RESUMEN
Some aspergilli are among the most cosmopolitan and ecologically dominant fungal species. One pillar of their success is their complex life cycle, which creates specialized cell types for versatile dispersal and regenesis. One of these cell types is unique to aspergilli-the Hülle cells. Despite being known for over a century, the biological and ecological roles of Hülle cells remain largely speculative. Previously reported data on in vivo Hülle cell formation and localization have been conflicting. Our quantification reveals that Hülle cells can occur at all locations on hyphae and that they show cellular activity similar to that seen with adjacent hyphae, indicating that they develop as intricate parts of hyphal tissue. In addition, we show that during sexual development associated with two parental strains, the typically multinucleate Hülle cells can inherit nuclei from both parents, indicating that they may serve as genetic backups. We provide an easy, reproducible method to study Hülle cell biology and germination with which we investigate the 90-year-old puzzle of whether and how Hülle cells germinate. We present clear evidence for the germination of Hülle cells, and we show that Hülle cells grow hyphae that develop into a spore-producing colony. Finally, we show that Hülle cell-derived colonies produce conidiospores faster than spore-derived colonies, providing evidence for an as-yet-undescribed developmental shortcut program in Aspergillus nidulans We propose that Hülle cells represent a unique cell type as specialized hypha-derived sexual tissue with a nucleus storage function and may act as fungal backup stem cells under highly destructive conditions.IMPORTANCE The in vivo identification of Hülle cells in cases of aspergillosis infections in animals and humans illustrates their biological relevance and suggests that they might be involved in pathogenicity. It is striking that aspergilli have developed and maintained a multinucleate nurse cell that is presumably energy-intensive to produce and is usually found only in higher eukaryotes. Our findings shed light on how the understudied Hülle cells might contribute to the success of aspergilli by acting not only as nurse cells under detrimental conditions (sexual development) but also as fungal backup stem cells with the capacity to produce genetically diverse spores in an accelerated manner, thereby substantially contributing to survival in response to predator attack or under otherwise severely destructive conditions. Our study solved the 90-year-old puzzle of Hülle cell germination and provides easy, reproducible methods that will facilitate future studies on biological and ecological roles of Hülle cells in aspergilli.
Asunto(s)
Aspergillus nidulans/citología , Aspergillus nidulans/fisiología , Proteínas Fúngicas/metabolismo , Hifa/citología , Aspergillus nidulans/genética , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Hifa/fisiología , Células Madre Multipotentes/citología , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
The bacterial secondary metabolite 2,4-diacetylphloroglucinol (DAPG) is of interest as an active ingredient of biological control strains of Pseudomonas fluorescens and as a potential lead pharmaceutical molecule because of its capacity to inhibit growth of diverse microbial and non-microbial cells. The mechanism by which this occurs is unknown and in this study the filamentous fungus Neurospora crassa was used as a model to investigate the effects of DAPG on a eukaryotic cell. Colony growth, conidial germination and cell fusion assays confirmed the inhibitory nature of DAPG towards N. crassa. A number of different fluorescent dyes and fluorescent protein reporters were used to assess the effects of DAPG treatment on mitochondrial and other cellular functions. DAPG treatment led to changes in mitochondrial morphology, and rapid loss of mitochondrial membrane potential. These effects are likely to be responsible for the toxicity of DAPG. It was also found that DAPG treatment caused extracellular calcium to be taken up by conidial germlings leading to a transient increase in cytosolic free Ca(2+) with a distinct concentration dependent Ca(2+) signature.
Asunto(s)
Antifúngicos/metabolismo , Calcio/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Neurospora crassa/efectos de los fármacos , Neurospora crassa/fisiología , Homeostasis/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neurospora crassa/crecimiento & desarrollo , Floroglucinol/análogos & derivados , Floroglucinol/metabolismo , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
Saccharomyces cerevisiae is a proven model to investigate the effects of small molecules and drugs on fungal and eukaryotic cells. In this study, the mode of action of an antifungal metabolite, 2,4-diacetylphloroglucinol (DAPG), was determined. Applying a combination of genetic and physiological approaches, it was established that this bacterial metabolite acts as a proton ionophore and dissipates the proton gradient across the mitochondrial membrane. The uncoupling of respiration and ATP synthesis ultimately leads to growth inhibition and is the primary toxic effect of DAPG. A genome-wide screen identified 154 DAPG-tolerant mutants and showed that there are many alterations in cellular metabolism that can confer at least some degree of tolerance to this uncoupler. One mutant, ydc1, was studied in some more detail as it displayed increased tolerance to both DAPG and the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) and appears to be unconnected to other tolerant mutant strains. Deleting YDC1 alters sphingolipid homoeostasis in the cell, and we suggest here that this may be linked to reduced drug sensitivity. Sphingolipids and their derivatives are important eukaryotic signal molecules, and the observation that altering homoeostasis may affect yeast response to metabolic uncoupling agents raises some intriguing questions for future studies.