application-membrane-cell-wall-selective-fluorescent-dyes-for-live

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

University of Innsbruck

Fungi interact with plants in various ways, with each interaction giving rise to different alterations in both partners. While fungal pathogens have detrimental effects on plant physiology, mutualistic fungi augment host defence responses to pathogens and/or improve plant nutrient uptake. Tropic growth towards plant roots or stomata, mediated by chemical and topographical signals, has been described for several fungi, with evidence of species-specific signals and sensing mechanisms. Fungal partners secrete bioactive molecules such as small peptide effectors, enzymes and secondary metabolites which facilitate colonization and contribute to both symbiotic and pathogenic relationships. There has been tremendous advancement in fungal molecular biology, omics sciences and microscopy in recent years, opening up new possibilities for the identification of key molecular mechanisms in plant-fungal interactions, the power of which is often borne out in their combination. Our fragmentary knowledge on the interactions between plants and fungi must be made whole to understand the potential of fungi in preventing plant diseases, improving plant productivity and understanding ecosystem stability. Here, we review innovative methods and the associated new insights into plant-fungal interactions. 

Friends or foes? Emerging insights from fungal interactions with plants.

Studying microbial interactions by MALDI mass spectrometry imaging (MSI) directly from growing media is a difficult task if high sensitivity is demanded. We present a quick and robust sample preparation strategy for growing fungi (Trichoderma atroviride, Rhizoctonia solani) on glass slides to establish a miniaturized confrontation assay. By this we were able to visualize metabolite distributions by MALDI MSI after matrix deposition with a home-built sublimation device and thorough recrystallization. We present for the first time MALDI MSI data for secondary metabolite release during active mycoparasitism.

Visualizing fungal metabolites during mycoparasitic interaction by MALDI mass spectrometry imaging.

Mycoparasitism is a lifestyle where one fungus establishes parasitic interactions with other fungi. Species of the genus Trichoderma together with Clonostachys rosea are among the most studied fungal mycoparasites. They have wide host ranges comprising several plant pathogens and are used for biological control of plant diseases. Trichoderma as well as C. rosea mycoparasites efficiently overgrow and kill their fungal prey by using infection structures and by applying lytic enzymes and toxic metabolites. Most of our knowledge on the putative signals and signaling pathways involved in prey recognition and activation of the mycoparasitic response is derived from studies with Trichoderma. These fungi rely on G-protein signaling, the cAMP pathway, and mitogen-activated protein kinase cascades during growth and development as well as during mycoparasitism. The signals being recognized by the mycoparasite may include surface molecules and surface properties as well as secondary metabolites and other small molecules released from the prey. Their exact nature, however, remains elusive so far. Recent genomics-based studies of mycoparasitic fungi of the order Hypocreales, i.e., Trichoderma species, C. rosea, Tolypocladium ophioglossoides, and Escovopsis weberi, revealed not only several gene families with a mycoparasitism-related expansion of gene paralogue numbers, but also distinct differences between the different mycoparasites. We use this information to illustrate the biological principles and molecular basis of necrotrophic mycoparasitism and compare the mycoparasitic strategies of Trichoderma as a "model" mycoparasite with the behavior and special features of C. rosea, T. ophioglossoides, and E. weberi.

Necrotrophic Mycoparasites and Their Genomes.

Mycoparasites, e.g. fungi feeding on other fungi, are prominent within the genus Trichoderma and represent a promising alternative to chemical fungicides for plant disease control. We previously showed that the seven-transmembrane receptor Gpr1 regulates mycelial growth and asexual development and governs mycoparasitism-related processes in Trichoderma atroviride. We now describe the identification of genes being targeted by Gpr1 under mycoparasitic conditions. The identified gene set includes a candidate, sfp2, encoding a protein of the fungal-specific Sur7 superfamily, whose upregulation in T. atroviride upon interaction with a fungal prey is dependent on Gpr1. Sur7 family proteins are typical residents of membrane microdomains such as the membrane compartment of Can1 (MCC)/eisosome in yeast. We found that GFP-labeled Gpr1 and Sfp2 proteins show partly overlapping localization patterns in T. atroviride hyphae, which may point to shared functions and potential interaction during signal perception and endocytosis. Deletion of sfp2 caused heavily altered colony morphology, defects in polarized growth, cell wall integrity and endocytosis, and significantly reduced mycoparasitic activity, whereas sfp2 overexpression enhanced full overgrowth and killing of the prey. Transcriptional activation of a chitinase specific for hyphal growth and network formation and strong downregulation of chitin synthase-encoding genes were observed in Δsfp2. Taken together, these findings imply crucial functions of Sfp2 in hyphal morphogenesis of T. atroviride and its interaction with prey fungi.

The Gpr1-regulated Sur7 family protein Sfp2 is required for hyphal growth and cell wall stability in the mycoparasite Trichoderma atroviride.

Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported in the last decade, the technique could not reach its full potential due to a set of limitations immanent to the samples themselves. Particularly, high background signals impede the proper performance of most single-molecule identification and localization algorithms. One option is to exploit the characteristic blinking of single molecule signals, which differs substantially from the residual brightness fluctuations of the fluorescence background. To pronounce single molecule signals, we used a temporal high-pass filtering in Fourier space on a pixel-by-pixel basis. We evaluated the performance of temporal filtering by assessing statistical parameters such as true positive rate and false discovery rate. For this, ground truth signals were generated by simulations and overlaid onto experimentally derived movies of samples with high background signals. Compared to the nonfiltered case, we found an improvement of the sensitivity by up to a factor 3.5 while no significant change in the localization accuracy was observable.

Article	Year
Friends or foes? Emerging insights from fungal interactions with plants. FEMS microbiology reviews\| PubMed ID: 26591004	2016
Visualizing fungal metabolites during mycoparasitic interaction by MALDI mass spectrometry imaging. Proteomics\| PubMed ID: 26959280	2016
Necrotrophic Mycoparasites and Their Genomes. Microbiology spectrum\| PubMed ID: 28281442	2017
The Gpr1-regulated Sur7 family protein Sfp2 is required for hyphal growth and cell wall stability in the mycoparasite Trichoderma atroviride. Scientific reports\| PubMed ID: 30104659	2018
Temporal Filtering to Improve Single Molecule Identification in High Background Samples. Molecules (Basel, Switzerland)\| PubMed ID: 30562966	2018

Susanne Zeilinger

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