104 research outputs found
Vectors for construction of translational fusions to beta-galactosidase
No full textThe use of lacZ as a reporter gene requires appropriate plasmid constructions to produce fusions of fungal promoter and translations initiation sequences to the E. coli lacZ gene. I have constructed three plasmids which may facilitate construction of lacZ fusions
Identification and functional analysis of secreted proteins from Magnaporthe grisea.
No full textSecreted proteins are the most likely candidates for being elicitors of resistance gene products in rice and other host plants. To identify the candidate secreted protein genes in M. grisea, the 5' ends of genes in the M. grisea EST databases, and genes from genomic sequences were analyzed using the SIGNALP-2.0 program
Glucose
No full textThis chapter reviews the regulation of glucose uptake and metabolism in filamentous fungi and highlights the similarities with and differences from mechanisms in yeast. Fungal glucose transporters are classified as high-affinity transporters if the Km for glucose is in the micromolar range and low-affinity transporters if Km for glucose is in the millimolar range. S. cerevisiae contains a large number of proteins that can transport glucose across the yeast cell membrane, 17 of which (Hxt1 through Hxt11p, Hxt13 through Hxt17p, and Gal2p) belong to the yeast glucose transporter family. The use of glucose analogues has been used to determine whether glucose sensing in fungi requires uptake and/or further metabolism of glucose. The role of hexokinases in glucose sensing was confirmed in studies using strains carrying mutations in the three genes encoding sugar-phosphorylating enzymes: HXK1, HXK2, and GLK2. Given the central role of glucose in carbon metabolism and the diverse nutrient sources used by different fungi, it is not surprising that differences in glucose transport, glucose metabolism, glucose signaling, and carbon catabolite repression have arisen through selection.Margaret E. Katz and Joan M. Kell
A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia
No full textCurrent approaches for obtaining homokaryons of asexually growing N. crassa rely on serial passages of macroconidia, which are usually multinuclear (Davis and de Serres 1970 Methods Enzymol. 17A:79- 143). This method is time consuming and labor intensive. We have devised a simple method for purifying viable uninucleate microconidia from conidiating strains of N. crassa grown on Westergaard and Mitchell synthetic crossing medium (SC) supplemented with iodoacetate (IAA). Rossier, Oulevey and Turian (1973 Arch. Mikrobiol. 91:345-353) showed that standing liquid cultures containing 1x SC and 1 mM IAA produced microconidia. We have modified their method to reliably obtain microconidia from 150 mm slants of solid agar medium (0.1 x SC/0.5% sucrose/2% agar/1 mM IAA). We have purified these microconidia free of macroconidia and mycelia using Millipore Durapore Millex 5 µm filters. We are using this technique for the one step purification of homokaryons following DNA-mediated transformation. These methods may be generally useful for studies with heterokaryons
The use of lacZ gene fusions in Neurospora crassa
No full textSystematic analyses of gene expression in diverse organisms have relied on genetic fusions in which the regulated expression of the product of the Escherichia coli lacZ gene, ß-galactosidase, is used to assay gene activity. Because there are low but readily detectable levels of ß-galactosidase in Neurospora crassa (e.g. Landman, Arch. Biochem. Biophys. 52:93-109, 1954), the use of E. coli lacZ as a reporter gene in this organism has not been extensively investigated. Here we report that lacZ fusion proteins can be used to analyze the regulation of two N. crassa genes, arg-2 and con-10. The levels of ß-galactosidase produced by strains carrying the fused genes indicate that they are developmentally regulated in a manner similar to the intact genes (Davis, Microbiol. Rev. 50: 280-313, 1986; Orbach, Sachs, and Yanofsky, J. Biol. Chem. in press 1990; Roberts, Berlin, Hager and Yanofsky, Mol. Cell. Biol. 8:2411-2418, 1988; Sachs and Yanofsky, in preparation)
Novos aspectos da patogenicidade de Magnaporthe grisea.
No full textO principal objetivo deste estudo foi identificar e analisar o papel que o gene PTH1 exerce na formação e maturação do apressório em M. grisea. Isolados de M. grisea mutantes (denominado pth1) para o gene PTH1 são incapazes de perfurar o tecido do hospedeiro para dar início à colonização e assim estabelecer uma interação patógeno-hospedeiro
Roles of Cytoskeletal Polarity in Fungal Spore Adhesion and Growth
No full textFilamentous fungi are complex microorganisms that reproduce via the production of spores and grow by producing elongated polarized cells called hyphae. Both processes are essential to the life cycle of these organisms. Spore production, dispersal, and adhesion are essential for dissemination of the organism. Spore adhesion is especially important to plant pathology, as fungi employ diverse means to adhere their spores to infection courts to begin the disease cycle. This adherence is required for infection to occur. As the primary means of fungal growth, hyphae are also incredibly important structures to understand. Hyphae require maintenance of polarized growth to properly extend the mycelium. Here, I investigated the role cytoskeletal proteins play in both spore adhesion and polarized hyphal growth using fluorescence microscopy. I found that conidial adhesion in C. graminicola is a polarized phenomenon in which an adhesive strip that localizes only to one face of the conidium is responsible for adhesion. This strip colocalizes with a polarized actin array that is present only in conidia detached from conidiophores. This discovery grants insight into the adhesion strategy of C. graminicola, as we hypothesize that the actin array plays a role in generation of this adhesive and that the polar nature of the strip grants conidia a dispersal benefit. Additionally, my research found that hyphal growth is strongly correlated with the localization of proteins associated with the endocytic collar. This correlation is strongest when determining the distance these proteins are behind the growing apex. Interestingly, the rate of endocytosis does not seem to correlate with the rate of hyphal growth, suggesting that it is the regulation of the location of endocytosis that plays the greatest role in growth rate, not the speed at which endocytosis occurs
Aspergillus nidulans swoQ Plays an Important Role in Polarized Hyphal Growth
No full textFilamentous fungi form polarized hyphae by concentrating the addition of new material at the apex of the cell. The mechanics of polarized hyphal growth are coordinated by the cytoskeleton which utilizes microtubules (MTs) and filamentous actin in order to facilitate vesicle trafficking. The cytoskeletal machinery implicated in the regulation of endocytosis and exocytosis during polarized growth is complex and has been studied extensively in recent years. Despite these efforts there are many gaps in the current growth model, and the exact role of the cytoskeleton on secretion and internalization mechanisms during polarized growth remains to be elucidated. In order to address this knowledge gap, this study investigates the contribution of cytoskeletal elements on polarized growth.
In this study, I identified the Aspergillus nidulans temperature-sensitive (TS) mutant, swoQ. A mutant allele in the swoQ gene results in abnormal polarity maintenance and cell morphogenesis in the swoQ strain, as evidenced by the presence of abnormally swollen cells. A forward genetic screen revealed the swoQ polarity defect was suppressed following transformation with the pRG3-47-1 library plasmid which encodes myoA, a class I myosin motor protein. A sub-clone of myoA was introduced into the swoQ mutant and wild-type which resulted in restoration of polarized growth in the polarity mutant and hyper-branching in both strains.
To further assess the influence of swoQ, the localization of various polarity markers including: FimA::GFP, Exo84::GFP, DnfA::GFP, and DnfB::GFP, were examined in the mutant strain. The localization of FimA::GFP and Exo84::GFP was abnormal in the swoQ mutant. FimA is an actin nucleating protein which works alongside MyoA, an essential myosin in A. nidulans. Together these proteins facilitate the formation of actin patches which drive endocytosis. Here I show that overexpression of MyoA has a polarization effect on growing cells and is able to suppress the growth defect of the swoQ mutant
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