Substrate Hydrophobicity and Adhesion of Uromyces Urediospores and Germlings

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.     

Deformation of Stomatal Guard Cell Lips and Microfabricated Artificial Topographies during Appressorium Formation by Uromyces

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.     

Superoxide Dismutase: A Differentiation Protein Expressed in Uromyces Germlings during Early Appressorium Development

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.     

Imaging of total calcium in urediospore germlings of Uromyces by ion microscopy.

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.     

The effect of pH and K+ on appressorium formation byUromyces appendiculatus urediospore germlings

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 Ca²⁺ and K⁺.     

The microtubule cytoskeleton in hyphae ofUromyces phaseoli germlings: Its relationship to the region of nucleation and to the F-actin cytoskeleton

Summary The microtubule and F-actin cytoskeleton of nondifferentiated germlings ofUromyces phaseoli was studied using immunofluorescence methodologies. The microtubules were oriented mostly parallel to the longitudinal axis of the hypha. Microtubule depolymerizing agents, such as cold, demecolcine, griseofulvin and nocodazole, were effective in destroying the microtubule network, but not the F-actin system. Repolymerization of microtubules, following release from these agents, occurred first in the hyphal apices and not near the nuclei or spindle pole bodies. It was concluded that the microtubule nucleating region in such fungal cells is located in the apical regions. Enhanced microtubule arrays were visualized following incubation of the cells in taxol, an agent known to favor microtubule polymerization.     

Adhesion of Cochliobolus heterostrophus Conidia and Germlings to Leaves and Artificial Surfaces

Braun, E. J., and Howard, R. J. 1994. Adhesion of Cochliobolus heterostrophus conidia and germlings to leaves and artificial surfaces. Experimental Mycology 18, 211-220. We have examined the nonspecific attachment of Cochliobolus heterostrophus germlings to a variety of surfaces (glass, cellophane, Mylar, polystyrene, Teflon, maize leaves) in an effort to more fully characterize this important stage of pathogenesis. Washing experiments showed that conidia began adhering to glass just prior to germ tube emergence, about 20 min after hydration and inoculation. By 50-60 min after inoculation, over 90% of the germinating conidia resisted washing and remained firmly attached. Similar results were obtained with the other surfaces. Both sodium azide and cycloheximide prevented attachment, indicating that metabolic activity was required for adhesion. Light microscopy and cryo scanning electron microscopy were used to document a temporal and spatial relationship between attachment, appearance of extracellular matrix materials, and germ tube emergence. Attachment of conidia to the substratum was correlated with the appearance of extracellular material exuded from the tips of conidia just prior to germination. The two-layered sheath of matrix materials associated with germ tubes also surrounded appressoria and appeared to aid in attachment of these structures to leaves and artificial surfaces. We conclude that extracellular matrix is produced and/or secreted within 20 min of hydration and serves in the nonspecific attachment of germlings to the substrate.     

Development of an apparatus and protocol for the efficient isolation of bacteria and yeasts that attach to Botrytis cinerea germlings

An apparatus and protocol for the efficient and consistent isolation of bacteria and yeasts with the ability to attach to germlings of Botrytis cinerea is described. The study focused on minimising microbial contamination by bacteria or yeasts which do not attach to the pathogen B. cinerea but which interact with materials used in the equipment and would otherwise be isolated along with target microbes. After development, the assay reduced the contamination rate to 1–2 cells per 100 added and this was found to be a satisfactory level for the selection of microbial attachers to fungal hyphae. One or more phenotypes of microbes that adhered to B. cinerea germlings were found in 97% of the 70 samples collected from phylloplane washings and processed using this assay.     

Recovery and development of parasexual fusion products inEnteromorpha linza (L.) J. Ag. (Ulvales,Chlorophyta)

Newly released zoospores fromEnteromorpha linza (L.) J. Ag. lack significant cellulose cell wall material and are suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca^{+ +}, PEG and centrifugation. Treated zoospores settled on glass cover slips within 3 h and were examined microscopically at 1000 ×. Presumptive fusion products were identified by their larger size and presence of twin chloroplasts and eyespots. Unfused zoospores adjacent to fusion cells were killed by 2–3 min exposure to blue light (410–490 nm) from a high pressure mercury illuminator. Unexposed fusion cells developed into uniseriate germlings within 10 days at which stage they could be readily identified at 60 × with a dissecting microscope and isolated by micropipette. Ten-day germlings from both unfused zoospores and fusion cells were stained with the DNA-localizing fluorochrome hydroethidine and relative nuclear DNA content determined with epi-(incident) UV illumination. All germlings were found to be uninucleate. Germlings from unfused zoospores had haploid nuclei with 1N = 10 and 1C and 2C levels of DNA, while germlings from fusion cells had diploid nuclei with 2N = 20 and 2C and 4C levels of DNA. These result are interpreted as evidence of karyogamy following parasexual zoospore fusions. Isolated diploid germlings, cultured for 10 weeks were found to conserve their 2N chromosome complements and elevated levels of nuclear DNA. Although most diploid germlings were morphologically similar to haploid control plants, some exhibited ‘gigas’ characteristics, including larger cells, chloroplasts, and nuclei. These results are discussed in terms of unique phenotypes that result when nuclear and organellar genes are combined in different ways.     

Flow cytometric sorting of the filamentous fungus Trichoderma reesei for improved strains

Metabolic measurements and screening of Trichoderma reesei have conventionally been performed during the hyphal stage of fungal development. To determine if flow cytometric measurements of protein expression could be made on germinating spores we created a gene construct, placing the Renilla reniformis green fluorescent protein gene under control of the cellobiohydrolase I (cbh1) promoter and terminator of T. reesei. This vector was transformed into T. reesei and GFP expression was measured in germlings by flow cytometry. Fluorescence associated with GFP expression was observed in germlings grown under conditions known to induce cellulases in Trichoderma. Spores were mutated using UV light and germinating spores were screened for increased GFP expression using high-speed cell sorting, to select for strains with genetic changes associated with increased protein expression. Secondary screens for cellulase production were conducted in microtitre plates. Flow cytometric screening of germinating spores expressing GFP yielded a mutant with improved ability to hydrolyse biomass.     

The involvement of F-actin inUromyces cell differentiation: The effects of cytochalasin E and phalloidin

Summary The role of F-actin in cell differentiation ofUromyces appendiculatus (bean rust fungus) germlings was examined by treating differentiating and nondifferentiating germlings with the actin-binding drugs cytochalasin E (CE) and phalloidin. Prolonged exposure of urediospores to 5×10^–3–5 × 10^–5 M CE induced nuclear division in up to 28–45% of the resulting germlings, whereas the rate of mitosis in established germlings exposed to these concentrations of CE was significantly lower (4–11%). Germlings treated with CE shifted from polarized apical growth to spherical expansion, cytoplasmic microfilaments were depolymerized, and nuclear inclusions became enlarged. Differentiating germlings exposed to a 10 minute pulse of 5×10^–6M CE before the initiation of septum formation prevented the establishment of the F-actin septal ring and growth of the crosswall delimiting the appressorium. Although these CE treatments resulted in morphological and nuclear events similar to those occurring during normal appressorium formation, transient microfilament depolymerization was not sufficient to induce differentiation. Phalloidin stabilized cytoplasmic microfilaments, especially posteriorly-located microfilaments, but did not affect differentiation, nor did it significantly inhibit the effects of CE.Abbrevations CE cytochalasin E - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - F-actin filamentous actin     

Micro-autoradiographic studies on host-parasite interactions

Tritium labeled uredospores of Uromyces phaseoli were produced be feeding the host, Phaseolus vulgaris, with ³H-orotic acid. These spores were allowed to germinate on and to penetrate into a bean leaf. 24 hrs after inoculation, the bean rust had formed the first haustorium. All fungal structures, including the fungus walls, were heavily labeled. No label could be detected in the cells that had come into contact with the hyphae. In the infected host cell, the haustorium was labeled heavily, but the sheath around the haustorium and the host cell remained free of label. These results indicate that no detectable amounts of label leach from the bean rust into the host at this stage of infection although it is known that the rust takes up many metabolites. Since the sheath remains free of label and all fungal structures are evenly labeled, it is concluded that the sheath is formed by the host.     

An investigation into the involvement of defense signaling pathways in components of the nonhost resistance of Arabidopsis thaliana to rust fungi also reveals a model system for studying rust fungal compatibility

Seventeen accessions of Arabidopsis thaliana inoculated with the cowpea rust fungus Uromyces vignae exhibited a variety of expressions of nonhost resistance, although infection hypha growth typically ceased before the formation of the first haustorium, except in Ws-0. Compared with wild-type plants, there was no increased fungal growth in ndr1 or eds1 mutants defective in two of the signal cascades regulated by the major class of Arabidopsis host resistance genes. However, in the Col-0 background, infection hyphae of U. vignae and two other rust fungi were longer in sid2 mutants defective in an enzyme that synthesizes salicylic acid (SA), in npr1 mutants deficient in a regulator of the expression of SA-dependent pathogenesis related (PR) genes, and in NahG plants containing a bacterial salicylate hydroxylase. Infection hyphae of U. vignae and U. appendiculatus but not of Puccinia helianthi were also longer in jar1 mutants, which are defective in the jasmonic acid defense signaling pathway. Nevertheless, haustorium formation increased only for the Uromyces spp. and only in sid2 mutants or NahG plants. Rather than the hypersensitive cell death that usually accompanies haustorium formation in nonhost plants, Arabidopsis typically encased haustoria in calloselike material. Growing fungal colonies of both Uromyces spp., indicative of a successful biotrophic relationship between plant and fungus, formed in NahG plants, but only U. vignae formed growing colonies in the sid2 mutants and cycloheximide-treated wild-type plants. Growing colonies did not develop in NahG tobacco or tomato plants. These data suggest that nonhost resistance of Arabidopsis to rust fungi primarily involves the restriction of infection hypha growth as a result of defense gene expression. However, there is a subsequent involvement of SA but not SA-dependent PR genes in preventing the Uromyces spp. from forming the first haustorium and establishing a sufficient biotrophic relationship to support further fungal growth. The U. vignae-Arabidopsis combination could allow the application of the powerful genetic capabilities of this model plant to the study of compatibility as well as nonhost resistance to rust fungi.     

Embryology of zygote and development of germling in Sargassum vachellianum Greville (Fucales,Phaeophyta)

Sargassum vachellianum Greville is one of the most important members in the subtidal seaweed flora along the coasts of the Eastern China Sea. In order to understand the embryology of zygotes and development of germlings in S. vachellianum, we performed artificial ripening, systematically observed the morphological changes and development of the fertilized eggs and germlings, and explored the effects of culture conditions on the growth of the germlings. The receptacles matured, and eggs and sperm were released after 1–5 days of culture at 21–24 °C under 60–90 μmol photons m^?2 s^?1 (14L:10D). Once fertilized, zygotes immediately detached from the female receptacles and began to germinate. Eight nuclei in the released fresh zygote soon begun to fuse, forming a large central nucleus, and underwent two horizontal divisions to produce a small “original rhizoid cell,” which eventually formed rhizoids after several divisions, and three other cells which eventually formed landmine-like germlings after several continuous divisions. The germlings then formed rhizoids and attached onto the bottom of the flask within 12 h of culture. The growth and development of the germlings younger than 20 days were significantly influenced by the culture temperature and light, with the optimal conditions being 21 °C and 40 μmol photons m^?2 s^?1. However, for germlings older than 30 days, the optimal conditions were 24 °C and 60 μmol photons m^?2 s^?1.     

Endocytosis of 1,3-β-glucans by broad bean cells at the penetration site of the cowpea rust fungus (haploid stage)

Te penetration hypha of basidiospore-derived infection structures of the cowpea rust fungus (Uromyces vignae Barclay) in epidermal cells of the nonhost, broad bean (Vicia faba L.), was studied with the electron microscope after high-pressure freezing and freeze substitution. After fungal invasion of the epidermis, a plug in the penetration hypha separated the infection structures on the cuticle from the intraepidermal vesicle of the fungus. The plug and the fungal cell wall reacted with a polyclonal 1,3-β-glucan antibody. The plug in the haploid stage seems to have a task similar to the septum formed in the diploid stage of the fungus. Around the penetration hypha, the plant wall stained darkly and a papilla was deposited by the plant. In the papilla, 1,3-β-glucans were labelled by a monoclonal and a polyclonal antibody. In the infected epidermal cell, clathrin-coated pits, coated vesicles, partially coated reticula and multivesicular bodies were found. The contents of the coated pits, coated vesicles, partially coated reticula and multivesicular bodies bound to monoclonal and polyclonal 1,3-β-glucan antibodies. Accumulation and uptake of this paramural material into the plant cell by endocytosis is concentrated at the fungal penetration site. It may influence the host-parasite interaction.     

Photoacclimation characteristics of Sargassum thunbergii germlings under different light intensities

The photosynthesis and growth responses of Sargassum thunbergii germlings to different light intensities (10, 60, and 300 μmol photons m^?2 s^?1) were investigated. Maximum photochemical efficiency (F _v/F _m), rapid light curves (RLCs), and photochemical and non-photochemical quenching (qP and NPQ) were estimated by a pulse amplitude-modulated fluorometer. The photosynthesis of S. thunbergii germlings exhibited different properties to optimize light capture and utilization. The excitation pressure (1???qP) was rapidly increased to approximately 0.27 showing that germlings responded to high light by chronic photoinhibition with an accumulation of closed reaction centers, which ultimately resulted in a slow growth. This was accompanied by a reduced F _v/F _m with time and a development of high capacity for NPQ. Although F _v/F _m in moderate-light germlings did not fully recover overnight, germlings demonstrated a less severe chronic photoinhibition considering the reduced degree of excitation pressure accumulation of approximately 0.15. The relative stability of photosynthetic capacity (rETR_max, E _k, and α) could endow germlings with the highest relative growth rate (RGR) of approximately 9.3 % day^?1 in moderate light. By contrast, low-light germlings demonstrated high F _v/F _m and F _o, corresponding high α collectively suggested greater efficiency of light absorption and energy transformation. Sustained increases in electron transport capacity (rETR_max and E _k) occurred in low-light germlings, which resulted in a stable RGR of over 8.2 % day^?1. Consequently, S. thunbergii germlings are considered to prefer low light regimes and have a relative capacity of moderate and high light tolerance. However, the light acclimation to oversaturating conditions is at the cost of slow growth to maintain survival.     

Identification and localization of calcium-dependent protease II inNeurospora crassa andUromyces appendiculatus

Summary The existence of Ca²⁺-dependent protease II in crude extracts ofNeurospora crassa andUromyces appendiculatus was demonstrated by immunoblotting using specific antibodies. In both extracts two immunoreacting bands were observed. The molecular mass of the major band inN. crassa corresponded to 37 kDa, while that inU. appendiculatus was 43 kDa, similar to that previously reported forAllomyces arbuscula. Immunofluorescence of the enzyme was predominantly localized in the apical regions of germlings and growing hyphae, suggesting a functional role for the enzyme in hyphal growth.     

Dynamics of the Establishment of Multinucleate Compartments in Fusarium oxysporum

Nuclear dynamics can vary widely between fungal species and between stages of development of fungal colonies. Here we compared nuclear dynamics and mitotic patterns between germlings and mature hyphae in Fusarium oxysporum. Using fluorescently labeled nuclei and live-cell imaging, we show that F. oxysporum is subject to a developmental transition from a uninucleate to a multinucleate state after completion of colony initiation. We observed a special type of hypha that exhibits a higher growth rate, possibly acting as a nutrient scout. The higher growth rate is associated with a higher nuclear count and mitotic waves involving 2 to 6 nuclei in the apical compartment. Further, we found that dormant nuclei of intercalary compartments can reenter the mitotic cycle, resulting in multinucleate compartments with up to 18 nuclei in a single compartment.     

Synthesis and antifungal activity of two novel spermidine analogues

Two spermidine analogues were synthesised and examined for antifungal activity. Both compounds used as 1 mM post-inoculation sprays reduced infection of barley seedlings by the powdery mildew fungus, Erysiphe graminis f.sp. hordei, infection of broad bean seedlings by the rust fungus, Uromyces viciae-fabae, and infection of apple seedlings by the powdery mildew fungus, Podosphaera leucotricha. Since these fungal pathogens cannot be cultured axenically, the effects of the two spermidine analogues on mycelial growth in vitro, as well as preliminary investigations on polyamine biosynthesis, were undertaken using the oat stripe pathogen, Pyrenophora avenae. Although neither compound affected radial growth of the fungus on plates, both analogues reduced fungal biomass in liquid culture substantially. The two spermidine analogues, used at a concentration of 1 mM, had no significant effect on the conversion of labelled ornithine into polyamines in P. avenae.