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PubMed1974

Production of hormone B by Achlya heterosexualis.

Sexual morphogenesis in Achlya, a filamentous water mold, is initiated by two hormones. Hormone A, or antheridiol, is a C-29 steroid. Four stereoisomers of antheridiol have been synthesized. The natural one (antheridiol 22S 23R) and its 7-deoxy 7-dihydro form, when added to an aerated culture of hermaphroditic Achlya heterosexualis, stimulated this mold to secrete twice as much hormone B as the untreated control. An unnatural stereoisomer (antheridiol 22R 23S) and fucosterol, however, did not stimulate strain 8-6 in the same way. Methods by which hormone B can be produced in sufficient yield for isolation and characterization are described.

Chemical Phenomena↗
PubMed1981

Electron microscopy of Achlya deoxyribonucleic acid sequence organization.

Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.

Chytridiomycota↗
PubMed1976

Lipid metabolism in Achlya: studies of lipid turnover during development.

The life cycle of Achlya involves germination of spores to form coenocytic somatic hyphae, followed by differentiation of hyphal tips into sporangia. From germination to release of new spores occupies 27-30 h. Total lipid made up 10% of dry weight in ungerminated spores. After germination, total lipid fell to 6% of dry weight in 15 h, then rose to 7.7% at the time of sporangium formation. Half of the initial loss of lipid took place within 2 h of germination. The ability of Achlya to incorporate [1-14C]acetate into lipid was maximal at the time of sporangium formation, and glycerides were the principal component of total lipid to become 14C-labelled at all stages of the life cycle. Fatty acid synthetase activity measured in cell-free extracts was low in spores and in actively elongating mycelium, but increased during idfferentiation to a level 15-fold greater than that in spores. Fatty acid oxidation, as estimated by the release of 14CO2 from 1-14C-labelled fatty acids, was also maximal at the time of sporangium formation.

Acetates↗
PubMed1976

Lipid metabolism in Achlya: changes in lipid composition during development.

Fractionation of total lipid extracted from Achlya sp. at various stages of its developmental cycle revealed that in spores total lipid was composed of 62% neutral lipid, 13% phospholipid, and 25% glycolipid. After germination, the proportion of neutral lipid rose slightly after 2 h then fell sharply to 10% after 8 h, whereupon it rose to 55% of total lipid after 30 h of growth, when sporulation was completed. Conversely, phospholipid rose to 77% of total lipid after 8 h, then declined to 40% after 30 h. Glycolipid was maintained at 10-20% of total lipid throughout the life cycle after spore germination. Quantitative determination of neutral lipid components by photo-reflectometry showed that triglycerides accounted for 20% of neutral lipid in spores, and free fatty acids made up 50%. During growth, the absolute levels of both components fell precipitously on germination, remained at low levels throughout vegetative growth, and rose at the time of sporulation. The fatty acid composition of total lipid, phospholipid, neutral lipid, and free fatty acid fractions extracted from vegetative and sporulating Achlya cells was determined. The principal fatty acids present in all fractions of both stages of the life cycle were hexadecanoic and octadecanoic acids. Hydroxyhexadecanoic acid, eicosatetraenoic acid, and an unidentified long-chain acid were completely absent from the phospholipids of vegetative cells, although they were found in significant quantities in lipid fractions from other stages of growth.

Fatty Acids, Nonesterified↗
PubMed2002

Proteolytic release of membrane-bound endo-(1,4)-beta-glucanase activity associated with cell wall softening in Achlya ambisexualis.

Branching and other cell wall softening events in fungi and oomycetes are thought to involve the activity of secreted enzymes, which are packaged in membrane vesicles and delivered to sites of cell expansion, there to work in a carefully regulated manner upon the structure of the wall. Here we demonstrate a latent endo-(1,4)-beta-glucanase activity in a mixed membrane fraction of the oomycete Achlya ambisexualis, which can be released by cysteine proteases with an increase of apparent activity. In addition, a similar endogenous process is strongly inhibited by the cysteine protease inhibitor iodoacetamide, while inhibitors of other types of proteases have a much smaller effect. Detergent treatment of membranes releases two glucanases detectable by electrophoretic activity staining, with apparent molecular masses of about 164 and 35 kDa. Proteolysis produces several activity bands, with major species having apparent molecular masses of about 149, 133, 48, 35, and 25 kDa. The ca. 35- and 25-kDa bands migrate in parallel with glucanases secreted during wall softening in vivo. We propose that the initiation of wall softening in Achlya involves the proteolytic processing and solubilization of at least some secreted endoglucanases. We also propose that the solubilization component of this process functions not just to provide the enzymes with access to wall matrix substrates but also may provide a mechanism for the eventual termination of their biological function.

Cell Membrane↗
PubMed2001

[Screening, cloning and sequence analysis of the gene for pyruvate kinase from Achlya bisexualis cDNA library].

The gene for pyruvate kinase, which is involved in glycolysis and catalyze the biochemical reaction from phosphoenolpyruvate to pyruvate, was screened, cloned and sequenced from non-photosynthetic Oomycetes, Achlya bisexualis cDNA library. The size of the gene is about 1.6 kb. The amino acid sequence of the gene was deduced using the GCG program. On basis of analysis by the on-line PSORT II program the deduced amino acid sequence showed that the pyruvate kinase expressed by the gene was located in the cytosol. This result indicated that it is possible that the process of glycolysis for the lower form of eukaryotic cell such as Achlya bisexuali may occur in cytosol, which is different from Liaud's report.

Amino Acid Sequence↗
PubMed2006

Achlya abortispora, a new oomycete isolated from water samples taken from a water reservoir in Morocco.

Achlya abortispora sp. nov. was found in water and floating organic matter taken form a dam near Rabat, Morocco. The new species is described and compared with other species of the genus. Distinguishing characteristics of A. abortispora are the production of long fusiform sporangia with achlyoid and aplanoid discharge of zoospores; smooth-walled spherical to club-shaped oogonia, which are usually lateral, but at times intercalary, containing 1 to 20 oospheres. The oogonia can also bear 1 to 5 appendages, which may indicate oogonial proliferation. Most of the oospheres do not mature and are thus abortive. The antheridial branches supplying the oogonia are predominantly diclinous, but at times these may be monoclinous and androgynous. Antheridial branches coil and wrap around the oogonia. Morphologic features of the oomycete and the sequence of the ITS region of its rDNA, as well as their comparison with related species, are discussed. This is the first report of the occurrence of a saprolegniaceous oomycete from Morocco.

Achlya↗
PubMed2004

Molecular cloning and characterization of two different cDNAs encoding the molecular chaperone Hsp90 in the oomycete Achlya ambisexualis.

The chaperone Hsp90 plays a key role in the maturation and activation of many 'client' proteins in eukaryotic cells. In the oomycete Achlya ambisexualis two populations of hsp90 transcripts that differ slightly in size (2.8 and 2.9 kb) are present in heat-shocked mycelia. Only the 2.8 kb transcripts are seen in vegetative mycelia and in mycelia undergoing antheridiol-induced differentiation. Two different hsp90 cDNAs were isolated and characterized. Although nearly identical, an additional eight nucleotide sequence was present at the end of the 3'UTR of one of the two cDNAs. RT-PCR analyses indicated that hsp90 transcripts containing the eight nucleotide extension, were present only in heat-shocked mycelia. Hsp90 transcripts lacking this sequence were present in vegetative mycelia and the levels of these transcripts increased in both heat-shocked and hormone-treated mycelia. Each hsp90 cDNA encoded a nearly identical Hsp90 protein. However, two Hsp90 proteins (86 and 84 kDa) were observed on immunoblots of mycelial proteins. Only one of these, i.e., the 86 kDa protein was detected by an anti-phosphoserine antibody, suggesting that the difference in mass of the two Hsp90 isoforms, was due at least in part, to different levels of phosphoserine residues.

3' Untranslated Regions↗
PubMed2005

A beta4 integrin-like protein co-localises with a phosphotyrosine containing protein in the oomycete Achlya bisexualis: inhibition of tyrosine phosphorylation slows tip growth.

We present immunocytochemical data that indicate the presence of, and a close association between beta4 integrin-like proteins and proteins containing phosphorylated tyrosine residues in the oomycete Achlya bisexualis. When hyphae were plasmolysed, these proteins were present in wall-membrane attachment sites where there was also F-actin. A combination of immunoblots, ELISA, and a coupled enzyme assay suggest that phosphorylation may occur by both autophosphorylation and through the action of a tyrosine kinase. Tyrphostins, which are inhibitors of tyrosine kinases, abolished the anti-phosphotyrosine staining, inhibited the kinase activity, slowed tip growth and affected the organisation of the actin cytoskeleton, in a dose-dependent manner. By analogy with the integrins and associated kinases of the metazoa we suggest that these proteins could contribute to the process of tip growth by providing a means of bidirectional signaling between the cell wall and the cytoplasm.

Achlya↗
PubMed2006

Invasive hyphal growth: an F-actin depleted zone is associated with invasive hyphae of the oomycetes Achlya bisexualis and Phytophthora cinnamomi.

We have compared F-actin patterns in invasive and non-invasive oomycete hyphae. In Achlya bisexualis an F-actin depleted zone is present in 70% of invasive but only 9% of non-invasive hyphae. In Phytophthora cinnamomi these figures are 74 and 20%, respectively. Thus, the F-actin depleted zone appears to be associated with invasive growth. TEM images indicate that it is unlikely to represent areas of vesicle accumulation. Measurements of turgor indicate no significant increase under invasive conditions (0.65 MPa (invasive) and 0.63 MPa (non-invasive)). Similarly we found no difference in burst pressures (1.04 MPa (invasive) and 1.06 MPa (non-invasive)), although surrounding agarose may lead to overestimates of invasive tip strength. An F-actin depleted zone has the potential, along with wall softening, to increase protrusive force in the absence of turgor increases. Staining of F-actin in hyphae under hyperosmotic conditions suggests that decreases in F-actin at growing tips may also enable non-invasive growth at very low turgor.

Achlya↗
PubMed2004

Two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis.

We show that two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis, that have been chemically fixed with a combination of methylglyoxal and formaldehyde and stained with Alexa phalloidin. In approximately one half of the hyphae examined, an F-actin depleted zone within the apical F-actin cap was observed. The remaining hyphae had a continuous apical cap. In live, growing hyphae two types of cytoplasmic organization were observed at the tips, one in which a clear zone was present which may correlate with the F-actin depleted zone, and one where no such clear zone existed which may represent the continuous cap. We suggest that the F-actin depleted zone may be a structural component of the actin network in a subpopulation of oomycete hyphae and may be comparable to similar F-actin depleted zones at the apices of other tip growing cells such as pollen tubes and root hairs. This observation has implications with regard to models of hyphal extension. Hyphae fixed with formaldehyde alone showed continuous apical F-actin caps. Our ability to resolve the F-actin depleted zone likely reflects the cross-linking capabilities of methylglyoxal. The methylglyoxal-formaldehyde combination fixative gave more stained hyphae, brighter staining and more complete staining of F-actin compared to formaldehyde alone.

Achlya↗
PubMed1998

Regulation of hsp90 and hsp70 genes during antheridiol-induced hyphal branching in the oomycete Achlya ambisexualis.

When mycelia of Achlya ambisexualis J. Raper strain E87 were undergoing antheridial branching, a marked increase was observed in the levels of transcript populations encoding the heat shock protein chaperone Hsp90 and transcript populations encoding three different Hsp70-family heat shock protein chaperones, respectively. Although up to 90% of hyphae in the hormone-treated thalli were undergoing antheridial branching, no similar increase in the level of transcripts encoding actin was observed. Nuclear run-on assays demonstrated that the observed antheridiol-induced increases in the levels of the chaperone RNAs resulted from increased transcription. Although not tested for function, the nucleotide sequence of the 5' flanking region of each of the two A. ambisexualis hsp90 genes revealed a diversity of sequences and motifs similar or identical to the sequences of known transcription factor response elements. Among these potential response element sequences observed in the A. ambisexualis genes were motifs observed also in animal steroid hormone response elements. Surrounding the primer-extension determined transcription start site of each A. ambisexualis hsp90 gene was a 16-nucleotide sequence that matched in 14 out of 16 nucleotides a sequence found in the transcription initiation region of many different oomycete genes.

Base Sequence↗
PubMed1986

Achlya mitochondrial DNA: gene localization and analysis of inverted repeats.

Mitochondrial DNA from four strains of the oomycete Achlya has been compared and nine gene loci mapped, including that of the ribosomal protein gene, var1. Examination of the restriction enzyme site maps showed the presence of four insertions relative to a map common to all four strains. All the insertions were found in close proximity to genic regions. The four strains also contained the inverted repeat first observed in A. ambisexualis (Hudspeth et al. 1983), allowing an examination by analysis of retained restriction sites of the evolutionary stability of repeated DNA sequences relative to single copy sequences. Although the inverted repeat is significantly more stable than single copy sequences, more detailed analysis indicates that this stability is limited to the portion encoding the ribosomal RNA genes. Thus, the apparent evolutionary stability of the repeat does not appear to derive from the inverted repeat structure per se.

Base Sequence↗
PubMed1977

Synaptonemal complexes in antheridia of Achlya ambisexualis E87.

This is the first report of longitudinal sections of synaptonemal complexes in oömycetous fungi. These indicators of meiosis were observed in antheridial nuclei of Achlya ambisexualis E87. They were attached to a platelike structure at the inner membrane of the nuclear envelope. The lateral elements were separated from each other by an average distance of 160nm. These results provide new ultrastructural evidence for gametangial meiosis in Oömycetes.

Fungi↗
PubMed1976

Microfilament bundles in antheridial nuclei of Achlya ambisexualis E 87.

This is the first report of intranuclear microfilaments within gametangial nuclei of oömycetous fungi. Longitudinal sections of four to six microfilaments were frequently observed in meiotic antheridial nuclei of Achlya ambisexualis. Each microfilament measured approximately 7--10 nm in diameter. Spindle tubules (25 nm in diameter) were also observed within some of the nuclei possessing microfilaments.

Cell Nucleus↗
PubMed1991

Molecular phylogenetic analysis of actin genic regions from Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta).

Actin genic regions were isolated and characterized from the heterokont-flagellated protists, Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta). Restriction enzyme and cloning experiments suggested that the genes are present in a single copy and sequence determinations revealed the existence of two introns in the C. costata actin genic region. Phylogenetic analyses of actin genic regions using distance matrix and maximum parsimony methods confirmed the close evolutionary relationship of A. bisexualis and C. costata suggested by ribosomal DNA (rDNA) sequence comparisons and reproductive cell ultrastructure. The higher fungi, green plants, and animals were seen as monophyletic groups; however, a precise order of branching for these assemblages could not be determined. Phylogentic frameworks inferred from comparisons of rRNAs were used to assess rates of evolution in actin genic regions of diverse eukaryotes. Actin genic regions had nonuniform rates of nucleotide substitution in different lineages. Comparison of rates of actin and rDNA sequence divergence indicated that actin genic regions evolve 2.0 and 5.3 times faster in higher fungi and flowering plants, respectively, than their rDNA sequences. Conversely, animal actins evolve at approximately one-fifth the rate of their rDNA sequences.

Actins↗
PubMed1984

Detection of a pheromone-binding protein in the aquatic fungus Achlya ambisexualis.

Sexual reproduction among the eukaryotic fungi of the genus, Achlya, is controlled by two steroid pheromones. Antheridiol is the pheromone constitutively produced by female cells that induces male sexual differentiation and development. A biologically active tritium-labeled derivative of antheridiol, [1,2-3H]7-deoxy-7-dihydro-antheridiol ( [3H]7dA), has been synthesized. Radioligand-binding studies have revealed the presence of a specific binding protein in the cytosol of male cells that may represent the endogenous receptor for antheridiol. Binding to this macromolecule was characterized by an apparent equilibrium dissociation constant and maximum binding capacity of approx. 7 X 10(-10) M and 1 100-2 000 fmoles/mg protein, respectively. Sedimentation analysis in sucrose gradients revealed that the binding protein distributes in the 8S region under low ionic strength and sodium molybdate-stabilized conditions. Under conditions of high ionic strength, in the presence or absence of 10 mM sodium molybdate, the binding site distributes in the 3.6S region of the gradient. Analysis of radioligand binding in the presence of other steroids and steroid hormones revealed that the binding is specific for antheridiol and its analog.

Centrifugation, Density Gradient↗
PubMed1986

Steroid hormone-induced changes in secreted proteins in the filamentous fungus Achlya.

In the fungus Achlya, sexual development in the male strain E87 is mediated by the steroid hormone antheridiol. Treatment of vegetatively growing cells of E87 with antheridiol resulted in changes in the [35S]methionine labeling of several secreted proteins. The most heavily labeled group of proteins observed in the secreted fraction from control cells appeared on one-dimensional gels as a prominent wide band which could be resolved into three closely spaced components with relative molecular weights (MWs) of 57, 54, and 50 kD, respectively. After hormone treatment the two lower MW proteins of the group were barely detectable. Concomitant with the observed reductions in the labeling of the 54 and 50 kD proteins was the increased labeling of a doublet of very prominent proteins with relative MWs of 44.4 and 43 kD, respectively. The results of experiments with Endoglycosidase H suggested that the 44.4 and 43 kD proteins seen in hormone-treated cultures, might result from the removal or reduced synthesis of high mannose oligosaccharide groups found on the 54 and 50 kD proteins normally seen in control cultures. Additional support for this suggestion was provided by the observation that the 54 and 50 kD proteins from control cultures appeared to bind to conA columns and to be eluted with alpha-methylmannoside, while there was little or no binding of the 44.4 and 43 kD proteins from hormone-treated cells. Although other possibilities are not excluded, the results are suggestive of a steroid hormone-induced alteration in glycoprotein processing. The functions of the observed hormonally-responsive secreted proteins as well as those of the secreted proteins in non-hormone-treated cultures, are not known at this time.

Acetylglucosaminidase↗