Dataset Information

The High Osmolarity Glycerol (HOG) Pathway Functions in Osmosensing, Trap Morphogenesis and Conidiation of the Nematode-Trapping Fungus Arthrobotrys oligospora.

ABSTRACT: Hog1, a mitogen-activated protein kinase (MAPK), has been identified in diverse fungal species, and it regulates various cellular processes, such as osmoadaptation, nutrient-sensing, and pathogenesis. However, the roles that Hog1 plays in nematode-trapping fungi were previously unclear. Here, we characterized orthologs of Saccharomyces cerevisiae Hog1 and membrane mucin Msb2 in the nematode-trapping fungus Arthrobotrys oligospora. We generated gene deletion mutants of HOG1 and MSB2 in A. oligospora, and characterized their roles in osmosensing, growth, and trap morphogenesis. We found that both hog1 and msb2 mutants were highly sensitive to high osmolarity. Predation analyses further revealed that hog1 and msb2 deletion caused a reduction in trap formation and predation efficiency. Furthermore, HOG1 is required for conidiation in A. oligospora, demonstrating its critical role in this developmental pathway. In summary, this study demonstrated that the conserved Hog1 and Msb2 govern physiology, growth and development in the nematode-trapping fungus A. oligospora.

SUBMITTER: Kuo CY

PROVIDER: S-EPMC7711997 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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The High Osmolarity Glycerol (HOG) Pathway Functions in Osmosensing, Trap Morphogenesis and Conidiation of the Nematode-Trapping Fungus Arthrobotrys oligospora.

Kuo Chih-Yen CY Chen Sheng-An SA Hsueh Yen-Ping YP

Journal of fungi (Basel, Switzerland) 20200927 4

Hog1, a mitogen-activated protein kinase (MAPK), has been identified in diverse fungal species, and it regulates various cellular processes, such as osmoadaptation, nutrient-sensing, and pathogenesis. However, the roles that Hog1 plays in nematode-trapping fungi were previously unclear. Here, we characterized orthologs of Saccharomyces cerevisiae Hog1 and membrane mucin Msb2 in the nematode-trapping fungus Arthrobotrys oligospora. We generated gene deletion mutants of HOG1 a ...[more]

PMID: 32992476

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Project description:Nematode-trapping (NT) fungi are a unique group of carnivorous microorganisms that can capture and digest nematodes by producing ingenious trapping devices (traps). Arthrobotrys oligospora, a representative NT fungus, can develop adhesive three-dimensional networks for nematode predation. Hyphal fusion is indispensable for the trap formation of A. oligospora. Here, we characterized an orthologous Ste12 protein (AoSte12) in A. oligospora via gene disruption, DNA affinity purification sequencing (DAP-Seq), and multi-omics approaches. The disruption of the Aoste12 gene caused an increase in hyphal fusion and resulted in defects in mycelial growth, conidiation, trap morphology, and stress resistance, as well as reducing the number of nuclei and lipid droplet accumulation. Moreover, transcriptome and DAP-Seq analysis revealed that AoSte12 was involved in cellular processes associated with growth, cell fusion, the tricarboxylic acid cycle, vesicles, actin filaments, and lipid metabolism. In addition, combining metabolome with transcriptome and DAP-Seq analysis indicated that AoSte12 was involved in the mitogen-activated protein kinase signaling pathway, lipid metabolism, and secondary metabolites. A yeast two-hybrid assay revealed that AoSte12 can interact with diverse proteins, such as the MAK-2 orthologue protein Fus3, the vacuolar sorting protein Pep3, and UDP-glycosyltransferase. Our results suggest that AoSte12 plays an indispensable role in hyphal fusion and thus regulates sporulation and trap morphogenesis. These results provide deep insights into the connection between hyphal fusion and trap formation in NT fungi. IMPORTANCE Nematode-trapping (NT) fungi are an important natural enemy of nematodes and can capture their prey by producing traps. Hyphal anastomosis and fusion are important for mycelial growth and the colony morphological development of filamentous fungi and are also crucial for the trap morphogenesis of NT fungi. Arthrobotrys oligospora can form complex three-dimensional networks (traps) when sensing the presence of nematodes. This study revealed that AoSte12 is indispensable for hyphal fusion and that it regulates mycelial growth, conidiation, trap morphogenesis, stress resistance, the number of nuclei, and lipid droplet accumulation in A. oligospora. In addition, DNA affinity purification sequencing, transcriptome, and metabolome analyses further revealed that AoSte12 is involved in the mitogen-activated protein kinase pathway, lipid metabolism, and secondary metabolism. Overall, these findings expand the important role of AoSte12 in NT fungus A. oligospora and provide a broad foundation for elucidating the regulatory mechanism of trap development and the lifestyle transitions of pathogenic fungi.

Project description:Autophagy is a conserved cellular recycling and trafficking pathway in eukaryotes that plays an important role in cell growth, development, and pathogenicity. Atg1 and Atg13 form the Atg1-Atg13 complex, which is essential for autophagy in yeast. Here, we characterized the roles of the Aolatg1 and Aolatg13 genes encoding these autophagy-related proteins in the nematode-trapping fungus Arthrobotrys oligospora. Investigation of the autophagy process by using the AoAtg8-GFP fusion protein showed that autophagosomes accumulated inside vacuoles in the wild-type (WT) A. oligospora strain, whereas in the two mutant strains with deletions of Aolatg1 or Aolatg13, GFP signals were observed outside vacuoles. Similar results were observed by using transmission electron microscopy. Furthermore, deletion of Aolatg1 caused severe defects in mycelial growth, conidiation, conidial germination, trap formation, and nematode predation. In addition, transcripts of several sporulation-related genes were significantly downregulated in the ΔAolatg1 mutant. In contrast, except for the altered resistance to several chemical stressors, no obvious differences were observed in phenotypic traits between the WT and ΔAolatg13 mutant strains. The gene ontology analysis of the transcription profiles of the WT and ΔAolatg1 mutant strains showed that the set of differentially expressed genes was highly enriched in genes relevant to membrane and cellular components. The Kyoto Encyclopedia of Genes and Genomes analysis indicated that differentially expressed genes were highly enriched in those related to metabolic pathways, autophagy and autophagy-related processes, including ubiquitin-mediated proteolysis and SNARE interaction in vesicular transport, which were enriched during trap formation. These results indicate that Aolatg1 and Aolatg13 play crucial roles in the autophagy process in A. oligospora. Aolatg1 is also involved in the regulation of asexual growth, trap formation, and pathogenicity. Our results highlight the importance of Aolatg1 in the growth and development of A. oligospora, and provide a basis for elucidating the role of autophagy in the trap formation and pathogenicity of nematode-trapping fungi.

Project description:AMP-activated protein kinase (AMPK), a heterotrimeric complex, can sense energy and nutritional status in eukaryotic cells, thereby participating in the regulation of multiple cellular processes. In this study, we characterized the function of the catalytic α-subunit (SNF1) and the two regulatory β- and γ-subunits (GAL83 and SNF4) of AMPK in a representative nematode-trapping fungus, Arthrobotrys oligospora, by gene knockout, phenotypic analysis, and RNA sequencing. The ability of the AMPK complex mutants (including ΔAosnf1, ΔAogal83, and ΔAosnf4) to utilize a nonfermentable carbon source (glycerol) was reduced, and the spore yields and trap formation were remarkably decreased. Moreover, AMPK plays an important role in regulating stress response and nematode predation efficiency. Transcriptomic profiling between the wild-type strain and ΔAosnf1 showed that differentially expressed genes were enriched for peroxisome, endocytosis, fatty acid degradation, and multilipid metabolism (sphingolipid, ether lipid, glycerolipid, and glycerophospholipid). Meanwhile, a reduced lipid droplet accumulation in ΔAosnf1, ΔAogal83, and ΔAosnf4 mutants was observed, and more vacuoles appeared in the mycelia of the ΔAosnf1 mutant. These results highlight the important regulatory role of AMPK in the utilization of carbon sources and lipid metabolism, as well as providing novel insights into the regulatory mechanisms of the mycelia development, conidiation, and trap formation of nematode-trapping (NT) fungi. IMPORTANCE NT fungi are widely distributed in various ecosystems and are important factors in the control of nematode populations in nature; their trophic mycelia can form unique infectious devices (traps) for capturing nematodes. Arthrobotrys oligospora is a representative NT fungi which can develop complex three-dimensional networks (adhesive networks) for nematode predation. Here, we demonstrated that AMPK plays an important role in the glycerol utilization, conidiation, trap formation, and nematode predation of A. oligospora, which was further confirmed by transcriptomic analysis of the wild-type and mutant strains. In particular, our analysis indicated that AMPK is required for lipid metabolism, which is primarily associated with energy regulation and is essential for trap formation. Therefore, this study extends the functional study of AMPK in NT fungi and helps to elucidate the molecular mechanism of the regulation of trap development, as well as laying the foundation for the development of efficient nematode biocontrol agents.

Project description:Nematode-trapping (NT) fungi can form unique infection structures (traps) to capture and kill free-living nematodes and, thus, can play a potential role in the biocontrol of nematodes. Arthrobotrys oligospora is a representative species of NT fungi. Here, we performed a time course transcriptome sequencing (RNA-seq) analysis of transcriptomes to understand the global gene expression levels of A. oligospora during trap formation and predation. We identified 5,752 unique differentially expressed genes, among which the rac gene was significantly upregulated. Alternative splicing events occurred in 2,012 genes, including the rac and rho2 gene. Furthermore, we characterized three Rho GTPases (Rho2, Rac, and Cdc42) in A. oligospora using gene disruption and multiphenotypic analysis. Our analyses showed that AoRac and AoCdc42 play an important role in mycelium growth, lipid accumulation, DNA damage, sporulation, trap formation, pathogenicity, and stress response in A. oligospora. AoCdc42 and AoRac specifically interacted with components of the Nox complex, thus regulating the production of reactive oxygen species. Moreover, the transcript levels of several genes associated with protein kinase A, mitogen-activated protein kinase, and p21-activated kinase were also altered in the mutants, suggesting that Rho GTPases might function upstream from these kinases. This study highlights the important role of Rho GTPases in A. oligospora and provides insights into the regulatory mechanisms of signaling pathways in the trap morphogenesis and lifestyle transition of NT fungi. IMPORTANCE Nematode-trapping (NT) fungi are widely distributed in terrestrial and aquatic ecosystems. Their broad adaptability and flexible lifestyles make them ideal agents for controlling pathogenic nematodes. Arthrobotrys oligospora is a model species employed for understanding the interaction between fungi and nematodes. Here, we revealed that alternative splicing events play a crucial role in the trap development and lifestyle transition in A. oligospora. Furthermore, Rho GTPases exert differential effects on the growth, development, and pathogenicity of A. oligospora. In particular, AoRac is required for sporulation and trap morphogenesis. In addition, our analysis showed that Rho GTPases regulate the production of reactive oxygen species and function upstream from several kinases. Collectively, these results expand our understanding of gene expression and alternative splicing events in A. oligospora and the important roles of Rho GTPases in NT fungi, thereby providing a foundation for exploring their potential application in the biocontrol of pathogenic nematodes.

Dataset Information

The High Osmolarity Glycerol (HOG) Pathway Functions in Osmosensing, Trap Morphogenesis and Conidiation of the Nematode-Trapping Fungus Arthrobotrys oligospora.

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The High Osmolarity Glycerol (HOG) Pathway Functions in Osmosensing, Trap Morphogenesis and Conidiation of the Nematode-Trapping Fungus <i>Arthrobotrys oligospora</i>.

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