Cell mass doubles in 2-4 hr during expone ntial growth , even in simple salt media with sucrose as the sole carbon source. The microorgani sm forms a mycelium of long hyphae durlng vegetative growth . The mitochondria can be isolated under relatively gentle condi tions since a few breaks in the threadlike hyphae are sufficient to cause the outflow of the organelles. This article describes two methods for the physical disruption of the hyphae : (I) The cell s are opened in a grind mill between two rotating corundum di sks. This is a continuous and fast procedure and allows large- and small-scale preparations of mitochondria. (2) Hyphae are ground with sand in a mortar and pestle. This procedure can be applied to microscale preparations of mitochondria starting with minute amounts of cells. Other procedures for the isolation of Neurospora mitochondria after the physical di sruption or the enzymatic degradation of the cell wall have been described elsewher

How does Epichloë festucae avoid the host defence response? : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand

Epichloë festucae is a filamentous fungus, which forms symbiotic associations with aerial tissues of Lolium and Festuca grass species. Chitin, a polymer of N-acetyl-Dglucosamine, is an important component of the fungal cell wall and a well-known pathogen associated molecular pattern (PAMP). Chitin promotes pathogen-triggered immunity (PTI) upon hydrolysis with plant chitinases and release of chitin oligomers. Therefore, to establish a stable and successful symbiosis, the endophyte needs to remain ‘hidden’ from the host immune system or actively suppress it. Confocal laser scanning microscopy (CLSM)-based analysis of leaf tissue infected with the E. festucae wild type strain and infiltrated with the chitin-specific molecular probe, WGA-Alexa Fluor-488, showed that only the septa of endophytic hyphae bound this probe while the entire cell wall was labelled in epiphyllous hyphae confirming previous observations that hyphal cell wall chitin is either masked or remodelled in endophytic hyphae. The aims of this project were (i) to test whether E. festucae LysM-containing proteins have a role in binding to or sequestering cell wall chitin oligomers and thereby preventing PAMPtriggered immunity and (ii) to analyse the composition of the cell wall of endophytic and epiphytic hyphae. An analysis of the E. festucae genome identified seven genes encoding proteins with LysM domains. Expression of two of these genes, lymA and lymB, increased in planta compared to in culture. Interestingly, both are divergently transcribed from chitinase encoding genes (chiA and chiB respectively), which also have increased expression in planta. Single gene deletion mutants of lymA, lymB, chiA and chiB as well as a double gene deletion ΔlymA/B were generated, and their plant interaction phenotype analysed. Plants infected with DlymA, DlymB or DchiA had the same plant-interaction phenotype as wild type whereas ΔchiB and ΔlymA/B mutants had defects in hyphal growth within the leaves. Analysis of hyphal cell wall structure using Chitin Binding Protein (CBP) and chitosan (CAP (Chitosan Affinity Protein) and OGA-488)-specific eGFP-based biosensors suggest that cell wall chitin is converted to chitosan in endophytic hyphae. This structural change is consistent with a lack of a defence response when E. festucae forms a mutualistic symbiotic association with L. perenne. Three E. festucae chitin deacetylase genes were identified (cdaA, cdaB and cdaC), and gene expression analysis showed cdaA expression is significantly increased in planta compare to in culture. Functional analysis of cdaA revealed that although plants infected with the ΔcdaA mutant had a similar whole plant interaction phenotype as wild type, they had an abnormal cellular phenotype. Patches of chitin were exposed along the endophytic hyphae confirming this mutant was unable to convert chitin to chitosan. However, hyphae in these plants still labelled with the chitosan biosensor OGA-488 demonstrating that despite the deletion of the cdaA, the hyphal cell wall of endophytic hyphae still contain chitosan suggesting that another chitin deacetylase, possibly CdaB has a redundant function in E. festucae. Collectively these results show that lymA, lymB and chiB are required for establishment of the symbiosis between E. festucae and L. perenne. In addition, this study shows that chitin is converted to chitosan in the hyphal cell wall of endophytic hyphae during the infection and colonisation of the host. The E. festucae chitin deacetylase gene cdaA is also essential for proper hyphal growth in planta and the symbiotic interaction

Candida albicans Yeast, Pseudohyphal, and Hyphal Morphogenesis Differentially Affects Immune Recognition

The authors thank Amy Whittington for preliminary experiments on in vitro induction of pseudohyphae and Mihai Netea for discussions. The authors also thank Michael Weig for the pga29 strains. Funding NG and AW were supported by the Wellcome Trust (086827, 075470, 097377, 101873, and 200208); the European Union ALLFUN (FP7/2007 2013, HEALTH-2010-260338), and the MRC Centre for Medical Mycology for funding (N006364/1). LM was supported by a SORSAS (Scottish Overseas Research Students Award Scheme) from the University of Aberdeen and Funding from the Rwandan Government.Peer reviewedPublisher PD

Contact-induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae

Acknowledgements We thank Marco Thiel for assistance with data interpretation, Peter Sudbery for the provision of strains and Jeremy Craven for useful discussions. This work was supported by a BBSRC-DTG to D. D. T., NIH award DK083592 to F. J. B. and P. A. J., and a Royal Society URF UF080611 and MRC NIRG 90671 to A. C. B.Non peer reviewedPublisher PD

DevA, a GntR-like transcriptional regulator required for development in streptomyces coelicolor

The gram-positive filamentous bacterium Streptomyces coelicolor has a complex developmental cycle with three distinct phases: growth of the substrate mycelium, development of reproductive structures called aerial hyphae, and differentiation of these aerial filaments into long chains of exospores. During a transposon mutagenesis screen, we identified a novel gene (devA) required for proper development. The devA mutant produced only rare aerial hyphae, and those that were produced developed aberrant spore chains that were much shorter than wild-type chains and had misplaced septa. devA encodes a member of the GntR superfamily, a class of transcriptional regulators that typically respond to metabolite effector molecules. devA forms an operon with the downstream gene devB, which encodes a putative hydrolase that is also required for aerial mycelium formation on R5 medium. S1 nuclease protection analysis showed that transcription from the single devA promoter was temporally associated with vegetative growth, and enhanced green fluorescent protein transcriptional fusions showed that transcription was spatially confined to the substrate hyphae in the wild type. In contrast, devAB transcript levels were dramatically upregulated in a devA mutant and the devA promoter was also active in aerial hyphae and spores in this background, suggesting that DevA might negatively regulate its own production. This suggestion was confirmed by gel mobility shift assays that showed that DevA binds its own promoter region in vitro

Experimental measurement of endocytosis in fungal hyphae.

The present study examines the notion that polarized exocytosis in the tips of growing hyphae creates an excess of plasma membrane and thus the need for its removal by endocytosis. To measure endocytosis experimentally, we developed a photobleaching (FRAP) procedure to count endocytic events in hyphae of Neurospora crassa carrying a fluorescent tag on the actin-binding protein fimbrin (FIM-1-GFP). Given 40 nm as the average diameter of endocytic vesicles, we calculated that about 12.5% of the plasma membrane discharged in the apex becomes endocytosed in the subapex. According to our calculations, the GFP-tagged hyphae of N. crassa, measured under the constrained conditions of confocal microscopic examination, needed about 8800 vesicles/min to extend their plasma membrane or about 9800/min, if we include predicted demands for cell wall growth and extracellular secretion. Our findings support the notion that exocytosis and endocytosis operate in tandem with the latter serving as a compensatory process to remove any excess of plasma membrane generated by the intense exocytosis in the hyphal tips. Presumably, this tandem arrangement evolved to support the hallmark features of fungi namely rapid cell extension and abundant secretion of hydrolytic enzymes