Regulatory Roles of Transcription Factor AOL-113 in Mycelial Growth， Stress Response， and Predatory Ability of Arthrobotrys oligospora

doi:10.13560/j.cnki.biotech.bull.1985.2025-0603

Abstract

Abstract:

Objective To investigate the biological function of the Zn(II)₂Cys₆-type transcription factor AOL-113 of Arthrobotrys oligospora. Method The ΔAOL-113 deletion strain and the CΔAOL-113 complementary strain were constructed by homologous recombination, and the phenotypes such as mycelial growth, nutrient utilization, environmental adaptability, conidial production and germination, as well as nematode-trapping efficiency were subsequently analyzed. Additionally, transcriptome analysis was conducted to identify differentially expressed genes between the WT strain and the ΔAOL-113 deletion strain under sodium acetate conditions. Result The transcription factor AOL-113 gene encoded GAL4 zinc finger protein and shared high homology with the transcription factor FacB. Compared to the wild-type (WT) strain, the ∆AOL-113 strain presented significantly reduced growth rates and a marked decrease in carbon source utilization, especially fatty acid metabolism. It is more sensitive to oxidative stress agents such as H₂O₂ and menadione. The ∆AOL-113 strain also showed diminished trap production and a lower rate of sticky network formation, which significantly impaired its ability to degrade and digest nematodes. The complementary strain showed no significant phenotypic differences compared to the WT strain. Transcriptome analysis further revealed that the deletion of the AOL-113 led to the down-regulation of key genes involved in multiple pathways such as oxidative phosphorylation, lipid metabolism, glyoxylate cycle, peroxisome function, galactose metabolism, and glycolysis, this affected multiple biological processes such as mycelial growth, carbon source utilization, stress resistance, and predation ability. Conclusion The transcription factor AOL-113 not only provides energy support for hyphal growth and trap formation by regulating cellular metabolism (especially acetic acid metabolism and fatty acid β-oxidation), but also participates in regulating the efficiency of nematode predation and digestion, providing new ideas for revealing the molecular mechanism of the transition of A. oligosporus from a saprophytic to a parasitic lifestyle and the development of biocontrol agents for livestock nematode diseases.

Key words: Arthrobotrys oligospora, Zn(II)?Cys?-type transcription factor, carbon source utilization, mycelial growth, predatory ability

SUN Yan-sen, WEI Li-xiang, LI Ruo-bing, ZHANG Cheng-zhi, NIE Yu-hang, LI Jie, CAI Xue-peng, QIAO Jun, MENG Qing-ling. Regulatory Roles of Transcription Factor AOL-113 in Mycelial Growth， Stress Response， and Predatory Ability of Arthrobotrys oligospora[J]. Biotechnology Bulletin, 2026, 42(2): 338-350.

Figures/Tables 9

Table 1 Specific primers used in the study

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	长度 Length （bp）	基因目的 Gene description
AOL-113-F/AOL-113-R	ATGTCGTTGAAACGGTCG/GTCATTCCAGCTTGATAGAACCG	3 213	AOL-113基因全长
AOL-113-5F-pUC19	GTACTGAGAGTGCACCATATGCGACAGCCTTGGAGAGTTTGAC	1 861	AOL-113基因5′端基因序列
AOL-113-5R-HygR	GTTACTGAAATCTCCAACGGTTAGGAATATGTTCGTCAATGAGG	1 861	AOL-113基因5′端基因序列
HygR-F-113-5R	GACGAACATATTCCTAACCGTTGGAGATTTCAGTAACGTTAAG	1 441	潮霉素抗性基因
HygR-R-113-3F	CTACCCGGTTGACCAAGGTCGGAGACAGAAGATGATATTG	1 441	潮霉素抗性基因
AOL-113-3F-HygR	CATCTTCTGTCTCCGACCTTGGTCAACCGGGTAGAGG	1 865	AOL-113基因3′端基因序列
AOL-113-3R-pUC19	GACCATGATTACGCCAAGCTTTGCACACGTACTACAAAGCAAGC	1 865	AOL-113基因3′端基因序列
CΔ AOL-113-5F-pUC19	CTGAGAGTGCACCATATGCTTCCTCTTCAATTCCTCTTACAAGG	3 482	CΔ AOL-113基因回补全长及5′端侧翼序列
CΔ AOL-113-5R-NeoR	GTGCAGATTATTTGGATTGACAAGTATATCCTTCACCCATGACC	3 482	CΔ AOL-113基因回补全长及5′端侧翼序列
NeoR-F-113-5R	GTGAAGGATATACTTGTTCAATCCAAATAATCTGCACCG	919	G-418抗性基因
NeoR-R-113-3F	CCATCTATGTCACCTCTACCCCAGGTTGGGCGTCGCTTG	919	G-418抗性基因
CΔ AOL-113-3F-pUC19	CAAGCGACGCCCAACCTGGGGTAGAGGTGACATAGATGGAACG	1 382	CΔ AOL-113 3′端基因序列
CΔ AOL-113-3R-pUC19	GACCATGATTACGCCAAGCTTCAGTTGTGAAGAGCTTCTCCCTTG	1 382	CΔ AOL-113 3′端基因序列
Δ AOL-113YZ-F/Δ AOL-113YZ-R	GTATACTTACCAACCTGCTCC/ GCTACTTCCTGATGATATCCTTC	3 827/2 209	野生型和缺失转化子菌株的验证
CΔ AOL-113YZ-F/CΔ AOL-113YZ-R	CTTCCTCTTCAATTCCTCTTACAAGG/ GGTCATGGTGAAGGATATACTTGT	3 445/2 301	回补株转化子菌株的验证

Fig. 1 Molecular characteristics and phylogenetic analysis of the Zn2(II)Cys6-type transcription factor AOL-113 of A. oligosporaA: Domain characteristic of transcription factor AOL-113. B: Phylogenetic analysis of different fungi based on transcription factor AOL-113orthologs

Fig. 2 Construction and screening of deletion strains and complementary strainsA: Electrophoresis detection results of deletion strains. M: DL-5000 DNA marker; 1: negative control; 2-3: positive clones of deletion strains. B: Electrophoresis detection results of complementary strains. M: DL-5000 DNA marker; 1: positive clones of complementary strains; 2-4: negative control

Fig. 3 Comparison of growth rate and mycelial cell lengthA: TG, YPSSA, TYGA, PDA and CMY media were incubated at 28 ℃ for 5 d for colony morphology observation and growth rate measurement. B: CFW staining of mycelia. Scale = 20 μm, the hyphal septa are denoted by white arrows. *P<0.05, **P<0.01. The same below

Fig. 4 Analysis of utilization capacity of different carbon, nitrogen sources and fatty acid

Fig. 5 Sensitivities to osmotic agents, cell wall-perturbing agents, oxidants agents, thermal agents and pH agents

Fig. 6 Comparison of sporulation rate, spore germination rate and fluorescence observation of conidiaA: Side view of the phloem peduncle. B: Determination of sporulation rate. C: Spore germination rate. D: Observation of conidial germination. E: Fluorescence observation of conidia and spore length determination

Fig. 7 Comparison of the trap formation and predatory abilityA: Trap formation and traps counts at different time points. B: Observations of nematode predation and determination of predation rate

Fig. 8 Analysis of DEGs between WT and Δ AOL-113 of Arthrobotrys oligospora exposed to sodium acetate (SA)A: Volcano plot of DEGs. Up-regulated DEGs in red; down-regulated DEGs in blue. B: GO enrichment analysis of downregulated DEGs. C: KEGG enrichment analysis of downregulated DEGs. D: Validation of transcriptomic sequencing using RT-qPCR

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