Experimental Mycology最新文献

Amir, R., Levanon, D., Hadar, Y., and Chet, I. 1995. Factors affecting translocation and sclerotial formation in Morchella esculenta. Experimental Mycology 19, 61-70. Morchella esculenta was grown on square split plates, forming sclerotia on one side and mycelium on the other. After the fungus ceased to colonize and before sclerotial initials appeared, [¹⁴C]3-O-methyl glucose was added to the edge of the plate on the mycelial side. The effect of various activities in the mycelium (source) and sclerotia (sink) on sclerotial formation and translocation were examined using inhibitors and water potential changes of the media. Sodium azide or cycloheximide applied separately to both sides inhibited both sclerotial formation and translocation, showing that processes in the source and sink depend on metabolic activities as well as protein synthesis. The use of nikkomycin inhibited sclerotial formation, without affecting translocation to the sclerotia. Since the hyphal tips swelled and burst, the translocated compounds were lost to the media. In a strain defective in sclerotial formation, used as a control, no translocation took place, showing that there is a connection between sclerotial formation and translocation. Reversal of the water potential gradient between the two media (lower on the mycelial side), reduced the formation of sclerotia and translocation to them. Translocation to Morchella sclerotia takes place via turgor driven mass flow, but is nevertheless affected by activities in both the source and the sink.

Rizwana, R., and Powell, W. A. 1995. Ultraviolet light-induced heterokaryon formation and parasexuality in Cryphonectria parasitica. Experimental Mycology 19, 48-60. The effect of ultraviolet-light on heterokaryon formation, vegetative compatibility, and parasexuality in Cryphonectria parasitica was examined. Heterokaryons of complementary auxotrophic strains could not be made by hyphal anastomosis if the strains belonged to different vegetative compatibility groups. Protoplast fusions overcame incompatibility of strains differing in the alleles of a single but not multiple vegetative incompatibility loci. Fusion of protoplasts from ultraviolet light-treated complementary auxotrophs increased heterokaryon formation by 10⁴ to 10⁵ using the strains differing in alleles of a single vegetative incompatibility gene but had no detectable effect on strains differing in multiple vegetative incompatibility genes. Vegetative compatibility tests of single conidial isolates resolved from these heterokaryons suggest that diploids had formed followed by the loss of one of the VIC alleles. Presence of both auxotrophic markers in some of these single conidial isolates confirms the occurrence of a parasexual cycle. These experiments demonstrate that ultraviolet-light can enhance heterokaryon formation and parasexuality in C. parasitica .

Rauscher, M., Mendgen, K., and Deising, H. 1995. Extracellular proteases of the rust fungus Uromyces viciae-fabae. Experimental Mycology 19, 26-34. On thigmo-inductive membranes the broad bean rust fungus Uromyces viciae-fabae differentiates complex infection structures including haustorial mother cells. Using this in vitro system, formation of extracellular proteases of the obligately biotrophic fungus was studied during infection structure differentiation. Enzyme activities occur when appressoria are formed, and extracellular washing fluids of substomatal vesicles, infection hyphae, and haustorial mother cells show complex protease patterns on polyacrylamide gels containing gelatin as substrate. The majority of the rust proteases can be classified as metallo-, including Ca²⁺-stabilized proteases. The presence of substrate is not required for synthesis of the enzymes. The extracellular proteases, in contrast to intracellular enzymes of this fungus, specifically degrade fibrous, hydroxyproline-rich proteins. Since such proteins are important in plants for cell wall stability and play a role in defense against fungal pathogens, the extracellular proteases of U. viciae-fabae may be involved in localized breaching of the host cell wall.

Nitrate-nonutilizing (nit) mutants of Glomerella graminicola were recovered by selecting chlorate-resistant sectors. Heterokaryons were formed by complementation between two different classes of nit mutants. Complementation groups were distinguished in nitrogen feeding tests and segregated as two, unlinked genes among random progeny of sexual crosses. The two genes are comparable to those encoding the nitrate reductase enzyme and one of a series of molybdenum cofactors in Aspergillus nidulans and Neurospora crassa. Heterokaryon tests were reliable indicators of allelic and dominance relationships between mutations with similar phenotypes. Vegetative compatibility (VC) between two strains of G. graminicola appeared to be regulated by approximately five unlinked VC loci, analogous to those described for other fungi.

Confocal microscopy in mycological research. Experimental Mycology 18, 275-293. Confocal microscopy is a revolutionary advance in light microscopy. Utilizing the latest laser, computer, and imaging technologies, the technique offers biologists a unique form of cell and/or subcellular visualization. The principal feature of confocal microscopy is that it permits “optical sectioning” rather than mechanical sectioning of both thin and relatively thick microscope specimens in fluorescence and reflection imaging modes; the resulting images have far better resolution and contrast than can be obtained with a conventional light microscope. This capability is achieved by using a monochromatic laser light in a scanning raster across a specimen. When a confocal pinhole is placed in the light path between the sample and the detector, most out-of-focus light is removed. Digitized confocal images may be further analyzed or improved by image processing and 3-D reconstruction. Laser scanning confocal microscopy can be successfully applied to a variety of fungal problems including cell dynamics, fungus-host interactions, organelle structure and function, or cytoskeletal localization and cytochemistry using an array of various reporters, in virtually any sample that can be viewed by conventional light microscopy. It is particularly advantageous for examining thick fungal samples or in host-pathogen interactions where the fungus is embedded deep within the host tissue. As with any technique, some artifacts and difficulties are inherently associated with laser scanning microscopy and computer processing of the resulting images. The advantages and disadvantages of confocal microscopy must be considered when determining its potential applications for specific mycological samples

The effect of substrate phosphorylation on the susceptibility to proteolytic cleavage by purified aminopeptidase from Saccobolus platensis was investigated using the model heptapeptide L-R-R-A-S-L-G. Phosphorylation of serine greatly altered the action of peptidase producing a fragment, A-S(P)-L-G, insensitive to further attack by the peptidase. The action of peptidase was tested on peptides generated by subtilisin digestion of fungal cytosolic proteins labeled in vivo with [³H]leucine and phosphorylated in vitro with the catalytic subunit of cyclic AMP-dependent protein kinase. Phosphopeptides were enriched by gel filtration through P-2 columns. After exhaustive exopeptidase degradation the peak of [³²p]phosphopeptides remained mostly unchanged. Removal of phosphate with alkaline phosphatase prior to treatment with peptidase produced a 12% liberation of [³H]leucine. The results support the idea that phosphorylation influences final protein processing.

We used fluorescence microscopy to examine the positioning of the two nuclei, the configuration of the mitotic apparatus, and the site of septation during conjugate division in eight dikaryons homozygous for either one α-or one of the seven β-tubulin mutations as well as the parental wild-type dikaryon of Coprinus cinereus. In the wild-type dikaryon, more than 90% of the mitotic apparatus had distinct asters, whereas in the mutants the frequency of the apparatus with distinct asters was more or less lowered. In one of the β-tubulin mutants, BEN 193, the defect of astral microtubules was most remarkable and 75% of the mitotic apparatus lacked astral microtubules, although spindle formation and elongation occurred normally. In BEN 193, the two nuclei were positioned normally during conjugate division in the majority of hyphal cells examined, despite the defect of astral microtubules; the leading nucleus divided in the clamp and the second nucleus in the main hypha just beneath the clamp. This result provided evidence against a direct involvement of astral microtubules in the positioning of the two nuclei during conjugate division. In BEN 193, however, septation in the clamp was disturbed in spite of the fact that a mitotic nucleus was positioned normally in the clamp. In the mutant, the actin ring, which forms prior to septation, was also disturbed in its angle in the clamp. These results strongly suggested that astral microtubules control septation in the clamp.

Structure and properties of casein kinase Its from Allomyces arbuscula phosphorylating serine residues. Experimental Mycology 18, 349–362. Two casein kinase (CK) activities have been purified from the aquatic fungus Allomyces arbuscula and have been referred to as CK IIA and CK IIB. Both enzymes have the properties characteristic of animal and yeast casein kinase II, i.e., tetrameric holoenzyme, ability to use ATP as well as GTP as donor of phosphate group in phosphotransferase reactions, inhibition of kinase activity by heparin, and inability to use basic protein like histone H1 as substrate. The two enzymes differ from each other by their affinity to DE-52-cellulose, CK IIA does not bind to DE-52 while CK IIB does. The kinetic parameters of the two enzymes also differ. The K_m of CK IIA has been found to be 7.7 μM, 20.83 μM, and 0.96 mM and that of CK IIB 22.2 νM, 41.7 ~LM, and 1.86 mM, for casein, ATP, and Mgt²⁺ , respectively. Both α and β subunits of CK IIA and CK IIB undergo autophosphorylation. Polylysine is inhibitory to autophosphorylation of a subunit whereas spermine, protamine, and histone H1 are stimulatory. Phosphoamino acid analysis showed that serine was the phospho-accepting amino acid. The phosphopeptide analysis showed that the trypsin recognition sites of the α and β subunits in CK IIA and CK IIB are different.