Parasexuality in asexual development mutants of Aspergillus nidulans

The parasexual cycle with parameiosis has been characterized previously by the occurrence of genetic recombination and haploidization inside heterokaryotic hyphae prior to conidial formation. The aim of current research was to characterize, through genetic and cytological analyses, an asexual development mutant strain of A. nidulans and to use it to obtain parameiotic segregants. Analyses showed the medusa phenotype of the B84 strain, whose mutant allele was mapped in the chromosome I. The heterokaryons B84(med)//G422(med+) and B84(med)//G839(brl) were formed in liquid MM+2% CM and inoculated in the appropriate selective media. Two mitotic segregant groups were obtained: aneuploids and haploid stable recombinants. Mitotic segregants, wild-types, and developmental mutants, which did not produce new visible mitotic sectors in the presence of Benomyl and which showed normal meiotic behavior during the sexual cycle, were classified as parameiotics.     

Aspergillus nidulans asexual development: making the most of cellular modules

Asexual development in Aspergillus nidulans begins in superficial hyphae as the programmed emergence of successive pseudohyphal modules, collectively known as the conidiophore, and is completed by a layer of specialized cells (phialides) giving rise to chains of aerial spores. A discrete number of regulatory factors present in hyphae play different stage-specific roles in pseudohyphal modules, depending on their cellular localization and protein-protein interactions. Their multiple roles include the timely activation of a sporulation-specific pathway that governs phialide and spore formation. Such functional versatility provides for a new outlook on morphogenetic change and the ways we should study it.     

FluG and flbA function interdependently to initiate conidiophore development in Aspergillus nidulans through brlA beta activation.

The Aspergillus nidulans fluG gene is necessary for the synthesis of a small diffusible factor that is required for the endogenously regulated induction of asexual sporulation that takes place during the development of an air-exposed colony. Previous work established that FluG is present at nearly constant levels throughout the Aspergillus life cycle, leading to the hypothesis that FluG factor is constitutively produced and development initiates after its concentration surpasses a fixed threshold. Here we show that overexpression of fluG can overcome the developmental block normally imposed on vegetative cells in submerged culture and leads to the formation of complex conidiophores that are remarkably similar to wild-tye conidiophores made by air- exposed colonies. This fluG-induced sporulation requires the activities of other early developmental regulatory genes including, flA, flB, flC, flD, flE, and brlA. The requirement for flbA in fluG-induced sporulation is particularly interesting because overexpression of flbA can also induce sporulation in submerged culture and this flbA activity requires fluG. The interdependence of fluG and flbA activities suggests a close relationship between the products of these two genes in controlling conidiophore development. In addition to the endogenous sporulation signal provided by fluG, several environmental factors, including air exposure, carbon or nitrogen stress, and increased osmolarity, can influence developmental activation. We demonstrate that each of these signals requires the brlA beta gene, but not brlA alpha, to initiate conidiophore development. We present a model to account for the complex genetic and environmental controls leading to the activation of brlA beta and sporulation.     

Sporulation competence in Aspergillus nidulans: a role for iron in development.

There is a difference in the response of DNA from mycelial extracts of Aspergillus nidulans to hot acid hydrolysis depending upon the state of sporulation competence. The DNA in incompetent culture mycelia is not hydrolyzable while the DNA in competent culture is hydrolyzable. The inhibition of DNA hydrolysis is due to the presence of iron. Although the concentration of iron decreases in mycelia during growth, there is sufficient iron present in competent mycelia to inhibit DNA hydrolysis. The change in DNA hydrolyzability may be the result of a change in intracellular iron distribution, or a change in an iron binding component. We suggest that these changes are related to the altered capacity for gene expression which occurs at the time of acquisition of sporulation competence.     

Maize ribosome-inactivating protein inhibits normal development of Aspergillus nidulans and Aspergillus flavus

The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion.     

Phosphopantetheinyl transferase CfwA/NpgA is required for Aspergillus nidulans secondary metabolism and asexual development

Polyketide synthases (PKSs) and/or nonribosomal peptide synthetases (NRPSs) are central components of secondary metabolism in bacteria, plants, and fungi. In filamentous fungi, diverse PKSs and NRPSs participate in the biosynthesis of secondary metabolites such as pigments, antibiotics, siderophores, and mycotoxins. However, many secondary metabolites as well as the enzymes involved in their production are yet to be discovered. Both PKSs and NRPSs require activation by enzyme members of the 4'-phosphopantetheinyl transferase (PPTase) family. Here, we report the isolation and characterization of Aspergillus nidulans strains carrying conditional (cfwA2) and null (DeltacfwA) mutant alleles of the cfwA gene, encoding an essential PPTase. We identify the polyketides shamixanthone, emericellin, and dehydroaustinol as well as the sterols ergosterol, peroxiergosterol, and cerevisterol in extracts from A. nidulans large-scale cultures. The PPTase CfwA/NpgA was required for the production of these polyketide compounds but dispensable for ergosterol and cerevisterol and for fatty acid biosynthesis. The asexual sporulation defects of cfwA, DeltafluG, and DeltatmpA mutants were not rescued by the cfwA-dependent compounds identified here. However, a cfwA2 mutation enhanced the sporulation defects of both DeltatmpA and DeltafluG single mutants, suggesting that unidentified CfwA-dependent PKSs and/or NRPSs are involved in the production of hitherto-unknown compounds required for sporulation. Our results expand the number of known and predicted secondary metabolites requiring CfwA/NpgA for their biosynthesis and, together with the phylogenetic analysis of fungal PPTases, suggest that a single PPTase is responsible for the activation of all PKSs and NRPSs in A. nidulans.     

Coordinate control of secondary metabolite production and asexual sporulation in Aspergillus nidulans

Microbial secondary metabolite production is frequently associated with developmental processes such as sporulation, but there are few cases where this correlation is understood. Recent work with the filamentous fungus Aspergillus nidulans has provided new insights into the mechanisms coordinating production of the toxic secondary metabolite sterigmatocystin with asexual sporulation. These processes have been shown to be linked through a common need to inactivate a heterotrimeric G protein dependent signaling pathway that, when active, serves to stimulate growth while blocking both sporulation and sterigmatocystin biosynthesis.