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Chapter 14 : Vacuoles in Filamentous Fungi
Category: Microbial Genetics and Molecular Biology; Fungi and Fungal Pathogenesis
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Using the ectomycorrhizal fungus Pisolithus tinctorius, four zones of vacuolar morphology from the growing hyphal tip were observed: (i) the apical zone, which has few or no vacuoles; (ii) the subapical zone with small ovoid-spherical vacuoles; (iii) the nuclear zone, where tubular vacuoles predominate; and (iv) the basal zone, where large spherical vacuoles are most common. This vacuolar structure is the product of live-cell imaging in microscopes utilizing fluorescent dyes that accumulate in vacuoles and green fluorescent protein (GFP)or red fluorescent protein (RFP)-tagged proteins targeted to vacuoles or vacuolar membranes. For biochemical studies in vitro, vacuoles can be isolated from filamentous fungi as a pure organellar fraction of round vesicles, approximately 0. 2 to 2 μ in diameter. For filamentous fungi, the study of formation and biogenesis of the vacuole is a nascent research area. Fungal vacuoles serve as storage reservoirs for high levels of phosphorus and nitrogen in the form of basic amino acids and polyphosphate (polyPi), respectively. The vacuolar system is highly variable in appearance at different locations within the mycelium and in response to different growth conditions. Several labs are introducing new approaches to study the dynamic behavior of the vacuolar system of filamentous fungi. Homologs of S. cerevisiae genes with known functions in vacuolar biogenesis, autophagy, and ion transport are excellent candidates for identifying the genes involved in these processes in filamentous fungi. Investigations performed in recent years demonstrate that the vacuole is a dynamic organelle with many important roles in metabolism, growth, and development.
Visualizing the vacuole with RFP and GFP. RFP (dsRED) or GFP was fused to proteins predicted to be in the membrane of the vacuole, using the plasmids pMF272 or pMF334 constructed by M. Freitag (Oregon State University, Corvallis). By transformation into N. crassa, the recombinant genes were targeted to the his-3 locus. Images were obtained by confocal microscopy (B. Bowman and E. J. Bowman, unpublished results). (A) RFP dsRED was fused to the N terminus of the CAX protein. (B) RFP dsRED was fused to the N terminus of the VAM-3 protein. In panels A and B the region shown is approximately 100 μm behind the hyphal tip. The vacuolar system consists of tubules and small vesicles. (C) GFP was fused to the C terminus of the A subunit of the V-ATPase (encoded by vma-1). The region shown is approximately 2 mm behind the hyphal tip. In this older part of the hypha, the fluorescence is localized to the membrane of a large vacuole and to many small vesicles. In this strain the vacuole appears as a network of tubules and small vesicles nearer the hyphal tip (not shown), as in panels A and B. The bar represents 10 μm, and the scale is the same for all panels.
Electron micrograph of the V-ATPase in vacuolar membranes. Vacuolar membranes were isolated from N. crassa, negatively stained, and examined by transmission electron microscopy as described previously ( Dschida and Bowman, 1992 ). A few of the V-ATPases are indicated by arrows. The globular head is 12 nm wide and is attached to the membrane by a 3-nm-wide stalk.
Two models of the V-ATPase. As shown on the left, the V-ATPase is composed of 14 different types of subunits, some of which are present in multiple copies. The diagram is modified from the model of the N. crassa V-ATPase in Venzke et al. (2005 ). The model on the right shows the major functional domains. The rotor portion of the enzyme is composed of subunits D, F, d, c, c’, and c”. The A and B subunits form the ATP binding sites and constitute the motor domain. It is not known for certain which subunits form the stator domain.