Nematode-trapping fungus (NTF) is a crucial predator of nematodes, which can capture nematodes by developing specific trapping devices. However, there is limited understanding of the role and mechanism of cell surface proteins attached to the surface of mycelia or trapping cells. Here, the effects of a putative GPI-anchored protein-encoding gene Aog185 on the growth and nematode-trapping efficiency of A. oligospora were investigated. Compared to the wild-type (WT) strain, the ΔAog185 mutant grew more slowly, exhibited a 20% decrease in conidiation, delayed conidial germination, generated fewer traps, attenuated nematode trapping efficiency, and was more sensitive to chemical stressors. Transcriptomic analysis indicated that a large number of transmembrane transport-related genes were differentially expressed between the WT and ΔAog185 mutant strains. Aog185 deletion could damage the intrinsic components of the membrane and cytoskeleton. Specifically, knockout of Aog185 disrupted transmembrane transport homeostasis during the phagocytosis, cell autophagy, and oxidative phosphorylation processes, which were associated with the fusion of cells and organelle membranes, transport of ions and substrates, and energy metabolism. Hence, the putative GPI-anchored protein-encoding gene Aog185 may contribute to the lifestyle switch of NTF and nematode capture, and the effect of Aog185 gene on cell transmembrane transport is considered key to this process. Our findings provide new insights into the mechanism of Aog185 gene during the process of nematode trapping by NTF.

1. Introduction

Plant-parasitic nematodes cause approximately >$150 billion worth of damage to agriculture each year worldwide [1]. The nematode-trapping fungus (NTF) is a natural predator of nematodes that forms specific mycelial structures called trapping devices to prey and digest nematodes [2]. The trap also belongs to a mechanism of fungus, allowing it to switch from saprophytic to predacious lifestyles [3]. Over the last 30 years, our understanding of the mechanisms of trapping and killing nematodes by NTF has improved tremendously, and the switch of strains from saprophytic to parasitic lifestyles has proven to be a key for trapping and killing nematodes [4]. In recent years, the identification of critical genes, proteins, metabolic pathways, and biological processes involved in the switch of the NTF lifestyle has become a popular research topic [5]. Compelling evidence suggests that multiple signaling pathways are activated in fungi by its nematode prey to further modulate downstream genes involved in distinct cellular processes, including catabolism, energy metabolism, transmembrane transport, biosynthesis of the cell wall and adhesive proteins, peroxisome biogenesis, stress responses, and cell division [6]. Therefore, the switch of strains from saprophytic to parasitic lifestyles is a global metabolic reprogramming. However, the critical factors affecting this reprogramming remains unknown.

Previous multiomics analyses revealed that many adhesion-associated protein-encoding genes were detected in the NTF genome, most of which were upregulated during the formation of traps [4, 7]. Liang et al. proved the important role of cell surface adhesins in the formation of traps and the lifestyle switching in NTF at the molecular level through the knockout of the adhesion-associated protein-encoding gene Mad1 in Arthrobotrys oligospora [8]. These studies indicate that the functional cell surface proteins are important for trapping nematodes. Glycosylated phosphatidylinositol-anchored proteins (GPI-APs) are a class of proteins that can be anchored to the eukaryotic cell membrane surface through the GPI structure at the carboxyl end [912]. Previous research has suggested that GPI-APs have important impacts on fungal adhesion, morphological transformation, and cell wall synthesis and play a vital role in mediating the adhesion process between fungi and host cells [13].

A. oligospora is a well-characterized NTF that captures nematodes with specialized traps. In this study, a putative GPI-AP gene Aog185 was identified in A. oligospora, and the effects of Aog185 on the lifestyle switch were assessed through gene knockout experiments. The potential mechanisms by which Aog185 affects cell growth and trapping efficiency were then revealed using comparative transcriptomics analysis.

2. Materials and Methods

2.1. Strains, Plasmids, and Culture Conditions

A. oligospora was supplied by American Type Culture Collection (ATCC 24927; VA, USA), and its fungal mutant strains were cultured on potato dextrose agar (PDA) medium in an incubator at 28°C. Plasmid pUC19 (TaKaRa Bio, Shiga, Japan) was maintained in E. coli strain DH5α. Plasmid pUC57-HygB was synthesized by General Biosystems (Anhui, China) for recombinant plasmid construction. Fungal mycelia were harvested into liquid TG (1% tryptone and 1% glucose) medium. The 0.75% agar TB3 (200 g sucrose, 3 g tryptone, and 3 g yeast extract diluted to 1 L) medium was utilized for protoplast regeneration. TYGA (1% tryptone, 1% glucose, 0.5% yeast extract, and 0.5% molasses) and CMA (17 g/L corn meal agar) were employed to determine the phenotypic traits of A. oligospora and mutant strains. Caenorhabditis elegans was maintained on NGM (NaCl, 0.3 g; peptone, 0.25 g; agar powder, 1.7 g; 1 mol/L MgSO4, 0.1 mL; 1 mol/L CaCl2, 0.1 mL; 5 mg/mL cholesterol ethanol solution, 0.1 mL; 1 mol/L KH2PO4-K2HPO4 buffer (pH 6.0), 2.5 mL per 100 mL) medium at 20°C.

2.2. Sequences and Phylogenetic Analysis of Aog185

The amino acid sequence of Aog185 was downloaded from GenBank (https://www.ncbi.nlm.nih.gov/). Conserved functional domains and partial biochemical properties were analyzed using the InterPro (http://www.ebi.ac.uk/interpro/) and pI/Mw tool (http://www.expasy.ch/tools/pi-tool.html), respectively. The orthologous proteins of GPI-anchored cell surface containing the actin-depolymerizing factor (ADF)/cofilin domain from different fungi were retrieved from GenBank, and a neighbor-joining tree for ADF/cofilin was established with Mega 7.0 software [14].

2.3. Deletion of Aog185 Gene

Aog185 gene was knocked out by homologous recombination as previously described [15]. PCR amplification was performed on 2 flanking fragments (a 2000-bp downstream and 2000-bp upstream) using the paired primers (Supplementary Table S1). The hygromycin B (HygB) resistance gene cassette in the PUC57-HygB plasmid was also amplified. The two homologous fragments, HygB cassette and linearized pUC19 vector were inserted into E. coli DH5α.

The combined plasmid pUC19-Aog185-HygB was then transformed into A. oligospora protoplast as previously described [16, 17]. The presence of HygB resistance gene in the transformants was detected using the agar containing hygromycin B (150 μg/mL). The putative transformants were verified by PCR assays using the specific primer pairs (Supplementary Table S1) and then sequenced.

2.4. Mycelial Growth, Conidiation, and Germination

Wild-type (WT) and mutant strains were maintained on CMA plates at 28°C for 6 days, and a 0.8 cm diameter hyphal disk was then cultured on PDA, TYGA, and CMA plates at 28°C for 6 days to assess the diameter of fungal growth every day at a fixed time point. The number of conidia (sterile water-rinsed, collected conidia of the previous CMA plates) and their conidial germination rates were determined and observed on water agar plates. All experiments were carried out in triplicate.

2.5. Stress-Tolerance Analysis

To determine the effects of both WT and mutants on the resistance of A. oligospora to chemical stressors, the colonies initiated with hyphal-mass disks (0.8 cm in diameter) were incubated for 6 days at 28°C on TG medium alone (control) or supplemented with chemical stressors as follows: (1) 0.1, 0.2, and 0.3 M NaCl for osmotic stress; (2) 0.01%, 0.02%, and 0.03% SDS for cell wall perturbation; (3) 5, 10, and 15 mM H2O2 for oxidative stress [16, 18]. The diameter and hyphal morphologies of each colony were examined, and the suppression ratio was calculated according to the where is the diameter of each colony grown for six days in the native TG plates supplemented without any chemical stressors, and is the diameter of each colony grown for six days in the TG plates supplemented with chemical stressors at different concentrations. All experiments were conducted in triplicate.

2.6. Trap Formation and Bioassays

To evaluate whether Aog185 can regulate trap formation in A. oligospora, both WT and mutant strains were cultured on water agar (WA, 15%) medium for 3 days at 28°C. Approximately 500 nematodes were then introduced into the cultures. After 8 h of incubation, the traps and captured nematodes in each plate were determined using an inverted fluorescence microscope and counted at specific time points [4].

2.7. Transcriptomic Analysis

The LNAAM was used as the culture medium for the shake-flask cultivation of A. oligospora prior to transcriptomic analysis. The hyphae of WT and Aog185 knockout strains were harvested at 0, 18, and 36 h after adding the nematode extract (NE) liquid. The collected hyphae were immediately frozen using liquid nitrogen and kept in a -80°C refrigerator. Three biological repeats were performed at each time point. The samples were transferred to the Shanghai Majorbio for transcriptome sequencing using an Illumina Novaseq 6000 sequencer. Statistical methods, such as fastx_toolkit, Sickle, SeqPrep, and fastp, were used to perform statistics and quality control of the tested sequences.

The clean reads were mapped by the complete reference using Bowtie2 v2.4.1 (http://bowtie-bio.sourceforge.net/index.shtml) [19]. RSEM v1.3.3 (https://www.biostat.wisc.edu/~cdewey/), kallisto v0.46.2, and Salmon v1.3.0 were used to measure the expression level of each gene [20]. Using the gene expression level of WT strain as a control, differentially expressed genes (DEGs) were detected in Aog185 mutant strain using the DESeq2 tool v1.24.0, and were considered DEGs. GO annotation of genes was performed using the eggNOG database (version 5.0). Phyper (a function of the R program) and Goatools were used for the GO enrichment analyses.

2.8. Statistical Analysis

The values are presented as . SPSS software v23.0 (SPSS Inc., IL, USA) was employed to process the data in this study, and the test was used to analyze and compare the differences between the groups. Levels of statistical significance were set at and . Prism 8.0 (GraphPad, CA, USA) was employed for image construction.

3. Results

3.1. Sequences and Phylogenetic Analysis of Aog185 in A. oligospora

Aog185 protein containing 1102 amino acids ( kDa) was identified in A. oligospora as an ortholog of GPI-anchored cell surface protein at the ADF/cofilin domain of Glarea lozoyensis and Pyronema omphalodes. Two conserved domains, ADF-H (IPR002108) and cofilin_ADF (PF00241), belonging to the ADF-H/gelsolin-like domain superfamily (IPR029006), were predicted in Aog185 protein through InterProScan. The Aog185 sequences were found to be more identical (68–78.9%) to those of NTF orthologs such as Drechslerella stenobrocha (78.9%), Drechslerella brochopaga (76.7%), and Dactylellina cionopaga (68%) than those of other filamentous fungi such as Aspergillus aculeatinus (37.9%) and Glarea lozoyensis (40.6%). A phylogenetic tree was constructed based on the homologous Aog185 protein of different fungi. The results showed that Aog185 proteins from these fungi could be clustered into two clades (A-Ι and A-II). The orthologous Aog185 sequences from three NTF species were grouped as A-II, while other fungi belonged to A-Ι (Figure 1).

3.2. Construction of Knockout Plasmids and Verification of ΔAog185 Mutant

To ligate the downstream and upstream homology arms of Aog185 (2000 bp each) and HygB resistance cassette (2121 bp), the seamless ligation primers were designed using the SnapGene software (Table S1). Figure S1 displays the principle of Aog185 gene knockout via homologous recombination. The linearized pUC19 cut by NdeI and PciI was first verified as the backbone vector for infusion cloning, and then transformed into E. coli. Seven clones were randomly chosen for PCR amplification using the corresponding primers (Aog185-1F/1R, Aog185-3F/3R, or HygB-2F/2R), and PCR data revealed that all 3 fragments were successfully ligated into the linearized pUC19. Five clones were simultaneously chosen for PCR verification using the respective primer Aog185-1F/3R, resulting in a 6121-bp amplicon, followed by DNA sequencing. The results confirmed that the knockout plasmid pUC19-Aog185-HygB was successfully constructed.

After transforming pUC19-Aog185-HygB into the protoplasts of A. oligospora, the transformants were chosen on TYGA plates containing 150 μg/mL HygB. Mycelial PCR indicated that an amplicon of 3114 bp was amplified from the ΔAog185 mutant, whereas a 4952-bp amplicon was generated from the WT strain (Figure S2A). For comparison, both 3114-bp and 4952-bp amplicons were produced for false-positive transformants, where Aog185 had been knocked out partially. As displayed in Figure S2A, only seven completely positive ΔAog185 mutants (No. 2, 3, 5, 11, 14, 16, and 26) were obtained. These ΔAog185 mutants were subsequently cultured on nonantibiotic TYGA medium for three generations. Mycelial PCR amplification and sequencing of PCR product were also carried out to confirm the correct transformants. As shown in Figure S2B, Aog185 gene in all these seven transformants was knocked out.

3.3. Effect of Aog185 on Conidiation and Growth

Compared to the WT strain, the growth of mutant strain was significantly lower on the CMA, TYGA, and PDA plates (Figure 2). In addition, the disruption of Aog185 gene could reduce conidiation. Conidial production in the ΔAog185 mutant ( conidia per plate) significantly reduced compared to that in the WT strain ( conidia per plate) (Figure S3A). After incubation on WA plates, the conidial germination rates of WT were remarkably increased compared to ΔAog185 mutant both at 2 h (61.45% and 55.64% for WT and ΔAog185 mutant, respectively) and 4 h (84.35% and 75.33% for WT and ΔAog185 mutant, respectively). Both WT and mutant strains showed 100% conidial germination rate after incubation for 12 h (Figure S3B). Thus, Aog185 gene plays crucial roles in enhancing conidiation, conidial germination, and growth of A. oligospora.

3.4. Vital Role of Aog185 in Stress Responses

The responses of WT and ΔAog185 mutant strains to three types of chemical stressors, such as an osmotic agent (NaCl), a cell wall-perturbing agent (SDS), and an oxidant (H2O2), were also studied. As compared to the native TG media without supplement of any chemical stressors, the suppression ratio of ΔAog185 mutant was significantly higher than the WT strain in TG media supplemented with NaCl at low concentrations ranging from 0.1 M to 0.2 M, whereas the suppression ratios for both the strains at high NaCl concentration of 0.3 M were not significantly different (Figure S4A and B). Similarly, compared to the native TG media, the suppression ratio of ΔAog185 mutant was significantly higher than the WT strain in TG plates containing >0.02% SDS, whereas both the strains showed similar suppression ratios at 0.03% SDS concentration (Figure S4C and D). As for chemical stressor H2O2, the WT strain and ΔAog185 mutant had similar suppression ratios at TG plates supplemented H2O2 at concentrations ranging from 5 mM to 15 mM (Figure S4E and F).

3.5. Aog185 Is Responsible for Trap Formation and Host Infection

Trap formation was induced by the addition of nematodes to WA plates. After 8, 12, and 24 h of incubation, traps were observed on the WT strain- and ΔAog185 mutant-WA plates (Figures 3(a) and 4(b)). After incubation for 8 h, the WT strain began to form immature traps containing a single ring, while no trap was found on the WA plate cultured with ΔAog185 mutant. Mature traps and 3D nets were formed after incubation for 12 and 24 h. In the plate incubation of the WT strain, approximately 11.7, 36.1, and 557.67 traps were observed after incubation for 8, 12, and 24 h, respectively. Meanwhile, only 0, 8.33, and 164.67 traps were observed in the plate where the ΔAog185 mutant was incubated (Figure 3(c)). Moreover, the nematicidal activities of WT and ΔAog185 mutant strains were also counted and measured at varying time points. There was no predation at 8 h after nematode addition in either strain. The WT strain captured 5.61% and 66.58% of nematodes after incubation for 12 and 24 h, respectively, whereas the ΔAog185 mutant captured 0 and 42.33% of the nematodes at the same time points, respectively (Figure 3(d)).

3.6. Effects of Aog185 on the Transcriptomics Landscape of A. oligospora

To explore the underlying mechanisms of Aog185, the transcriptomic data were compared between WT and Aog185 knockout strains at 0, 18, and 36 h after NE stimulation. The results of transcriptomic analysis showed that 26, 376, and 122 DEGs were identified at 0, 18, and 36 h, respectively. Venn analysis displayed the number of DEGs in each gene set and the overlapping relationship of genes between gene sets and identified common and unique genes between gene sets (Figure 4(a)). Compared with WT strains, 9, 285, and 84 genes were upregulated in Aog185 knockout strains at 0, 18, and 36 h, respectively, while 17, 91, and 38 genes were downregulated at the same time points, respectively ( and ). Data of these DEGs are presented in Table S6.

Based on the differential expression analysis, it was clearly observed that the effect of Aog185 on A. oligospora appeared at 18 h after NE stimulation. GO enrichment analyses were performed based on the DEGs identified at 18 h, and the data are presented in Figure 4(b). As shown in Figure 4(b), the DEGs at 18 h were mainly the integral component of membrane (), intrinsic component of membrane (), and membrane part () and involved in the biological process of transmembrane transport (). Aog185 gene knockout had a significant impact on the cell transmembrane transport system, thereby regulating biological processes and pathways such as cellular oxidative phosphorylation, phagosomes, and autophagy, which are highly associated with transmembrane transport.

4. Discussion and Conclusions

In the present study, a putative GPI-anchored protein-encoding gene Aog185 was shown to contribute to mycelial development, hyphal growth, conidiation, 3D trap formation, pathogenicity, and stress responses. These results are consistent with the multiomics analysis results reported previously [4, 7]. However, Liang et al. pointed out that knockout of the cell surface adhesin-coding gene AoMad1 in A. oligopora could promote the formation of adhesive networks [8], which are inconsistent with the results of this study. Therefore, the effects of potential cell surface proteins on NTF growth and trapping were different. The main reasons may be related to the intrinsic mechanisms of regulating cell global metabolism. Liang et al. demonstrated that the cell surface adhesin-coding gene AoMad1 could influence NTF growth and trapping mainly through the regulation of nitrogen metabolism-related gene expression [8]. Our transcriptomics analysis indicated that the DEGs in the Aog185 mutant were mainly involved in transmembrane transport-related metabolic pathways and biological processes, particularly affecting cell membrane components, ion transmembrane transport-related oxidative phosphorylation, and cell reorganization-related cell autophagy and endocytosis.

In this study, Aog185 was identified as a GPI-AP gene, which has been shown to be involved in membrane protein transport, cell adhesion, cell wall formation, and cell surface protection [21, 22] and plays a key role in cell invasion [23]. This explains why knockout of Aog185 inhibited the growth, development, and pathogenicity of A. oligospora. In addition, Aog185 protein contains the actin-depolymerizing factor (ADF)/cofilin domain, and ADF/cofilin is able to promote actin disassembly [24] and remodeling of the actin cytoskeleton [2527]. Deletion of Aog185 may influence the reorganization of actin cytoskeleton, which further results in growth retardation and sporulation capacity reduction, and eventually causes cells to be more sensitive to chemical stressors. In addition, it has been reported that the actin cytoskeleton and actin-associated protein Crn1p are involved in the trap formation of A. oligospora [28]. ADF/cofilin proteins play important roles in controlling the temporal and spatial extent of actin dynamics, which play key roles in mediating host-pathogen interactions [29]. Aog185 has been proposed to be involved in the depolymerization of actin. Deletion of Aog185 may indirectly impede the depolymerization and remodeling of actin and ultimately decrease trap formation and nematicidal activity. Overall, the Aog185 mutation changes the intrinsic components of the membrane and cytoskeleton and further disrupts the homeostasis of intrinsic transmembrane transport.

Phagocytosis is a specialized process by which cells internalize large particles or a target organism. During phagocytosis, the membrane expands by cytoskeletal rearrangement to recognize foreign particles and ultimately forms a membrane-bound vacuole known as the phagosome [30]. Autophagy is an evolutionarily conserved catabolic pathway that employs lysosomes/vacuoles to degrade and recycle aging organelles and proteins in both yeast and mammalian cells. Autophagy is necessary for trap formation in A. oligospora [31, 32]. Both phagocytosis and cell autophagy depend on cell membrane fusion and transmembrane transport, while phagocytosis and autophagy are two important processes for microbial invasion. Chen et al. suggested that autophagy is initiated in A. oligospora during trap formation [33]. The effects of Aog185 knockdown on transmembrane transport homeostasis further disturbed cell phagocytosis and autophagy, resulting in different growth and trapping rates between the Aog185 mutant and WT strains.

The vacuolar H+-ATPase (vATPase) is a multisubunit enzyme that plays key roles in regulating both cell phagocytosis and autophagy processes. In the fusion of phagosome with endosomes or autophagosome with vacuolar, vATPase is the critical rate-limiting enzyme [33, 34]. The enzyme vATPase transports protons across biological membranes by utilizing the energy from ATP hydrolysis [3537]. In addition, ATPase is a critical enzyme involved in the oxidative phosphorylation system and plays a vital role in regulating proton transmembrane transport [38]. In this study, ATPase was significantly upregulated in the Aog185 knockout strain, indicating that Aog185 affects the phagosome/autophagy pathway and ATPase mediates oxidative phosphorylation. The effect of Aog185 on proton transmembrane transport can further dispute cell self-cleaning and energy metabolic homeostasis and regulation of cell growth, trapping, and stress responses.

Our transcriptomics analysis also found that some genes were associated with the mitogen-activated protein kinase (MAPK) pathway [3941] and mitophagy [42, 43], thereby regulating the adaptation of cells to environmental stress and development of morphology [44]. The DEGs also significantly changed in the Aog185 knockout strain, including PBS2 and myotubularin-related phosphatase (MTMR) 6_7_8. PBS2 has been identified earlier in genetic screening for polymyxin B resistance genes, which integrates with MAPK pathways and is involved in stress signal transmission [45]. MTMR is associated with autophagosome formation, and knockdown of MTMR3 increases autophagosome formation [46]. MTM1, MTMR1-4, MTMR6-8, and MTMR14 are phosphatidylinositol 3-phosphatases that dephosphorylate the D-3 positions of PI(3)P and PI(3,5)P2 in vitro [47, 48]. MTMR6_7_8 (Ymr1p) is a yeast myotubularin-related PI(3)P phosphatase that plays an essential role in regulating phosphatidylinositol 3-phosphate [49]. Our study showed that MTMR6_7_8 was significantly downregulated after Aog185 knockout, indicating that Aog185 is involved in the autophagy pathway, phosphatidylinositol signaling system, and inositol phosphate metabolism in A. oligospora.

Data Availability

The data used to support the findings of this study are included within the supplementary information file.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.


This research was funded by the Outstanding Talents Program of Anhui University, China (Y.W.), Open Fund for Discipline Construction, Institute of Physical Science and Information Technology, Anhui University (Y.W.), and Natural Science Foundation of China (Y.W., grant numbers: 31470218 and 31770066).

Supplementary Materials

Figure S1: the principle of construction of gene knockout plasmid of Aog185. Figure S2: screening and verification of ΔAog185 transformants. Figure S3: comparison of conidiation and conidial germination rates between the WT strain and the ΔAog185 mutant. Figure S4: comparison of stress tolerance of A. oligospora to osmotic agents, cell wall-perturbing agents, and oxidative agents. Table S1: list of primers used in this study. Table S2: quality check of total RNA. Table S3: summary of sequencing reads after filtering. Table S4: summary of genome mapping. Table S5: function annotation statistics table. Table S6: table of statistical results of expression differences. (Supplementary Materials)