Molecular cloning and selection of genes regulated in Aspergillus development

Over 350 clones homologous to poly(A)+ RNAs that are significantly more prevalent in conidiating cultures of Aspergillus nidulans than in somatic cells have been selected from a recombinant DNA library formed between nuclear DNA and lambda Charon 4A. The procedure used for this selection involved in situ hybridization to a cDNA probe which had been selectively depleted of sequences represented in somatic cells by complement hybridization. Five of these clones have been characterized further. All but one encoded poly(A)+ RNAs that were at least ten times more prevalent in conidiating cultures than in somatic cells. One clone hybridized to a single, developmentally regulated RNA. The three others were complementary to several RNAs having different molecular weights, each of which was more prevalent in condiating cultures than in vegetative cells. These results and quantitative aspects of the selection procedure suggest that developmentally controlled poly(A)+ RNA coding regions may not be distributed randomly in the Aspergillus genome.     

Protein changes during the asexual cycle of Neurospora crassa.

A method for synchronizing conidiation and isolating large numbers of cells at discrete stages of conidia development is described. Using two-dimensional gel electrophoresis, we analyzed the protein profiles of mycelia, aerial hyphae, and conidia and observed that the concentration of 14 polypeptides increase and 38 decrease during the asexual cycle. Twelve polypeptides were present in extracts of aerial hyphae or conidia, but not mycelia, suggesting that they may be conidiation specific. The protein profiles of mutants defective in conidiation were also analyzed. Differences were detected in the two-dimensional profiles of protein extracts from fluffy and wild-type aerial hyphae. Polyadenylated RNA isolated from wild-type mycelia and conidiating cultures was translated in vitro in a rabbit reticulocyte lysate. Differences were detected in the polypeptide products specified by the two RNA populations, suggesting that changes in steady-state levels of polyadenylated RNAs also occur during conidiation.     

Accumulation of Ergot Alkaloids During Conidiophore Development in Aspergillus fumigatus

Production of ergot alkaloids in the opportunistic fungal pathogen Aspergillus fumigatus is restricted to conidiating cultures. These cultures typically accumulate several pathway intermediates at concentrations comparable to that of the pathway end product. We investigated the contribution of different cell types that constitute the multicellular conidiophore of A. fumigatus to the production of ergot alkaloid pathway intermediates versus the pathway end product, fumigaclavine C. A relatively minor share (11 %) of the ergot alkaloid yield on a molar basis was secreted into the medium, whereas the remainder was associated with the conidiating colonies. Entire conidiating cultures (containing hyphae, vesicle of conidiophore, phialides of conidiophore, and conidia) accumulated higher levels of the pathway intermediate festuclavine and lower levels of the pathway end product fumigaclavine C than did isolated, abscised conidia, indicating that conidiophores and/or hyphae have a quantitatively different ergot alkaloid profile compared to that of conidia. Differences in alkaloid accumulation among cell types also were indicated by studies with conidiophore development mutants. A ?medA mutant, in which conidiophores are numerous but develop poorly, accumulated higher levels of pathway intermediates than did the wildtype or a complemented ?medA mutant. A ?stuA mutant, which grows mainly as hyphae and produces very few, abnormal conidiophores, produced no detectable ergot alkaloids. The data indicated heterogeneous spatial distribution of ergot alkaloid pathway intermediates versus pathway end product in conidiating cultures of A. fumigatus. This skewed distribution may reflect differences in abundance or activity of pathway enzymes among cell types of those conidiating cultures.     

Association of laccase activity with conidiation in an aflatoxigenic strain of Aspergillus parasiticus

Abstract The relationship between laccase activity and asexual development in Aspergillus parasiticus was established. A. parasiticus produced a laccase activity similar to that reported for Aspergillus nidulans . Laccase activity appeared only in conidiating cultures and was absent from vegetative cultures. Shaking of the cultures inhibited conidiation and suppressed laccase activity. The composition of the media affected the degree of conidiation and the specific activity of laccase. Ammonium sulphate as sole nitrogen source was suppressive, whereas glutamate was highly stimulatory to both conidiation and laccase production. The addition of citrate was also stimulatory to conidia production and, to a lesser degree, laccase activity. There appears to be no quantitative correlation between laccase activity and the number of conidia produced.     

Conidiogenous cell development inHelminthosporium carbonum

We devised a procedure to propagate selectively the vegetative and asexual-reproductive states ofHelminthosporium carbonum so that we could characterize morphological and subcellular events associated with the onset of conidiation. Solidified agar media were uniformly inoculated with an overlay of conidia suspended in molten agar. After the overlay solidified, it was covered with a sheet of Miracloth. When incubated in the dark, cultures produced abundant aerial hyphae that grew through the Miracloth layer and conidiation was suppressed for 48 to 50 h. Hyphae were easily harvested from the surface of the Miracloth with a spatula. When cultures were placed in the light after 38 h of growth in the dark, differentiation was detected in 90% of the hyphal tips within 8 to 10 h. The initial response of the hyphal tips, comprising early stages in conidiophore development, was rapid and highly synchronized. The behavior of nuclei during conidiogenous cell development and the initiation of conidia was similar to that reported for other fungi that form blastic conidia. One-dimensional gel electrophoresis ofin vitro translation products confirmed differences in poly(A) RNA populations from dark-grown and light-induced cultures.     

Evaluation of the potential role of water in spread of conidia of the Neotyphodium endophyte of Poa ampla

Neotyphodium endophytes are asexual, filamentous fungi, mutualistically associated with diverse cool season grasses. Infected seeds and vegetative organs of infected host plants are the only known modes of propagation of the asexual endophytes. In the last decade certain Epichloë and Neotyphodium-infected grass species have been shown to have epiphyllous structures of the endophytes, hyphae, conidiophores, and conidia, growing on leaf blades. The production of epiphyllous conidia suggests the possibility that some of these endophytes may have the ability for plant-to-plant spread. The objective of this study was to determine the possible mechanisms involved in liberation and dispersal of asexual spores of Neotyphodium growing in vitro and epiphyllously on leaves of Poa ampla. Our results indicate that water dispersal is the most likely means of dissemination of conidia of the Neotyphodium sp. Wind generated by dry compressed air does not dislodge the conidia from slide cultures or from P. ampla leaves.     

The chsA gene from Aspergillus nidulans is necessary for maximal conidiation

A fragment from the open reading frame of the cloned chsA gene from Aspergillus nidulans was deleted and replaced with the argB gene. The resulting construct was used to replace the wild-type chsA gene in an argB deletion strain. The growth and morphology of the vegetative hyphae from the resulting chsA disruptant strain were indistinguishable from those of a wild-type strain but the chitin content of the hyphae from the disruptant was reduced to approximately 90% of that of wild-type. The disruptant showed reduced ability to produce the asexual spores (conidia) that are formed by differentiated aerial hyphae called conidiophores. The ability to form undifferentiated aerial hyphae was not impaired in the disruptant. The conidiophores and conidia produced by the disruptant were indistinguishable from those of wild-type. Conidium formation by the disruptant grown on a variety of media was reduced to about 30% of the wild-type. A chsE null strain did not show a defect in conidiation but a strain in which both chsA and chsE were inactivated produced about 3% of the conidia of wild-type. That finding supports the hypothesis that chsA and chsE encode a partially redundant function necessary for conidiophore development.     

Activation of Balbiani ring genes inChironomus tentans after a pilocarpine-induced depletion of the secretory products from the salivary gland lumen

We have constructed recombinant plasmid libraries containing complementary DNA (cDNA) inserts made to poly(A)⁺ RNA isolated from two stages of Dictyostelium development. The procedure utilized for the cloning allows the excision of the cDNA inserts free of vehicle sequences. The two libraries were screened for inserts complementary to moderately abundant and abundant poly(A)⁺ RNA whose genes are differentially modulated during Dictyostelium development. Several of these plasmids were then further examined by hybridization techniques to determine the reiteration frequencies of their genes, the relative rate of complementary RNA synthesis during development, and the relative accumulation and disappearance of complementary RNA during the Dictyostelium life cycle. RNA complementary to two sequences was found to accumulate from approximately one molecule per cell during vegetative growth to several hundred molecules during preaggregation.     

The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.     

TheAspergillus nidulans geneschsA andchsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, andchsC) fromAspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, namedchsD, fromA. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 ofSaccharomyces cerevisiae and Chs3 ofCandida albicans. Disruption ofchsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption ofchsA andchsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption ofchsC andchsD caused no defect. Thus it appears thatchsA andchsD serve redundant functions in conidia formation.     

Autolytic hydrolases affect sexual and asexual development of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Radial growth, asexual sporulation, and cleistothecia formation as well as extracellular chitinase and proteinase formation of Aspergillus nidulans were monitored in surface cultures in order to study the physiological role of extracellular hydrolase production in carbon-stressed cultures. We set up carbon-stressed and carbon-overfed experimental conditions by varying the starting glucose concentration within the range of 2.5 and 40 g/L. Glucose starvation induced radial growth and hydrolase production and enhanced the maturation of cleistothecia; meanwhile, glucose-rich conditions enhanced mycelial biomass, conidia, and cleistothecia production. Double deletion of chiB and engA (encoding an extracellular endochitinase and a β-1,3-endoglucanase, respectively) decreased conidia production under carbon-stressed conditions, suggesting that these autolytic hydrolases can support conidia formation by releasing nutrients from the cell wall polysaccharides of dead hyphae. Double deletion of prtA and pepJ (both genes encode extracellular proteases) reduced the number of cleistothecia even under carbon-rich conditions except in the presence of casamino acids, which supports the view that sexual development and amino acid metabolism are tightly connected to each other in this fungus.     

Protein analysis in a pleomorphically deteriorated strain of the insect-pathogenic fungus Metarhizium anisopliae

Pleomorphic deterioration is a process where a fungal isolate loses the ability to produce conidia during repeated subculturing. We have previously isolated strains of the entomopathogenic fungus Metarhizium anisopliae that have irreversibly lost the ability to produce conidia and only produce mycelia when grown on agar. Gel electrophoresis was used to examine differences in intracellular protein patterns (urea-soluble proteins and urea-insoluble proteins (i.e., hydrophobins)) in conidiating and mycelial cultures of M. anisopliae. Two major proteins present in a conidiating culture and one from a mycelial culture were N-terminally sequenced but showed no homologies to known proteins. The presence of hydrophobins in conidiating and mycelial cultures was also examined, and it was shown that these proteins were abundant in conidiating cultures but not in mycelial cultures. We also used primers designed from regulatory genes involved in conidiation in Aspergillus nidulans. The amplified fragments were not homologous to A. nidulans genes.     

The Role,Interaction and Regulation of the Velvet Regulator VelB in Aspergillus nidulans

The multifunctional regulator VelB physically interacts with other velvet regulators and the resulting complexes govern development and secondary metabolism in the filamentous fungus Aspergillus nidulans. Here, we further characterize VelB’s role in governing asexual development and conidiogenesis in A. nidulans. In asexual spore formation, velB deletion strains show reduced number of conidia, and decreased and delayed mRNA accumulation of the key asexual regulatory genes brlA, abaA, and vosA. Overexpression of velB induces a two-fold increase of asexual spore production compared to wild type. Furthermore, the velB deletion mutant exhibits increased conidial germination rates in the presence of glucose, and rapid germination of conidia in the absence of external carbon sources. In vivo immuno-pull-down analyses reveal that VelB primarily interacts with VosA in both asexual and sexual spores, and VelB and VosA play an inter-dependent role in spore viability, focal trehalose biogenesis and control of conidial germination. Genetic and in vitro studies reveal that AbaA positively regulates velB and vosA mRNA expression during sporogenesis, and directly binds to the promoters of velB and vosA. In summary, VelB acts as a positive regulator of asexual development and regulates spore maturation, focal trehalose biogenesis and germination by interacting with VosA in A. nidulans.