The Role of Transcription Factor AoMsn2 in the Hog1 Signaling Pathway in Fungal Growth, Development, and Pathogenicity

Background

Plant-parasitic nematodes cause significant annual losses to agriculture, while nematode-trapping fungi (NT fungi), due to their ability to capture nematodes by forming specialized trapping structures, have gradually become model organisms for studying the interaction between fungi and nematodes. Arthrobotrys oligospora is a typical NT fungus that captures and kills nematodes by forming adhesive networks. Previous studies have shown that the high-osmolarity glycerol (Hog1) signaling pathway plays a key role in osmoregulation and nematocidal activity in A. oligospora. However, the functions of downstream transcription factors of the Hog1 signaling pathway in NT fungi remain unclear. Therefore, this study aimed to investigate the functions and potential regulatory network of AoMsn2, a downstream transcription factor of the Hog1 signaling pathway in A. oligospora.

Source of the Paper

This research was conducted by Qianqian Liu, Kexin Jiang, Shipeng Duan, Na Zhao, Yanmei Shen, Lirong Zhu, Ke-Qin Zhang, Jinkui Yang, and others, affiliated with the School of Life Sciences, Yunnan University, and the Key Laboratory for Microbial Diversity of Southwest China, Ministry of Education. The study was published in the Journal of Advanced Research in 2025, with the title “Identification of a Transcription Factor AoMsn2 of the HOG1 Signaling Pathway Contributes to Fungal Growth, Development and Pathogenicity in Arthrobotrys oligospora.”

Research Process

1. Sequence and Phylogenetic Analysis of the AoMsn2 Gene

The researchers first obtained the AoMsn2 gene (Aol_s00076g216) from the A. oligospora genome through an NCBI BlastP search. By calculating the isoelectric point and molecular weight, they found that AoMsn2 has a molecular weight of 55.12 kDa and an isoelectric point of 5.79. Subsequently, through multiple sequence alignment and phylogenetic analysis, the researchers discovered that AoMsn2 exhibits high sequence similarity (87.9%–96.2%) with Msn2 homologs from other NT fungi, while showing low similarity (24.2%–29.0%) with homologs from filamentous fungi.

2. Construction of AoMsn2 Knockout Mutants

To investigate the function of AoMsn2 in A. oligospora, the researchers constructed AoMsn2 knockout mutants using a homologous recombination strategy. First, they amplified the flanking sequences of the AoMsn2 gene by PCR and ligated them to a hygromycin resistance gene (hph), successfully constructing the AoMsn2 mutants through yeast transformation and protoplast transformation techniques. The construction of the mutants was further validated by Southern blot analysis.

3. Phenotypic Experiments

The researchers conducted detailed observations and quantitative analyses of mycelial growth, conidiation, trap formation, and nematode predation efficiency in wild-type (WT) and AoMsn2 mutants on PDA, TYGA, and TG media. The results showed that mycelial growth was significantly slowed in AoMsn2 mutants, while conidial yield and germination rates were significantly reduced. Additionally, trap formation and nematode predation efficiency were severely affected.

4. Stress Response Experiments

To explore the role of AoMsn2 in stress responses, the researchers cultured WT and mutant strains on media containing various stressors (e.g., hydrogen peroxide, NaCl, sorbitol) and observed their growth. The results revealed that AoMsn2 mutants exhibited significantly increased sensitivity to oxidative and osmotic stress.

5. Lipid Metabolism and Autophagy-Related Experiments

Through BODIPY staining and transmission electron microscopy (TEM), the researchers found that lipid droplets (LDs) were significantly accumulated in AoMsn2 mutants, with an increase in LD size. Moreover, the mutants showed restricted growth on media containing different fatty acids, indicating that AoMsn2 plays an important role in lipid metabolism. Additionally, MDC staining and transcriptomic analysis revealed that AoMsn2 is also involved in the regulation of autophagy pathways.

6. Transcriptomic and Metabolomic Analysis

To further uncover the regulatory network of AoMsn2, the researchers conducted RNA sequencing and metabolomic analysis on WT and mutant strains. The results showed that multiple metabolic pathways (e.g., glycerolipid metabolism, fatty acid synthesis) were significantly downregulated in AoMsn2 mutants, while the synthesis of the unique secondary metabolite Arthrobotrisins was significantly increased.

7. Yeast Two-Hybrid Assay

Through yeast two-hybrid assays, the researchers identified interactions between AoMsn2 and several proteins (e.g., Aohog1, AoMcm1, AoArc18) and mapped the protein interaction network of AoMsn2.

Results and Conclusions

1. AoMsn2 Regulates Mycelial Growth and Development

The study found that mycelial growth was significantly slowed in AoMsn2 mutants, with swollen hyphal cells and reduced septal distances. Additionally, the number of nuclei in mutant cells was significantly reduced, indicating that AoMsn2 plays an important role in mycelial growth and morphological development.

2. AoMsn2 Regulates Conidiation and Spore Germination

The conidial yield and germination rates of AoMsn2 mutants were significantly reduced, and the expression of multiple genes related to conidiation was significantly downregulated, suggesting that AoMsn2 plays a critical role in conidiation.

3. AoMsn2 Regulates Trap Formation and Nematode Predation Efficiency

The ability to form traps was significantly reduced in AoMsn2 mutants, and nematode predation efficiency was severely affected, indicating that AoMsn2 plays an important role in nematode predation.

4. AoMsn2 Regulates Stress Responses

AoMsn2 mutants exhibited significantly increased sensitivity to oxidative and osmotic stress, suggesting that AoMsn2 plays a crucial role in fungal stress responses.

5. AoMsn2 Regulates Lipid Metabolism and Autophagy

Significant accumulation of lipid droplets was observed in AoMsn2 mutants, and the mutants showed restricted growth on media containing fatty acids, indicating that AoMsn2 is involved in lipid metabolism. Moreover, autophagy pathways were affected in the mutants, suggesting that AoMsn2 plays a key role in autophagy regulation.

6. AoMsn2 Regulates Secondary Metabolite Synthesis

Metabolomic analysis revealed a significant increase in the content of Arthrobotrisins in AoMsn2 mutants, indicating that AoMsn2 regulates the synthesis of secondary metabolites.

Significance and Value of the Study

This study is the first to systematically reveal the multifunctionality of AoMsn2 in A. oligospora, clarifying its crucial roles in mycelial growth, conidiation, trap formation, nematode predation efficiency, as well as lipid metabolism and autophagy. Through transcriptomic and metabolomic analysis, the researchers further uncovered the potential regulatory network of AoMsn2, providing important molecular insights into the morphogenesis and environmental adaptation mechanisms of NT fungi. Additionally, the research findings offer theoretical support for the development of effective nematode biocontrol strategies.

Research Highlights

1. The multifunctionality of AoMsn2 in A. oligospora has been systematically revealed for the first time.
2. Through transcriptomic and metabolomic analysis, the potential regulatory network of AoMsn2 was uncovered.
3. The study provides important molecular insights into the morphogenesis and environmental adaptation mechanisms of NT fungi.
4. The findings offer theoretical support for the development of effective nematode biocontrol strategies.