Comparative inhibitory effects of cytochalasins on the gametangial differentiation inAllomyces arbuscula

In contrast to cytochalasin D which selectively prevents differentiation of female gametangia, cytochalasins A, B and E also masculinizeAllomyces arbuscula by preventing septation between female and male gametangia, thereby allowing the dominant expression of the male characteristics.     

Studies in the life cycle of Allomyces javanicus Kniep

Summary The life cycle of Allomyces javanicus was studied with the hanging drop method under laboratory conditions. The isolate has a life cyrle similar to that already described in A. javanicus and A. arbuscula. Planonts from resistant sporangia do not germinate directly to produce gametophytic plants, but on the contrary, the latter germinate to produce plants like their immediate parents. Female and male gametes growing in the gametangia of relatively young sexual mycelia develop into asexual mycelia after conjugation, but some of the female gametes after germination develop into asexual mycelia without conjugation.     

Fertilization-induced Chemotaxis in the Zygotes of the Watermold Allomyces

Zygotes obtained by self-fertilization of Allomyces macrogynus, strain Burma 3 and of A. arbuscula, strain Ceylon 1, behave chemotactically as do their respective zoospores. All the swarmers respond to an equimolar mixture of L-leucine and L-lysine with the response enhanced by the addition of L-Proline. The A. arbuscula swarmers also respond moderately to a mixture of L-proline with certain amino acids other than leucine and lysine whereas those of A. macrogynus do not. The gametes are not chemotactically responsive to the amino acids. Within no more than five minutes subsequent to fertilization, the zygotes become chemotactically active. The genetically-derived, approximately 95 % male or female isolates do not appear to form zygotes when crossed. The few zygotes observed in a series of attempted crosses appear to be the result of selfing by the contaminating opposite sex in each of the highly unisexual strains.     

STRUCTURE AND DEVELOPMENT OF HYPHAL SEPTA IN ALLOMYCES

Development of hyphal septa (pseudosepta) in Allomyces macrogynus begins with the formation of five or more discontinuous pieces of wall material that project inward from the hyphal wall. Lateral fusion of these projections leaves a central pore in the septum that is later filled in by centripetal deposition of wall material. However, lateral fusion of the projections is not complete; peripheral pores remain in the rim of the mature septum. The position of cytoplasmic microtubules corresponds to the position of actively moving cellular particles and organelles. Allomyces reticulatus and A. arbuscula have similar septa.     

Anomalies physio-morphologiques de la souche virosée (Bali 1) d'Allomyces arbuscula

An escape reaction of the hyphae from virus-containing strain Bali 1 of A. arbuscula was detected when intra- and interspecific Allomyces cultures were opposed on solid media. Hyphae of this virus-containing strain proliferate into witch-broom structures only detectable on solid media in which bunches of rhizoid-like structures remain more superficial than those of the control cultures, suggesting that metabolism of the Bali 1 strain is altered into a more aerobic type.     

STUDIES ON THE GENETIC CONTROL OF RESISTANT SPORANGIUM FORMATION IN ALLOMYCES

The diploid sporophyte of the phycomycetous fungus Allomyces arbuscula bears two types of sporangia: thin-walled, colorless, ephemeral zoosporangia (ZS) and thick-walled, dark-brown, resistant sporangia (RS). Normal wild-type cultures (strain Portugal IE) under standard conditions produce approximately 90% of their total sporangia as RS. These RS give the cultures a dark-brown color. A mutant was induced with UV irradiation in which the ratio of ZS to RS was shifted so that only 20% of the total sporangia are RS. These cultures are a pale, tan color. Hybrids between the mutants and wild-types produce ca. 65% RS and are also intermediate in the color of the culture. Meiotic segregation in the RS of the hybrid sporophytes gives gametophytes half of which when selfed produce mutant sporophytes and half of which produce wild-type sporophytes. The shift from RS to ZS formation is thus considered to be the result of a one-gene mutation at a locus ‘R.’ The haploid gametophytes of wild-type strains have in addition to male and female gametangia a small number (2-4%) of RS. In mutant gametophytes the percent RS has dropped to 0.1-0.2%. The proposed genotypes at the ‘R’ locus in Allomyces arbuscula are: wild-type sporophytes (RR), hybrid sporophytes (Rr), mutant sporophytes (rr), wild-type gametophytes (R) and mutant gametophytes (r).     

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.     

Developmentally regulated proteases in Allomyces arbuscula

A calcium-requiring neutral protease has been detected in the vegetative mycelia of Allomyces arbuscula. The half maximum activation of the enzyme required 0.7 mM and 2.8 mM Ca²⁺ in the crude and partially-purified preparation, respectively. Coinciding with differentiation of zoosporangia, there is a massive induction of another neutral protease which does not require Ca²⁺ for its activity and is of the serine type.     

Chemotaxis and transport of amino acids in Allomyces arbuscula

Among a number of amino acids tested, l-lysine and l-arginine are the principal attractants in the chemotaxis of the zygotes of Allomyces arbuscula. The reaction can be stimulated to a greater or lesser extent by a number of compounds chemically related to l-leucine. No relationship between transport of attracting amino acids and their effect on chemotaxis has been found.     

Zoospore Chemotaxis in the Watermold Allomyces

The chemotactic response of the mitospores and meiospores of Allomyces macrogynus and A. abuscula to casein hydrolysate was shown to he caused by the combined action of leucine and lysine in the hydrolysate. The testing was done by counting the zoospores that attached to a membrane through which substances diffused downward. The action of leucine and lysine was shown to be synergistic and to be specific for the L, forms. The optimum concentration above the membrane was 5 × 10^-2M for each amino acid. An effect was detectable down to approximately 10^-55 M. The addition of L-proline to the mixture increased the response. Proline in combination with leucine caused good attachment, although less than that by leucine and lysine, of the zoospores of A. arbuscula but not of those of A. macrogynus.     

The effect of actinomycin D on the formation of gametangia and mitosporangia in Allomyces

The effect of actinomycin on gametangium and mitosporangium production in Allomyces arbuscula and A. macrogynus has been investigated. Male gametangium production was not more sensitive to actinomycin than female development. Actinomycin at 20 g/ml added at the commencement of induction was completely inhibitory. The process became insensitive to actinomycin just before the first septum was laid down.     

Untersuchungen zur Entwicklung des Phycomyceten Allomyces arbuscula

Polysomes were isolated both from growing gametophytes of Allomyces arbuscula and from gametangia prepared from mycelia at different periods during gametogenesis. Analysis of polysomes by sucrose gradients showed that ribosomes present in the gametangia monosome pool were shifted into polysomes. This shift was found to be correlated with gametangia differentiation. The ribosome distribution remained virtually unchanged during the early stage of gamete formation. In mature gametes and swarming zygotes a low level of polysomes was detected. Labeling of rRNA by ³²PO₄ demonstrated a de novo synthesis of monosomes throughout the period of gametangia differentiation. No incorporation of ³²PO₄ was found to be present in ribosomes prepared from gametangia after onset of gamete formation. On the basis of these labeling experiments it is concluded that radioactivity in polysomes extracted from mature gametes and swarming zygotes can be attributed in part to conserved mRNA.Synchronous formation of gametangia was induced by transferring the vegetative mycelia from growth medium into a low salt buffer. Under these conditions the incorporation of either ³²PO₄ or ³H-uridine into RNA, particularly into rRNA, was found to be markedly decreased. This obviously indicates a shutdown of RNA synthesis. rRNA from induced mycelia examined by polyacrylamide gel electrophoresis was found to be severely degraded. In contrast to this, rRNA isolated from ribosomes of developing gametangia and from gametes exhibited no degradation products. It is suggested that endonucleases cause rRNA hydrolysis in the hyphal cytoplasm during gametangia differentiation. Ribosomes compartmentalized in gametangia seem to be inaccessible to nucleases during the later process of gametogenesis.

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Abkürzungen MAK Methyl-Albumin-Kieselgur - PAA Polyacrylamid - stains all 4,5,4,5-Dibenzo-3,3-diäthyl-9-methylthiacarbocyaninbromid (Serva, Heidelberg)     

Host-parasite relations betweenAllomyces andRozella

Summary Zoospores of the obligately parasitic chytridRozella allomycis encyst all over the hyphae of a susceptible host, the water-moldAllomyces arbuscula, but many of them fail to penetrate. At the sites of successful penetration lomasomes occur, and the inner layer of the host cell-wall grows and invaginates around a papilla, through the center of which the parasite enters. This host response resembles instances of normal, localized inward growth of fungal cell-walls. The response may also be related to a defense reaction of walled cells which form callosities to block the invasion of fungi.Rozella appears to utilize this response and to depend upon it—probably in order to enter the host cell with minimal disruption. A similar relationship may control the penetration of various obligately parasitic plant-pathogens into their hosts.     

Polyribosomes in different stages of the life cycle of the water mold Allomyces arbuscula

Synchronous gametogenesis in the water mold Allomyces arbuscula is blocked by actinomycin D added at the onset of the process. Formation of the male gametangium can be selectively inhibited by administering actinomycin one hr after the induction of gametogenesis. The polyribosome pattern obtained after density gradient centrifugation remains virtually unchanged throughout gametogenesis until a stage immediately preceding maturation of the gametes. When ribosomes from gametes and swarming zygotes are analyzed on gradients, some RNase-sensitive material is found to band in the heavier portion of the gradient. Its presence suggests that some messenger RNA associated with ribosomes is conserved in the swarming cells. During gametogenesis RNA is de novo synthesized and becomes associated with the polyribosomes.     

Untersuchungen zur Entwicklung des PhycomycetenAllomyces arbuscula

InAllomyces arbuscula formation of gametes occurs within 80 min in isolated gametangia. Gametogenesis shows to be sensitive to cycloheximide at 50 and 100 g/ml, while actinomycin D at 25 and 50 g/ml fails to inhibit gametogenesis. Synthesis ofrRNA can be profoundly inhibited by 3×10^-3 M 5-fluorouracil prior to gametogenesis, without any effect on differentiation of gametes. It is shown by polyacrylamide-gel electrophoresis that during gametogenesis radioactive phosphate is incorporated intotRNA, but not intorRNA. The results indicate that formation of gametes is dependent uponmRNA already present in the gametangia before induction of gametogenesis. It is concluded further that protein synthesis on cytoplasmic ribosomes is finished 20–30 min after induction and thatrRNA synthesis seems not to be a prerequisite for the differentiation of gametes.

     

Water- and temperature-dependence of DNA damage and repair in the fruticose lichen Cladonia arbuscula ssp. mitis exposed to UV-B radiation

The induction of cyclobutane pyrimidine dimers (CPDs) by ultraviolet‐B radiation (UV‐B, 280–315 nm) and repair mechanisms were studied in the lichen Cladonia arbuscula ssp. mitis exposed to different temperatures and water status conditions. In addition, the development and repair of CPDs were studied in relation to the different developmental stages of the lichen thallus podetial branches. Air‐dried lichen thalli exposed to UV‐B radiation combined with relatively high visible light (HL, 800 μmol m^?2 s^?1; 400–700 nm) for 7 days showed a progressive increase of CPDs with no substantial repair, although HL was present during and after irradiation with UV‐B. Fully hydrated lichen thalli, that had not been previously exposed to UV‐B radiation for 7 days, were given short‐term UV‐B radiation treatment at 25°C, and accumulated DNA lesions in the form of CPDs, with repair occurring when they were exposed to photoreactivating conditions (2 h of 300 μmol m^?2 s^?1, 400–700 nm). A different pattern was observed when fully hydrated thalli were exposed to short‐term UV‐B radiation at 2°C, in comparison with exposure at 25°C. High levels of CPDs were induced at 2°C under UV‐B irradiation, without significant repair under subsequent photoreactivating light. Likewise, when PAR (300 μmol m^?2 s^?1) and UV‐B radiation were given simultaneously, the CPD levels were not lowered. Throughout all experiments the youngest, less differentiated parts of the lichen thallus – namely ‘tips’, according to our arbitrary subdivision – were the parts showing the highest levels of CPD accumulation and the lowest levels of repair in comparison with the older thallus tissue (‘stems’). Thus the experiments showed that Cladonia arbuscula ssp. mitis is sensitive to UV‐B irradiation in the air‐dried state and is not able to completely repair the damage caused by the radiation. Furthermore, temperature plays a role in the DNA damage repairing capacity of this lichen, since even when fully hydrated, C. arbuscula ssp. mitis did not repair DNA damage at the low temperatures.     

Cell wall formation in zoospores of Allomyces arbuscula

Carbohydrate composition was determined in isolated cell walls of meiospores of Allomyces arbuscula after incubation for 15 min (encysted meiospores: cysts), 150 min (germlings: cysts + rhizoids) and 24 h (cysts + rhizoids + hyphae). The principal constituent in all cell wall samples is chitin, accounting for about 75% of the recovered carbohydrates. In addition, cell walls of all stages examined contain polysaccharides which release galactose, glucose, mannose, arabinose, xylose, fucose, and rhamnose on acid hydrolysis. While different developmental stages show minor quantitative changes in chitin, the ratio of galactose to glucose decreases sharply during differentiation of ungerminated cysts into germlings with rhizoids and hyphae. The increase in glucose is accompanied by a decrease in the amount of xylose and/or fucose and of galactose.List of Abbreviation TFA trifluoroacetic acid     

Some observations on the mode of division of somatic nuclei of Mucor and Allomyces

Summary A series of successive photographs of the division of the living nucleus in a germinating sporangiospore of Mucor fragilis has been obtained. In this sequence the nucleus is seen to divide directly by elongation and constriction. The nucleolus divides at the same time and in the same way. These observations agree with the finding, first made by Léger (1896) and several times confirmed since then, that the nuclei of Mucorales apparently divide without first arranging their chromosomes in a metaphase plate and without the help of a spindle apparatus.In stained preparations of Mucor chromosomes are not normally visible as separate entities but they can be clearly seen in Feulgen preparations of dividing somatic nuclei of Allomyces arbuscula. In contrast to Mucor the nucleolus of Allomyces is dissolved during division. The chromosomes seem to sort themselves out on their own and form new nucleoli. Metaphase plates and spindles have not been encountered.To Professor Dr. E. G. Pringsheim, teacher and friend, on his 80th bithday.     

Molecular phylogeny of Allomyces macrogynus: Congruency between nuclear ribosomal RNA- and mitochondrial protein-based trees

We have sequenced the nuclear and mitochondrial small subunit rRNA genes (rns) and the mitochondrial genes coding for subunits 1 and 3 of the cytochrome oxidase (cox1 and cox3, respectively) of the chytridiomycete Allomyces macrogynus. Phylogenetic trees inferred from the derived COX1 and COX3 proteins and the nuclear rns sequences show with good bootstrap support that A. macrogynus is an early diverging fungus. The trees inferred from mitochondrial rns sequences do not yield a topology that is supported by bootstrap analysis. The similarity and the relative robustness of the nuclear rns and the mitochondrial protein-derived phylogenetic trees suggest that protein sequences are of higher value than rRNA sequences for reconstructing mitochondrial evolution. In addition, our trees support a monophyletic origin of mitochondria for the range of analyzed eukaryotes. Correspondence to: B. Franz Lang