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Colletotrichum graminicola, like many plant pathogenic fungi develop appressoria on germling apices, to facilitate penetration of their host. Induction of these structures occurs after contact with the host surface has been established by the germling. Surface contact and subsequent development of appressoria by germlings of C. graminicola was assessed using interference-reflection microscopy (IRM) and microfabricated pillared silicon substrata. Observations with IRM revealed that under low nutrient conditions, 90% of the germlings developed appressoria once they established 4.5 microm of continuous contact with the substratum. Substrata bearing pillars < or =5 microm in width supported < or =10% appressoria; however, as pillar width was increased the percentage of appressoria formed increased in a sigmoid fashion to a maximum of 80%. The percentage of appressoria produced experimentally on these surfaces was compared to data sets generated from a model designed to calculate the probability of appressorium development on similar pillar arrays at various germ tube contact lengths. These results indicate that germ tubes of C. graminicola require more than 4microm of continuous contact with a hydrophobic substratum for induction of appressoria.

Pycnidiospores of Phyllosticta ampelicida germinate only after they become attached to a hydrophobic substratum. They do not readily attach or germinate on hydrophilic surfaces or on nutrient-rich media. The properties of the spore surface that function in attachment and signal mediation for germination were investigated in this study. Pycnidiospores were surrounded by a sheath that bound Concanavalin A (Con A)-TRITC and only weakly bound wheat germ agglutinin (WGA)-FITC. Galanthus nivalis lectin-FITC selectively bound primarily to the apical region of the sheath associated with the appendage. Con A, but not WGA or GNL, disrupted spore attachment. The Con A effect on spore attachment was significantly reduced when the lectin was pre-mixed with methyl-D-α-mannoside. The sheath as well as the associated appendage was removed with either Novozym 234 or crude β-glucuronidase. Such sheathless spores were initially adhesion incompetent but, by 40 min, the spores regained their ability to adhere to hydrophobic surfaces through the secretion of material that had an affinity toward colloidal gold. Non-treated pycnidiospores surrounded by sheath remained spatially separate; however, sheathless spores readily agglutinated. Addition of cations enhanced attachment to both hydrophobic and hydrophilic surfaces presumably through the masking of electronegative groups on the sheath or by reduction of the ordered water-shell surrounding the sheath. In particular, Ca 2+ induced both attachment and germination on normally non-inductive hydrophilic surfaces, suggesting an additional role for Ca 2+ in signal mediation for germination.

Calcium has been implicated in growth and appressorium formation of urediospore germlings of the bean rust fungus, Uromyces appendiculatus. Using ion microscopy, a mass spectrometry-based imaging technique, intracellular stores of calcium were analyzed by direct imaging of total calcium in frozen freeze-dried germlings. Calcium concentration was calculated by ratioing and spatially registering (40)Ca to (12)C signals. Intracellular distributions of total potassium, sodium, magnesium, and carbon were similarly imaged in the same germlings for a direct comparison of their localizations to total calcium. Calcium was remarkably heterogeneous with highest concentrations (2 to 10 mM) in the mid-region of the germling between the nuclei and the apex. A similar distribution of Ca(2+) (assessed using Fluo-3) was also noted sequestered in organelles in live germlings. Distributions of remaining elements (K, Na, Mg, and C) were mostly homogeneous throughout the cytoplasm and nuclei of the fungal cell. The K/Na ratio ranged from 17 to 31.

Germlings of Uromyces appendiculatus respond to topographical stimuli and develop appressoria. During this period several genes are expressed, one of which is Inf 24. Germlings were microinjected with a sense and antisense fragment of the ORF of Inf 24. Sense ( Inf 24-S), 5′-ATG AGT GCT TCA TCG CAG CAG CAG-3′ fragment did not affect germling response to the topographical stimuli and the developed appressoria. Microinjection of antisense ( Inf 24-RC), 5′-CTG CTG CTG CGA TGA AGC ACT CAT-3′ blocked gene transcription and appressoria were rarely formed. Injection of Inf 24-RC into already formed appressoria did not inhibit continued development of subsequent infection structures, e.g. penetration peg, sub-stomatal vesicle.

Pycnidiospores of Phyllosticta ampelicida, the causal agent of black rot of grape, were found to germinate only on substrata on which they were firmly attached. Such surfaces were poorly wettable and had advancing contact angles (straight thetaa) formed by a water drop of80deg;, e.g., grape leaf, polystyrene, Teflon, polycarbonate, collodion, and glass treated with the silanes n-octadecyltrichlorosilane, dimethyldichlorosilane, or diphenyldichlorosilane. When pycnidiospores were deposited on more wettable surfaces they did not attach firmly and did not germinate. Such highly wettable surfaces had straight thetaa/= 40deg; and were represented by heat-treated glass, cellophane, nutrient- and water-agars, polystyrene treated with UV-irradiation or sulfuric acid, and glass silanized with n-2-aminoethyl-3-aminopropyltrimethoxysilane, n-(trimethoxysilylpropyl)ethylenediamine triacetic acid trisodium, or 3-aminopropyltriethoxysilane. Adhesion of pycnidiospores was assessed with and without a hydraulic shearing force. Pycnidiospore adhesion occurred over several minutes in distilled deionized water, unless it was first acidified, which decreased attachment time to0.03 s. Attachment of pycnidiospores treated with sodium azide, formaldehyde, or boiled in water for 10 min was similar to nontreated conidia. Possible mechanisms of adhesion of the conidia to surfaces include hydrophobic and ionic interactions.

Lamboy, J. S., Staples, R. C., and Hoch H. C. 1995. Superoxide dismutase: A differentiation protein expressed in Uromyces germlings during early appressorium development. Experimental Mycology 19, 284-296. Germlings of the bean rust fungus Uromyces appendiculatus detect penetration sites on the surface of the host leaf by thigmosensing topographical features. Within 2-4 min after the apex of a urediospore germ tube encounters the cuticular lip of a stomate, the germling ceases polarized growth and begins to swell over the aperture. The mechanism by which the cells detect topographical signals is not understood; however, previous experiments indicated that the initiation process does not involve de novo gene expression. In order to detect posttranslational modifications, the protein profiles of induced and noninduced germlings were compared at the earliest stages of appressorium formation, and a 21-kDa differentiation protein was identified by a shift in isoelectric point. The N-terminal amino acid sequence exhibited homology with superoxide dismutase (SOD), and antibodies to a synthetic peptide fragment of the respective sequence recognized cooper/zinc isozymes of SOD in electroblots of native gels. Electroelution of the active enzyme bands and separation by SDS-PAGE indicated that the 21-kDa protein is a component of a tetrameric 85-kDa SOD.

Terhune, B. T., and Hoch, A. C. 1993. Substrate hydrophobicity and adhesion of Uromyces urediospores and germlings. Experimental Mycology 17, 241-252. Adhesions of urediospores and urediospore germlings of Uromyces appendiculatus, the bean rust pathogen, to various substrata was evaluated with regard to surface wettability. A range of surface wettabilities, or conversely hydrophobicities, was obtained by coating glass or quartz substrates with various organosilanes. Adhesion of urediospores or germlings was evaluated after the spore or germling laden-silanized surfaces were washed. Both urediospores and germlings adhered most tenaciously to surfaces with wettability ratings less than 30. Such surfaces were polystyrene and glass treated with dimethyldichlorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichorosilane, and diphenyldichlorosilane. The degree of germling contact to the various surfaces correlated closely with hydrophobicity and with the adhesion of germlings. Induction of appressoria on quartz substrates bearing inductive topographies (0.5-μm-deep grooves) was also closely associated with the degree of hydrophobicity.

Correa, A., Jr., and Hoch, H. C. 1993. Microinjection of urediospore germlings of Uromyces appendiculatus. Experimental Mycology 17, 253-273. Urediospore germlings of Uromyces appendiculatus were microinjected with a variety of materials to assess the applicability of microinjection methodologies for studying fungal cell growth and development. Until now, hyphal cells have not been successfully microinjected without first being pretreated with osmotica to lower the turgor pressures of the cells. We describe the development of a hydraulicly actuated system and protocols for successfully microinjecting Uromyces cells on a routine basis. Synthetic fluorocarbon and silicone fluids, various fluorophore-conjugated dextrans, rhodamine-conjugated phalloidin, rhodamine 123, cyclic AMP, and cytochalasin E were all microinjected into living urediospore germlings that grew or responded in ways consistent with expected pharmacological effects of the injected material. Germlings that were microinjected with synthetic fluids maintained the ability to sense and respond to inductive topographies for the formation of appressoria. Materials were injected at volumes of ≤0.5 to ≤400 fl. The results of this investigation indicate that fungal cells can be successfully microinjected with a variety of materials and, importantly, continue to function as normal cells.

Terhune, B. T., Bojko, R. J., and Hoch, H. C. 1993. Deformation of stomatal guard cell lips and microfabricated artificial topographies during appressorium formation by Uromyces. Experimental Mycology 17, 70-78. The inductive signal, stomatal guard cell lips or 0.5-μm-high plastic ridges, for appressorium formation in urediospore germlings of Uromyces appendiculatus was examined for signs of physical deformation during the course of appressorium development. The normally erect stomatal guard cell lips were usually observed prostrate at most stages of appressorium development; and, there were no persistent or significant indentations into the fungal cell that might have been caused by the topographical features. To further evaluate the events that occurred at the lip-appressorium interface in situ, polycarbonate and/or polystyrene ridges (0.5 μm high and 0.25 μm wide), mimicking stomatal guard cell lips of Phaseolus vulgaris, were cast on specifically microfabricated silicon templates. These artificial lips induced appressoria and became deformed approximately 30 min after initial contact by the germ tube apex as recorded and observed with time-lapsed video light microscopy. The collapsed nature of the ridges was further evaluated by both transmission and scanning electron microscopy. These results suggest that mechanical forces imposed by a combination of cell turgor pressure and adhesion of the appressorium to the substrate were responsible for deformation of the inductive topography.

Uromyces appendiculatus germlings attached to a plastic surface and differentiating in response to contact with inductive ridges formed appressoria most efficiently in the pH range 5.5–6.5. Formation of appressoria was inhibited by increased concentrations of Ca 2+ and K + .

Appressorium development in Uromyces appendiculatus was studied to establish temporal and spatial parameters possibly important in signaling mechanisms in this fungus. It was determined that urediospore germling apices sensed inductive topographies, e.g., precisely shaped ridges 0.5 μm high by 2.0 μm wide, within 4 minutes after initial contact. The area in the germling that senses such topographies was located on the substrate side of the cell and within 6.0 μm of the apex. These conclusions were based on observed changes in the rate of germling growth and nuclear migration as well as the timing of apical tip swelling that began 4 minutes after contact. DNA synthesis during appressorium development occurred only after the first nuclear division and was probably in preparation for the second mitosis that normally occurs in the substomatal vesicle. Treatments with the DNA synthesis inhibitors hydroxyurea and actinomycin D reduced the DNA content of individual nuclei only after nuclear division. These results indicate that the nuclei in urediospore germlings were already in the G2 phase when germination began. Formation of a septum during appressorium development was initiated 45–50 minutes after contact of the germling with the ridge and was completed within 60 minutes after all of the cytoplasm had entered into the swollen apex.

Germlings ofUromyces appendiculatus induced by exposure to 28.5°C heat for 1.5 h developed infection structures at nearly the same rate as those induced thigmotropically. Typically, the microtubules began to rearrange within 10 minutes of the start of heat treatment; nuclear migration followed. Mitosis started after 36 minutes. Appressorium development was accompanied by the appearance of six heat shock proteins, but not by the thigmotropic-specific proteins always observed during contact-induced development.

Ten millimolar cyclic AMP (cAMP) or cyclic GMP (cGMP) induced bean rust uredospore germlings to undergo one round of mitosis and to form septa, processes normally associated with appressorium formation. To assess the possibility of cyclic nucleotide regulation of bean rust development, we used an 8-azido-[32P]cAMP photoaffinity probe to identify three cyclic nucleotide binding peptides. The peptides bound either cAMP or cGMP. The phosphorylation of one peptide in uredospore germling extracts by [γ-32P]ATP was stimulated by either 1 μM cAMP or cGMP, but only in the presence of 10 mM Na2MoO4, a phosphatase inhibitor. Uredospores contain about 1500 and 23 pmol cAMP and cGMP/g dry wt, respectively, as determined by radiobinding assays.

Light microscopic localization of N-acetyl-β- d -glucosaminidase activity by the hydrolysis of α-naphthyl N-acetyl-β- d -glucosaminide and by the subsequent coupling of the released α-naphthol with Fast Red B and p-nitrobenzene diazonium tetrafluoroborate was obtained in Mucor racemosus. Enzyme activity appeared as discrete spherical sites, from d -glucosaminide was obtained with cell-free extracts of M. racemous, Rhizoctonia solani, Corticium sp., and Phycomyces nitens; however, cytochemical localizations of enzyme activity were observed only in M. racemosus: Four cytochemical substrates and 11 coupling agents were investigated. Only α-naphthyl N-acetyl-β- d -glucosaminide was satisfactory as a substrate. Fast Red B and p-nitrobenzene diazonium tetrafluoroborate were the only coupling reagents that yielded stable discrete reaction products.

Induction of differentiation of uredospore germlings of the bean rust fungus (Uromyces phaseoli) by K+ offers a new approach to the study of the differentiation process. Plastic membranes, required to induce infection structures thigmotropically, often interfere with biochemical analyses, and these may now be omitted from experimental protocols when this is advantageous. In this study, germling metabolism was surveyed for changes which occur in response to potassium salts. It was found that K+ induced a small but significant shift in metabolism toward an increased rate of protein synthesis at the expense of sugars. Studies on respiration showed that a shift occurred about the time of appressoria development. An increased ionic flux was not a part of the inductive effect. Continued synthesis of protein was required for appearance of the appressoria. DNA replication and mitosis were also induced by the K+ ion. Although the induction of infection structures by either chemicals or contact stimuli starts mitosis, the two processes may be different.

This review provides a general overview of a range of fluorochromes available with a brief description of their properties, uses and limitations in the study of fungi and other cells. Attention is also drawn to ways of optimizing visualization of images in the fluorescent microscope as well as to new developments in this field

Uredospores of the bean rust fungus ( Uromyces phaseoli ) germinating on either agar or water buffered with Tris-HCl or phosphate buffers were induced to form complete infection structures (appressoria, peg, and vesicles) by a variety of potassium salts. The optimum pH was 7.0, and the ED 50 in 2.5 m M Tris-HCl was about 5 m M K + . With 50 m M K + , 72% of the germ tubes formed infection structures. Differentiation was accompanied by nuclear division. At low levels of K + , Ca 2+ potentiated the stimulation by potassium ion, but alone, calcium ion did not induce differentiation. The start of nuclear division and development of appressoria began between 8 and 9 hours after sowing spores onto potassium phosphate-buffered media. Uredospores of the wheat stem rust fungus ( Puccinia graminis f. sp. tritici ) were only slightly stimulated to differentiate by K + , since about 5% of the germ tubes formed infections structures. The optimum pH for response by the wheat rust fungus was between 6.2 and 6.6, but the ED 50 was not established.

Nuclear division occurred at significantly greater frequency in uredospore germlings of Uromyces phaseoli (Pers.) Wint. stimulated with a heat shock or by sonication, provided that the spores were germinated on membranes. Nuclear division also occurred in significant numbers of germlings exposed to one of several antimicrotubule and antimicrofilament drugs. Small numbers of often irregularly shaped infection structures usually developed after any of these treatments. Failure of the spores to respond to chemical or physical stimuli when germinated on water suggests that association with a compatible surface activates a stimulus receptor in the germ tube. Nuclear division in the spores in response to treatments by antimicrotubule and antimicrofilament agents subsequent to germination on membranes suggests that depolymerization of the cytoplasmic microtubules and microfilaments in the germ tube induced nuclear division in the rust uredospore germlings.

Toroidal swellings of dolipore septa of Laetisaria arvalis were typical of Homobasidiomycetidae septa when preserved by conventional chemical fixation procedures using glutaraldehyde followed by osmium tetroxide. The toroidal swellings were electron translucent and were delimited by a plasmalemma, both of which were very irregular in outline. Toroidal swellings of similar septa preserved by freeze-substitution were approximately one-third the size of conventional chemically fixed specimens. Furthermore, freezesubstituted-preserved toroidal swellings were moderately electron dense with a finely granular matrix delimited by a plasmalemma and were smooth in outline. Based on the results of the two preservation procedures as well as certain modifications of them, and on the results of light microscopic examinations of septal complexes before and after chemical fixation, it was concluded that conventional chemical fixation procedures induce artifactual enlargement of toroidal swelling.

Nuclear division was efficiently induced in uredospores of the bean rust fungus ( Uromyces phaseoli ) germinated in the presence of comparatively high concentrations (10 −2 M ) of cAMP or several of its derivatives (Bt 2 cAMP and Su 2 cAMP). Synthesis of nuclear DNA was also started; however, appearance of appressoria, a cell differentiation event, was inhibited. At a lower concentration (10 −8 M ) of cAMP appressoria developed but at a low frequency (5.7%). This cell differentiation was also accompanied by nuclear division. Two inhibitors of cAMP phosphodiesterase (3-isobutyl-1-methyl xanthine, IBMX; 4-(3-butoxy)-4-methylbenzyl)-2-imidazolidinone, Ro. 20–1724), studied in anticipation that the compounds would cause the internal levels of cAMP to rise, readily induced both nuclear division and cell differentiation. Nuclei in germlings induced to differentiate by contact with inducive surfaces divided beginning about 2.5 h after the start of germination, while mitosis began after 6 and 10 h in germlings treated with Bt 2 cAMP and IBMX, respectively. Putative stimulators of adenylate cyclase (NaF, Forskolin, adenosine, 2′-deoxyadenosine) induced mitosis in 8, 9, 13, and 43% of the germlings, respectively. Adenine, 3′-deoxyadenosine (Cordycepin), and 5′-adenosine monophosphate stimulated nuclear division in 9, 52, and 4.2% of the germlings, respectively. Although clearly mitogenic, the fact that high levels of cAMP also inhibited differentiation compared to the cell differentiation induced in the presence of phosphodiesterase inhibitors suggests that cAMP metabolism is closely regulated in uredospores.

Uredospore germlings of the bean rust fungus Uromyces appendiculatus display two contact-sensitive responses on leaves or certain synthetic surfaces: a specific orientation of germ tubes and an induction of appressoria at leaf stomates or scratched surfaces. Germ tube orientation, nuclear division (used as a marker for appressorium formation) and germling adhesion on scratched “Parafilm” was reduced by the proteolytic enzyme, pronasc E (ED 50 = 20–50 μg ml −1 ), but not by the heat denatured proteolytic enzyme. Forty micrograms of pronase E per millilitre significantly reduced nuclear division of germlings incubated on either a hydrophilic or a hydrophobic surface. Pronase E reduced adhesion regardless of whether the enzyme was applied during growth or after attachment had occurred, although higher concentrations were required after attachment had occurred. The data suggest that extracellular protein is required for germling adhesion to a substratum and that adhesion is required for germ tube orientation and thigmodifferentiation. We partially characterized the composition of the bean rust extracellular material and analyzed for proteins on SDS-polyacrylamide gels. Intact germlings have six predominant extracellular peptides detected by 125 I labelling. Spent culture fluid contains several different peptides; four of the five major peptides in the spent culture fluid are glycosylated.

Digital video microscopy in conjunction with the DNA-binding fluorescent probe 4′,6-diamidino-2-phenylindole was used to determine the relative DNA content of single nuclei in germ tubes of uredospores of the bean rust fungus (Uromyces phaseoli), a parasite of bean plants (Phaseolus vulgaris). The uredospores develop a series of infection structures in response to contact stimuli, and the first structure to appear, the appressorium, occupies the stomatal opening. This study was made to determine the time after the start of germination on an inductive surface that DNA replication and mitosis occur. It was found that the start of DNA replication detected by increased nuclear fluorescence power of some individual nuclei in the population was nearly coincident with mitosis between 2.0 and 2.5 h after the start of germination and that the appressorium was completed about 30 min later. The fluoresence power of nuclei was about the same before DNA replication began as at the end of mitosis; hence the germ tube nuclei were in the G1 phase of the cell cycle before mitosis.

Calmodulin has been isolated from uredospores of the bean rust fungus and purified to apparent homogeneity as judged using sodium dodecyl sulfatepolyacrylamide gels. The protein substituted for bovine calmodulin as an activator of Ca 2+ -dependent cyclic nucleotide phosphodiesterase. Its molecular mass was 15.6 kDa in the presence of Ca 2+ and 17.0 kDa in its absence. Thep I was 4.4. The amino acid composition was generally similar to those of calmodulin from other fungi.