Fus3 regulates asexual development and trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora

Meihua Xie; Na Bai; Xuewei Yang; Yankun Liu; Ke-Qin Zhang; Jinkui Yang

doi:10.1016/j.isci.2023.107404

. 2023 Aug 7;26(8):107404. doi: 10.1016/j.isci.2023.107404

Fus3 regulates asexual development and trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora

Meihua Xie ^1,², Na Bai ¹, Xuewei Yang ¹, Yankun Liu ¹, Ke-Qin Zhang ¹, Jinkui Yang ^1,^3,^∗

PMCID: PMC10440713 PMID: 37609635

Summary

Mitogen-activated protein kinase (MAPK) Fus3 is an essential regulator of cell differentiation and virulence in fungal pathogens of plants and animals. However, the function and regulatory mechanism of MAPK signaling in nematode-trapping (NT) fungi remain largely unknown. NT fungi can specialize in the formation of “traps”, an important indicator of transition from a saprophytic to a predatory lifestyle. Here, we characterized an orthologous Fus3 in a typical NT fungus Arthrobotrys oligospora using multi-phenotypic analysis and multi-omics approaches. Our results showed that Fus3 plays an important role in asexual growth and development, conidiation, stress response, DNA damage, autophagy, and secondary metabolism. Importantly, Fus3 plays an indispensable role in hyphal fusion, trap morphogenesis, and nematode predation. Moreover, we constructed the regulatory networks of Fus3 by means of transcriptomic and yeast two-hybrid techniques. This study provides insights into the mechanism of MAPK signaling in asexual development and pathogenicity of NT fungi.

Subject areas: Microbial physiology, Microbiology, Molecular biology

Graphical abstract

Highlights

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Fus3 is required for cell fusion, trap morphogenesis, and nematocidal activity
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Fus3 plays a crucial role in asexual growth, stress response, and secondary metabolism
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Fus3 regulates DNA damage, mitochondrial morphology, and autophagy
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Fus3 impairs diverse phenotypic traits by regulating downstream targets

Microbial physiology; Microbiology; Molecular biology

Introduction

The mitogen-activated protein kinase (MAPK) cascade is a key intracellular signal sensor that transmits information using the phosphorylation/dephosphorylation cycle of proteins.¹ There are five MAPK pathways in Saccharomyces cerevisiae, which are involved in the regulation of mating, invasive growth, cell-wall integrity, and ascospore formation.² In the MAPK Fus3/Kss1 cascades pathway, extracellular pheromones bind to cell surface receptors, triggering a series of intracellular signaling responses. This MAPK cascade pathway is mainly involved in regulating the cell-to-cell communication, cell fusion, appressorium development, pathogenicity, and secondary metabolism of pathogenic fungi.²^,³ For example, Fus3 and orthologs are required for the completion of cell fusion in S. cerevisiae and Neurospora crassa,⁴^,⁵ the yeast MAPK Fus3 mediates sexual cell fusion, whereas the Fus3 ortholog (MAK-2) mediates vegetative (asexual) cell fusion in N. crassa. In Botrytis cinerea, Bmp1 (homologous to Fus3) is essential for plant infection, and the mutant produced normal conidia and mycelia, and was nonpathogenic for carnation flowers and tomato leaves.⁶ This pathway is also required for pathogenicity in Claviceps purpurea and Stagonospora nodorum.⁷ In Magnaporthe grisea, Pmk1 (homologous to Fus3) regulates appressorium formation, and is required for penetration of the host epidermis and infection growth.⁸ In Aspergillus nidulans, MpkB (homologous to Fus3) plays a role in mycelial growth, conidia, conidial viability, sexual development, and autolysis.⁹ Fus3-Kssl-like MAPK genes in Pyrenophora teres¹⁰ and Cochliobolus heterostrophus¹¹ affect conidiation. Furthermore, Fus3 regulates the production of secondary metabolites in several species of Aspergillus, such as A. fumigatus,¹² A. flavus,¹³^,¹⁴^,¹⁵ and A. nidulans.¹⁶^,¹⁷ These findings suggest that Fus3 plays an important role in vegetative and sexual cell-cell fusion, fungal growth, development, secondary metabolism, and pathogenicity.

Nematode-trapping (NT) fungi are carnivorous microbes that can produce different types of trapping devices (traps) to prey on nematodes.¹⁸ NT fungi can live a saprophytic life, but when there are inducers such as nematodes in the environment, their vegetative hyphae are specialized to form unique traps, and their lifestyle changes from the saprophytic stage to the parasitic stage.¹⁹^,²⁰ Arthrobotrys oligospora is a model NT fungus for studying the interaction between fungi and nematodes, and can produce adhesive three-dimensional hyphal networks to prey on nematodes.²¹ The mature hyphal networks consist of 5–8 mycelial loops, and the formation of the traps is the result of the branching, bending and fusion of the hyphae, and is also a prerequisite for A. oligospora and other NT fungi to capture the nematodes.²² The formation of traps is a complex process in which multiple pathways such as cell wall biosynthesis, signal transduction pathways, and peroxisomal protein synthesis are involved.²³

In recent years, increasing studies have demonstrated that heterotrimeric G-proteins (G-protein) and related signaling pathways play a crucial role in the mycelia growth, trap formation, and pathogenicity of NT fungi, such as A. oligospora.²⁴^,²⁵^,²⁶^,²⁷^,²⁸ For example, deletion of the G-protein β-subunit-encoding gene nearly abolished trap formation²⁴; and the cAMP-PKA signaling regulated hyphal growth, conidiation, trap morphogenesis, stress tolerance, and autophagy.²⁸ In addition, deletion of hog1 resulted in hypersensitivity to hyperosmolarity, defective conidia formation, and reduced trap formation and predation efficiency.²⁹ Similarly, with disruption of the three components (AoBck1, AoMkk1 and AoSlt2) of the cell-wall integrity MAPK cascade, all of the ΔAobck1, ΔAomkk1, and ΔAoslt2 mutants showed severe defects in vegetative growth, and lost the ability to produce mycelial traps for nematode predation.³⁰^,³¹ Recently, an orthologous Fus3 was identified in A. oligospora (strain TWF154), where deletion of fus3 abolished nematode-induced trap morphogenesis and impaired the growth of hyphae.³² However, the function and related regulatory network of Fus3 remain largely unknown in A. oligospora and other NT fungi.

In this study, we characterized an orthologous Fus3 in A. oligospora (strain ATCC24927) using multi-phenotypic analysis and multi-omics approaches. Surprisingly, we found that the Δfus3 mutant could not produce three-dimensional networks, but rather a specialized spiral hyphal coil, due to the abolishment of hyphal fusion, which is different from previous report³²; meanwhile, the absence of fus3 caused a severe defect in conidiophore development, stress response, DNA damage, mitochondrial morphology, and secondary metabolism. In addition, we probed the regulatory networks and interacting proteins of Fus3 using transcriptomic and yeast two-hybrid (Y2H) techniques. Our results suggest that Fus3 plays an important role in asexual development, stress response, cell fusion, pathogenicity, mitochondrial development, DNA damage, and secondary metabolism.

Results

Sequence analysis of Fus3

The orthologous fus3 gene was retrieved from the fungus A. oligospora, which encodes a 350-amino acid polypeptide with a predicted molecular mass of 40.66 kDa and an isoelectric point of 6.90. Phylogenetic analysis indicated that Fus3 orthologs from several NT fungi (A. oligospora, Drechslerella stenobrocha, Dactylellina haptotyla, and Arthrobotrys flagrans) were clustered in a single clade (Figure S1A). Sixteen Fus3 orthologs from different fungi all contained active sites of Ser/Thr protein kinase (IPR008271) and protein kinase domain (IPR000719) (Figure S1B). Furthermore, Fus3 showed a high similarity (more than 89.7%) to other fungal orthologs, apart from S. cerevisiae, while it shared a similarity of 60.3% with S. cerevisiae (Figure S1C).

Fus3 regulates mycelial growth, cell nucleus number, and endocytosis

The gene fus3 was disrupted by a homologous recombination strategy, and transformants were verified by PCR with primers yzfus3-5f and yzfus3-3r and Southern blotting with the restriction enzyme StuI (Table S1; Figures 2A–2C). The gene fus3 was disrupted by a homologous recombination strategy, and transformants were verified by PCR with primers yzfus3-5f and yzfus3-3r (Table S1; Figures S2A and S2B). Subsequently, the genomic DNA of the wild-type (WT) and corresponding transformants was digested with the restriction enzyme StuI and used in Southern blotting analysis, and a single band of the expected size appeared in the WT and Δfus3 mutant strains (Figure S2C). Finally, we obtained two positive transformants (Δfus3-71 and Δfus3-72).

Comparison of conidial production and conidiophore morphology between the WT strain and Δ*fus3* mutants

(A) Conidiophores of WT strains and Δ*fus3* mutants were observed by light microscopy. Bar: 100 μm.

(B) Conidial yields of the WT strain and Δ*fus3* mutants.

(C) Morphology of conidiophores and spores. Bar: 10 μm.

(D) Comparison of morphologically normal and abnormal spores in the WT strain and Δ*fus3* mutants.

(E) Conidial morphology and nuclei were visualized by CFW and DAPI staining. Bar: 5 μm.

(F) The number of nuclei in spores.

(G) Relative transcription levels (RTLs) of sporulation-related genes in Δ*fus3* in the mutant and WT strain at different time points.

(H) Heatmap of gene expression involved in the mitotic cell cycle process and sporulation in WT and Δ*fus3* mutant strains.

Error bars in (B, D, F, and G): Data are represented as mean ± SD. The asterisk in (B, D, F, and G) indicates a significant difference between mutants and the WT strain (Tukey’s HSD, p < 0.05).

Compared with WT, the mycelial growth rate of the Δfus3 mutant was remarkably decreased on the PDA, tryptone-glucose agar (TG), and tryptone yeast-extract glucose agar (TYGA) media, and Δfus3 mutant exhibited a reduction in the number of aerial mycelia (Figures 1A and 1B). The hyphae of the Δfus3 mutant contained more hyphal septa than WT hyphae, and some hyphae also swelled and deformed (Figures 1C and 1D). The hyphal cells of the Δfus3 mutant had a reduced number of nuclei compared to WT (Figures 1E and 1F). To probe whether Fus3 plays a role in endocytosis, we stained the fungal hyphae with the lipophilic styryl dye FM4-64, after 10 min of staining with the lipophilic styryl dye FM4-64, the WT hyphal cells were filled with the dye solution, whereas only the membrane portion of the hyphal cells in the Δfus3 mutant was stained (Figure 1G). Further observation by transmission electron microscopy (TEM) also found that the WT contained more endocytic structures than the Δfus3 mutant (Figure 1H).

Comparison of mycelial growth, morphology, septa, cell nuclei, and endocytic structures in WT and Δ*fus3* mutants

(A) The colony morphology after growth on TYGA plate at 28°C for 5 days.

(B) Colony diameters of WT and Δ*fus3* mutant strains after growth on TG, PDA, and TYGA plate for 7 days.

(C) Comparison of mycelial cell length of the WT and Δ*fus3* mutant strains. Error bars: SD from 61 replicates.

(D) Mycelial morphology after culturing on CMY plate for 7 days and staining with 20 μg/mL calcofluor white (CFW). Arrows point to hyphal septa. Scale bar: 10 μm.

(E and F) Nuclei in WT and Δ*fus3* mutant hyphae were visualized after staining with CFW and DAPI. White and pink arrows indicate septa and nuclei, respectively. Scale bar: 10 μm.

(G) Mycelia of WT and Δ*fus3* mutant strains were stained with FM4-64 at 10 min. Scale bar: 10 μm.

(H) The endocytic structures in WT and Δ*fus3* mutants were observed using transmission electron microscopy.

Arrows point to hyphal endocytic structures. Scale bar: 0.5 μm. Error bars in (B, C, and F): Data are represented as mean ± SD. The asterisk in (B, C, and F) indicates a significant difference between mutants and the WT strain (Tukey’s HSD, p < 0.05).

Fus3 regulates conidiophore morphology and conidia yield

Deletion of fus3 resulted in a significant reduction in the number of conidiophores compared to the WT (Figure 2A), and a corresponding reduction in conidiation, which was reduced by approximately 99.5% (Figure 2B). In addition, the apex of the WT conidiophores had multiple conidia (1–16), while the apex of the Δfus3 mutant conidiophores only had one or two conidia (Figure 2C). This reduction in conidiation of the mutant is likely a combination of less conidiophores and less conidia per conidiophore. At the same time, 90.8% ± 2.2 of the conidia in the Δfus3 mutant changed remarkably in morphology (Figure 2D). In addition, the number of nuclei in the Δfus3 mutant (9–17 nuclei in each conidium) conidia was reduced compared with the WT strain (10–26 nuclei per conidium) (Figures 2E and 2F). The transcription of eight putative sporulation-related genes (abaA, aspB, flbC, fluG, nsdD, rodA, velB, and vosA)³³ between the WT and Δfus3 mutant strains was determined using real-time quantitative PCR (RT-qPCR) at different growth stages. Compared with WT, both rodA and abaA were downregulated at three time points, and abaA was the most remarkably downregulated (Figure 2G).

To probe the influence of Fus3 on mitotic process and conidiation, we compared the transcriptional levels of genes involved in mitotic cell cycle process and conidiation between the Δfus3 mutant and WT strain by RNA-sequencing. Analysis of clustering heatmaps showed that 16 genes involved in mitotic cell cycle process were upregulated and four were downregulated, such as DNA-repair protein Rad2 (AOL_s00007g552), serine/threonine protein kinases Ar1 (AOL_s00043g508) and Chk1 (AOL_s00080g44) were upregulated, while actin-related protein 2/3 complex subunit 1A (AOL_s00076g149) was downregulated. There were 15 putative sporulation-related genes upregulated and five downregulated, including upregulation of flbA, fluG, flbB, and flbC, and downregulation of flbD, abaA, stuA, vosA, and veA (Figure 2H; Tables S2 and S3).

Fus3 regulates multiple stress responses

The mycelial growth of the WT and Δfus3 mutant strains was inhibited on TG medium supplemented with osmotic agents (NaCl and sorbitol), oxidants (H₂O₂ and menadione), and cell wall-disturbing agents (Congo red and SDS) (Figure 3A). The influence of these stressors on the WT and Δfus3 mutant strains was determined by relative growth inhibition (RGI).³⁴ After exposure to 0.25 M sorbitol, the RGI value of the Δfus3 mutant increased by 25.4%, compared to that of the WT strain. Similarly, the RGI value, after exposure to 0.05 mM menadione and 5 mM H₂O₂, increased by 27.7 and 48.2%, respectively. The RGI value, after exposure to 0.01% SDS and 60 μg/mL Congo red, increased by 14.6 and 16.0%, respectively (Figure 3B). The cell wall synthesis-related gene gls was remarkably downregulated in the Δfus3 mutant on days 5 and 7 compared to WT, whereas hex, gfpa, trs, and glu were significantly upregulated on day 7 (Figure 3C). In addition, the transcriptional levels of seven genes associated with oxidative stress response were compared between the WT and Δfus3 mutant strains by RT-qPCR, and the glutathione S-transferase (glt) gene was downregulated in Δfus3 mutant at three time points compared with WT strains, while cat1 and glr were significantly upregulated on day 7 (Figure 3D).

Chemical stress tolerance of WT and Δ*fus3* mutants

(A) Colonial morphology of fungal strains treated with chemical stressors.

(B) Relative growth inhibition (RGI) of fungal colonies grown on TG plates supplemented with different chemicals.

(C) Relative transcription levels (RTLs) of genes involved in cell wall biosynthesis in Δ*fus3* mutants and WT strain at different time points.

(D) RTLs of genes involved in oxidative stress response in Δ*fus3* mutants and WT strain at different time points.

Error bars in (B–D): Data are represented as mean ± SD. The asterisk in (B–D) indicates a significant difference between mutants and the WT strain (Tukey’s HSD, p < 0.05).

Fus3 regulates hyphal fusion, trap morphogenesis, and pathogenicity

Hyphal fusion can be observed in the WT strain during spore germination and vegetative growth, whereas hyphal fusion is abolished in the Δfus3 mutant, and the hyphae are coiled (Figures 4A and 4B). Woronin bodies (WBs) are a specialized class of peroxisome-derived organelles that quickly plug the septal pore upon injury of the hypha, avoiding the excessive loss of cytoplasm.³⁵ Further TEM observation of the hyphae showed that the septal pores of the WT hyphae were sealed with WBs, whereas about 50% septal pores of the Δfus3 mutant were not sealed with WBs (Figure 4C). In addition, the traps produced by the WT and Δfus3 mutant strains were observed by means of scanning electron microscopy (SEM) after induction with nematodes for 48 h, and the WT strain produced many traps containing multiple mycelial loops through hyphal fusion, and most of the added nematodes were captured and digested. However, the Δfus3 mutant was not able to produce typical traps, instead producing a small number of hyphal coils due to the abolishment of hyphal fusion, and only few nematodes were captured. In addition, TEM observation showed that the WT trap cells contained many electron-dense (ED) bodies, whereas the number of ED bodies in the Δfus3 hyphal cells was significantly reduced (Figure 4D). Further comparison of the traps produced by the WT and the hyphal coil produced by the Δfus3 mutant showed that the WT produced 13.5 and 38.5 traps per cm² at 24 and 36 h post induction (hpi), respectively, whereas the Δfus3 mutants only produced 0.1 and 0.2 hyphal coils per cm² at 24 and 36 hpi, respectively (Figure 4E). Correspondingly, the nematode predation rate of the WT reached 21.7%, 45.9%, 81.1% and 93.1% at 12, 24, 36 and 48 hpi, respectively, whereas the nematode predation ability of the Δfus3 mutant was almost abolished at 12, 24 and 36 hpi, and only about 3.9% of the nematodes were captured at 48 hpi (Figure 4F).

Comparison of hyphal fusion, trap formation, and nematocidal activity of WT and Δ*fus3* mutants

(A) Spore germination after 48 h post incubation and stained with calcofluor white and then observed with a microscope. Pink arrows indicate hyphal fusion. Bar: 20 μm.

(B) Mycelial morphology of WT and Δ*fus3* mutants cultured on PDA medium at 28°C for 7 days. Pink arrows indicate hyphal fusion, white arrows indicate hyphal physical contact or hyphal branching of the Δ*fus3* mutants. Bar: 20 μm.

(C) Woronin bodies in the hyphal cells of WT and Δ*fus3* mutant strains were observed by TEM. White, pink, and blue arrows point toward the hyphal septum, Woronin body, and connected cytoplasm, respectively. Bar: 1 μm.

(D) Comparison of trap morphology after being induced with nematodes for 48 h by SEM and TEM. Pink and blue arrows point toward the traps and hyphal coils, respectively.

(E) Trap cells of WT and Δ*fus3* mutants were observed by TEM. Pink arrows point toward ED bodies. Bar: 0.5 μm.

(F) Traps produced at 24 and 48 hpi.

(G) Percentages of nematodes captured by WT and Δ*fus3* mutant strains at different time points.

Error bars in (F and G): Data are represented as mean ± SD. The asterisk in (F and G) indicates a significant difference between mutants and the WT strain (Tukey’s HSD, p < 0.05).

Fus3 regulates DNA damage repair and mitophagy

The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay revealed increased DNA fragmentation in the hyphae and spores of the Δfus3 mutant compared with that in those of the WT strain (Figures 5A–5C). TEM observation revealed that mitochondria were deformed in the Δfus3 mutant (Figure 5D), and the expression levels of mitochondrial distribution and morphology proteins (MD342 and MDM10)³⁶ were upregulated compared with WT (Figure 5E). Under starvation conditions, TEM observation showed that some autophagosomes were distributed in the hyphal vacuoles of WT, whereas the vacuoles of the Δfus3 mutant were larger than those of WT, and there was mitophagy in the vacuoles (Figure 5F). In addition, an analysis of clustering heatmaps revealed that the transcription of genes involved in autophagy and mitophagy was remarkably altered between the WT and the Δfus3 mutant strains, with 24 genes being upregulated and 14 genes being downregulated in autophagy, whereby vacuolar protein sorting proteins VpsB (AOL_s00215g592) and DigA (AOL_s00215g688) and cAMP-dependent protein kinase (AOL_s00054g80) were upregulated, while inositol hexaphosphate kinase Kcs1 (AOL_s00215g763), serine proteases (AOL_s00170g103), and serine proteinase PepC (AOL_s00004g122) were downregulated. In mitophagy, 31 genes were upregulated, and 12 genes were downregulated, with mitochondrial inheritance component Mdm12 (AOL_s00193g178), DNA polymerase gamma (AOL_s00215g177), and mitochondrial outer membrane protein Mdm10 (AOL_s00076g313) being upregulated, while small nuclear ribonucleoprotein D3 (AOL_s00215g169), protein phosphatase 2C (AOL_s00097g512), and autophagy-related protein 11 (AOL_s00004g301) were downregulated (Figure 5G; Tables S4 and S5).

Comparison of DNA damage, mitochondrial morphology, and autophagy in WT and Δ*fus3* strains at the vegetative growth stage

(A) Hyphae of WT and Δ*fus3* strains grown on CMY medium at 28°C for 10 days were stained with DAPI and TUNEL. Bar: 20 μm.

(B) Conidia of WT and Δ*fus3* strains stained with DAPI and TUNEL and examined by fluorescence microscopy. Bar: 20 μm.

(C) Comparison of mycelial cells and conidial apoptosis in WT and Δ*fus3* strains (the data are presented as the mean ± SD from three independent experiments, ∗p < 0.05).

(D) Mitochondrial morphology in WT and Δ*fus3* mutant hyphal cells was observed by TEM. Pink arrows point toward mitochondria. Bar: 0.5 μm.

(E) Comparison of transcription of genes involved in mitochondrial membrane in WT and Δ*fus3* strains. Gene *md342* encodes mitochondrial distribution and morphology protein 342 and *mdm10* encodes mitochondrial distribution and morphology protein 10. CK indicates the standard (which has an RTL of 1) for the statistical analysis of the RTL of each gene in a deletion mutant compared to that in the WT strain (Tukey’s HSD, ∗p < 0.05).

(F) TEM observations of autophagy in WT and Δ*fus3* strains. Pink arrows point toward mitochondria. Bar: 0.5 μm.

(G) Heatmap of gene expression involved in autophagy and mitophagy in WT and Δ*fus3* strains.

Transcriptomic insights into the regulatory role of Fus3

Transcriptional profiles of WT and Δfus3 mutant strains were compared on the basis of RNA sequencing. Quality control alignment of sequencing data and statistical analysis of sequence alignment showed that there was no contamination in related experiments, and the genes in each group were effectively expressed (Table S6). Principal component analysis showed that deletion of fus3 resulted in different gene expression patterns between the WT and the Δfus3 mutant strain (Figure S3). During vegetative growth, the Δfus3 mutant had 2,200 and 1,564 differentially expressed genes (DEGs) compared to the WT strain, respectively (Figure 6A). Gene Ontology (GO) enrichment of the above DEGs found that the upregulated DEGs were mainly involved in biological processes such as RNA processing, proteasomal protein catabolic process, actin polymerization or depolymerization, DNA-directed 5′-3′ RNA polymerase activity, positive regulation of RNA metabolic process, maturation of SSU-rRNA, glucan biosynthetic process, integral component of nuclear inner membrane, and integral component of organelle membrane. The downregulated DEGs were mainly involved in processes such as glucose metabolic processes, carboxylic acid catabolic processes, metal ion transport, translation, nucleoside metabolic processes, cytoplasmic translation, negative regulation of apoptotic processes, ribonucleotide metabolic processes, and propionate metabolic processes (Figure 6B; Table S7). Further Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that compared to WT, the Δfus3 mutant exhibited alterations in protein synthesis and processing (ribosome, RNA transport, and protein export), energy (glycolysis/gluconeogenesis, citrate cycle, and glyoxylate and dicarboxylate metabolism), autophagy processes (autophagy, mitophagy, and phagosome), oxidative phosphorylation, MAPK signaling, cell cycle, endocytosis, peroxisome, and proteasome (Figure S6C).

Comparison of transcriptional profiles in WT and Δ*fus3* mutant strains at the vegetative growth stage

(A) Numbers of differentially expressed genes (DEGs) in the Δ*fus3* mutant versus WT strain.

(B) Gene ontology (GO) enrichment of up- and downregulated DEGs, shown using Cytoscape Cluego. Red shapes indicate upregulation, and blue shapes indicate downregulation.

(C) KEGG pathway enrichment of the DEGs (p < 0.05) at the vegetative growth stage.

During the trap formation and nematode predation stages, the transcription level of the Δfus3 mutant at each time point changed significantly compared with WT, that is, the Δfus3 mutant had 1984, 1974, and 1917 upregulated and 2082, 1733 and 1271 downregulated genes at 12, 24, and 36 hpi, respectively (Figure 7A). In addition, the Δfus3 mutant had 41.8% (1698/4066), 26.2% (972/3707), and 23.5% (749/3188) DEGs at 12, 24, and 36 hpi in response to nematode challenge compared to WT, respectively. Among these DEGs, those related to the regulation of transcription DNA templates, intracellular protein transport, regulation of calcium ion transport, protein modification by small protein conjugation or removal, and phosphatidylinositol dephosphorylation were significantly enriched at 12 hpi; those involved in the purine nucleoside triphosphate metabolic process, mitotic DNA integrity checkpoint signaling, DNA metabolic processes, peptide biosynthetic processes, nucleotide–sugar biosynthetic processes, and mitochondrial large ribosomal subunits and DNA strand elongation involved in DNA replication were significantly enriched at 24 hpi; and those participating in tetrahydrofolate metabolic processes, methionine metabolic processes, ammonium transmembrane transporter activity, alditol metabolic process, glucan biosynthetic process, and cellular glucan metabolic processes were enriched at 36 hpi. Furthermore, 974 co-expressed genes at three time points were involved in fatty acid biosynthetic processes, deoxyribonuclease activity, telomere maintenance, sterol metabolic processes, negative regulation of actin filament polymerization, DNA ligase (ATP) activity, carboxypeptidase activity, nitrate assimilation and gamma-aminobutyric acid metabolic process (Figure 7B). Further KEGG enrichment analysis showed that RNA transport, proteasome, peroxisome, glyoxylate, and dicarboxylate metabolism, and SNARE interactions, as well as vesicular transport pathways, were significantly enriched at 12 hpi; ribosome, oxidative phosphorylation, mismatch repair, and thiamine metabolism pathways were significantly enriched at 24 hpi; and starch and sucrose metabolism and methane metabolism pathways were significantly enriched at 36 hpi (Figure 7C).

Comparison of transcriptional profiles of WT and Δ*fus3* strains during trap formation

(A) DEGs in the Δ*fus3* mutant compared with those in WT at 12, 24, and 36 hpi. URD, upregulated DEGs; DRD, downregulated DEGs.

(B) Gene ontology (GO) enrichment of the Δ*fus3* mutant compared with that in WT at 12, 24, and 36 hpi, shown using Cytoscape Cluego.

(C) KEGG pathway enrichment of the DEGs (p < 0.05) at12, 24, and 36 hpi.

Fus3 is involved in the regulation of secondary metabolism

The compounds in the fermentation broth of the WT and Δfus3 mutant strains were compared by liquid chromatography-mass spectrometry (LC-MS). Volcano plot analysis showed that the metabolites of the Δfus3 mutant had upregulated 2,344 compounds and downregulated 2,912 compounds compared with WT (Figure 8A). The top 20 upregulated compounds include ethyl myristate, hexadecasphinganine, 4-indolecarbaldehyde, and 2-hydroxycaproic acid (Table S8), and the top 20 downregulated compounds include delcosine, norethisterone acetate, and retosiban (Table S9). The compound peaks of the Δfus3 mutant were significantly different from the WT at multiple retention time points in the negative metabolic profile (Figure 8B). In addition, the arthrobotrisins (diagnostic fragments ion at m/z 139, 393, and 429 under negative ion conditions) peak area was reduced in the Δfus3 mutant compared to the WT (Figures 8C and 8D). Correspondingly, the transcript levels of related genes in the gene cluster AOL_s00215g associated with biosynthesis of arthrobotrisins in the Δfus3 mutant were also altered from those in WT, and the transcription of these genes in the Δfus3 mutant was downregulated at the vegetative growth stage (0 h) (Figure 8E). KEGG analysis of the differentially expressed compounds revealed that they were enriched in pathways such as metabolic pathways and biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and the biosynthesis of ubiquinone and other terpenoid–quinone (Figure S4).

LC-MS analysis of WT and Δ*fus3* strains

(A) Volcanic map of metabolites differentially produced by WT and Δ*fus3* strains.

(B) HPLC profiling of WT and Δ*fus3* strains.

(C) Negative ion metabolic profiles of WT and Δ*fus3* strains; the red box indicates the peak area of arthrobotrisins.

(D) Analysis of peak area of arthrobotrisins. (the data are presented as the mean ± SD from three independent experiments, ∗p < 0.05).

(E) Transcription heatmap of gene cluster *AOL_s00215g* associated with arthrobotrisin biosynthesis.

Prediction and analysis of proteins interacting with Fus3

A total of 278 proteins (Fus3 and its putative targets) were analyzed using STRING³⁷ and then visualized using Cytoscape³⁸ (Table S10). Fus3 can genetic interaction with these proteins, which are involved in the MAPK signaling pathway, cell cycle, meiosis, basal transcription factors, nucleotide excision repair, DNA replication, ubiquitin mediated proteolysis, mismatch repair, homologous recombination, non-homologous end-joining, and base excision repair (Figure 9A). These genes that are involved in cell communication were then further analyzed using ClueGo in Cytoscape, and it was found that these genes were mainly involved in the response to extracellular stimuli, the regulation of signal transduction, membrane docking, the regulation of cellular processes, mitochondrial transmembrane transport, regulation of calcium ion transport, signal peptide processing, membrane fusion, and cell morphogenesis (Figure 9B). In addition, several predicted interacting proteins were further verified by Y2H, showing that Fus3 interacts with transcription factor Ste12 (AOL_s00097g514), developmental regulator FlbA (AOL_s00215g516), and protein tyrosine phosphatases Pyp1 (AOL_s00043g69) and Ptp (AOL_s00169g50) (Figure 9C).

Analyses of regulatory network and target genes of Fus3

(A) Interconnected gene regulatory networks of Fus3.

(B) GO enrichment associated with cellular communication, shown using Cytoscape Cluego.

(C) Analysis of protein interactions using yeast two-hybrid screening with β-D-galactosidase.

Discussion

In yeasts and several filamentous fungi, Fus3/Kss1 is an indispensable component of the MAPK signaling cascade, which has been identified to regulate sexual and asexual development, cell fusion, host infection/virulence, and/or stress response, and production of secondary metabolites.²^,³^,¹³ In this study, we identified an orthologous Fus3 in A. oligospora, the sixteen selected Fus3 homologous proteins share high sequence identity, indicating that Fus3 is evolutionarily conserved in fungi. Deletion of fus3 was found to affect mycelial growth, conidiation, and trap formation, which is similar to previous reports³²; Furtherly, we also found that the Fus3 loss impaired the number of cell nuclei, endocytosis, stress response, hyphal fusion, DNA damage, mitochondrial morphology, autophagy, and secondary metabolism.

The disruption of fus3 caused severe defects with respect to hyphal development, including slowed radial growth and a decrease in the number of aerial hyphae, which was similar to what was observed in the Δfus3 mutant of the Trichoderma brevicrassum.³⁹ However, the Δfus3 mutant of A. flavus,¹³ A. oryzae,⁴⁰ and B. cinerea⁶ only displayed slower growth and exhibited no changes in number of aerial hyphae. Meanwhile, our results differed from those obtained for the Δfus3 mutants (Δpmk1) in M. grisea⁸ and Metarhizium robertsii,⁴¹ in which Fus3 is nonessential for vegetative growth. In addition, in this study, the hyphal septum of the Δfus3 mutant increased compared with WT, and some hyphae were inflated. Furthermore, transcriptome analysis revealed that the expression of genes involved in glycolysis/gluconeogenesis, citrate cycle, and glyoxylate and dicarboxylate metabolism was altered in the Δfus3 mutant. These findings indicate that Fus3 is critical for vegetative growth; however, its functional activity may vary among fungal species.

The main way for filamentous fungi to reproduce is to produce conidia, and the ability to produce conidia is one of the key factors determining the fecundity and fitness of filamentous fungi.⁴² The conidiophore of A. oligospora consists of one to six nodules, and conidia are densely attached to denticles with nodular protrusions.⁴³ In this study, deletion of fus3 resulted in only 1–2 spores being attached on the conidiophore, and spore yield was significantly reduced, with similar results having been reported in other fungal species. For example, the conidial yield of the Δfus3 mutant in A. flavus was significantly reduced; in addition, the conidia head and conidiophore were maldeveloped and sterile, the sclerotia yield of the Δfus3 was severely decreased compared with the WT.¹³ The Δfus3 of A. oryzae exhibited severe conidiation defects, and did not form any sclerotia.⁴⁰ The conidial yield of the Δfus3 mutant in M. robertsii was significantly reduced.⁴¹ However, our results differ from those obtained for the Δfus3 mutant (Δtbmk) in the T. brevicrassum, which did not change conidiation.³⁹ These findings indicate that Fus3 play a critical role in sporulation and morphological development of conidiophores in A. oligospora and most filamentous fungi.

Fungi can respond rapidly to external environmental stimuli or stress, and regulate a series of cellular activities of cells to adapt to environmental changes.⁴⁴ In this study, under hypertonic stress, the growth of the Δfus3 mutant was inhibited; these changes are similar to those of the Δfus3 mutant in M. robertsii,⁴¹ whereas the Δtbmk mutant of the T. breve showed no significant response to high osmolarity conditions caused by 0.5 M NaCl.³⁹ Under oxidative stress, the growth of the Δfus3 mutant was remarkably inhibited, with these changes being similar to those in the Δfus3 mutant (Δtbmk) in the T. breve under oxidative stress.³⁹ Meanwhile, no significant differences in growth rate with respect to stress of 0.01% H₂O₂ were observed between the Δfus3 mutant and M. robertsii WT strain.⁴¹ At the same time, the expression levels of genes glt, cat1, and glr were significantly changed in Δfus3 mutant compared with WT. In addition, the growth of the Δfus3 mutant was inhibited under cell wall-perturbing stress, and these changes were similar to the increased sensitivity of the Δfus3 mutant to Congo red in T. breve.³⁹ In M. robertsii, the Δfus3 mutant was significantly inhibited on PDA plates containing Congo red.⁴¹ Correspondingly, the transcripts of several genes related to cell wall biosynthesis were significantly altered in the Δfus3 mutant. Overall, these results indicate that Fus3 is important for environmental adaptation in A. oligospora and most of fungi.

Communication and cell fusion occurring between hyphae in colonies are important for strengthening the colony structure and supporting the development of complex structures such as aerial hyphae and sexual reproduction structures.³ In this study, the deletion of fus3 resulted in defects in hyphal cell fusion consistent with those reported in previous studies on the involvement of Fus3 in cell fusion in fungi. The Fus3 orthologs were shown to be essential for cell fusion in S. cerevisiae,⁴ N. crassa,⁵ A. nidulans,⁴⁵ and A. oryzae.⁴⁰ In N. crassa, the Fus3 ortholog (MAK-2) is essential for germling and hyphal fusion, which normally takes place during germination and colony development.⁵ Importantly, hyphal cell fusion plays a specific role in trap formation in NT fungi, as in the NT fungus Duddingtonia flagrans, deletion of sofT (hyphal anastomosis gene) resulted in defects in the ring closure of traps due to the anastomoses present in normal vegetative hyphae being completely inhibited.⁴⁶ Similarly, deletion of fus3 resulted in defects in trap cell fusion, such that the Δfus3 mutant was not able to produce typical three-dimensional networks to capture nematodes, and could only form spiral hyphal coils. In addition, WBs were unable to seal the damaged septal pores, and the number of ED bodies in the trap cells of the Δfus3 mutant was reduced. ED bodies are unique structures in the trap cells of NT fungi, and are organelles associated with peroxisomes that provide energy for invading hyphae.⁴⁷^,⁴⁸ WBs are peroxisome-derived dense-core vesicles unique to several filamentous ascomycetes, and have been shown to be involved in regulating trap formation in A. oligospora.⁴⁹ Recently, two peroxisome biogenesis genes, Aopex1 and Aopex6, were identified in A. oligospora, absence of Aopex1 and Aopex6 resulted in a failure to produce traps and conidia.⁵⁰ In addition, transcriptome analysis found that the absence of fus3 resulted in changes in processes associated with cell communication, membrane fusion, and regulation of calcium ion transport. Moreover, the nematode predatory efficiency was significantly reduced due to the inability of the Δfus3 mutant to produce typical traps. Similarly, the Δku70 fus3 mutant of A. oligospora (strain TWF154) completely lost the ability to produce traps,³² and the Δpmk1 mutant (an fus3 homolog) of the rice blast fungus M. grisea failed to complete the formation of mature appressoria and failed to grow invasively in rice plants.⁸ In addition, these effects are consistent with those caused by fus3 mutations in M. robertsii,⁴¹ C. heterostrophus,¹¹ Botrytis cinerea.⁶ These results suggest that Fus3 plays an important role in cell communication and hyphal fusion in A. oligospora and other fungi, and Fus3 plays a conserved and indispensable role in fungal pathogenesis.

In recent years, comparative transcriptome analysis has been broadly used to reveal the gene expression changes between fungi and their hosts.¹³^,⁵¹^,⁵²^,⁵³^,⁵⁴ In this study, the absence of fus3 resulted in several genes that are involved in regulation of multiple cellular processes being altered at the transcriptional level, suggesting that Fus3 is a multifunctional regulator involved in key cellular processes during vegetative growth and trap formation. In transcriptome analysis, mitotic cell cycle process, autophagy, and mitophagy were found to be associated with hyphal growth, sporulation, and the DNA repair process. DNA repair is an important biological process that maintains genome stability and protects DNA from damage and mutation, including mismatch, base excision, and nucleotide excision repair.⁵⁵ In our results, the partial hyphal cells of the Δfus3 mutant were enlarged and deformed, and the hyphae and spores of the Δfus3 mutant exhibited considerable DNA damage. Furthermore, mitophagy is an autophagic process that selectively sequesters and degrades damaged or incomplete mitochondria, and is involved in maintaining the integrity of mitochondrial network function and cellular homeostasis.⁵⁶ Consistently, in this study, the transcriptome revealed that an integral component of the organelle membrane changed after fus3 disruption, phenotypic analysis revealed that mitochondrial membranes of the Δfus3 mutant exhibited considerable damage, and we also observed mitophagy in Δfus3 mutant cells. In addition, transcriptome analysis also revealed changes in cell fusion, consistent with the failure of the Δfus3 mutant hyphal cells to fuse in our phenotypic experiments. Similarly, transcriptome analysis showed that endocytosis and the exocytic process were significantly enriched. Correspondingly, FM4-64 staining and TEM observation showed that the Δfus3 mutant exhibited an altered endocytosis process and endocytosis structures compared with WT.

A regulatory network of Fus3 was constructed on the basis of phenotypic and transcriptome analysis, and further Y2H validation found that AoSte12, AoFlbA, AoPyp1, and AoPtp are potential targets of Fus3. Among them, transcription factor Ste12 has been reported to be the downstream target of Fus3 in several fungi, such as S. cerevisiae, N. crassa,⁵⁷ M. grisea,⁵⁸ and A. flavus.¹³ FlbA encodes a G protein signaling regulator (RGS) domain protein, which negatively regulates vegetative growth.⁵⁹ In our recent work, deletion of AoflbA resulted in a severe defect in the conidiation and trap formation of A. oligospora.⁶⁰ In addition, Ptp and Pyp1 are involved in cell signal transduction, regulating cell growth, differentiation, metabolism, gene transcription and other processes.⁶¹ In fission yeast, Pyp1 act as negative regulators of mitosis, controlling cell size at division and negatively regulating nutrient monitoring pathways.⁶² Therefore, Fus3 can interact with diverse transcription factors and regulators, thus regulating multiple cellular and biological processes in A. oligospora.

It has been shown that Fus3 orthologs are involved in the secondary metabolism of several filamentous fungi, such as A. fumigatus,¹² A. flavus,¹³^,¹⁴ and T. brevicrassum.³⁹ In this study, the metabolites of the Δfus3 mutant were significantly altered compared with WT. In addition, the content of arthrobotrisins in the Δfus3 mutant, a special group of metabolites identified in A. oligospora and other NT fungi, was found to be reduced by means of metabolome analysis, and the expressional levels of gene cluster AOL_s00215g associated with biosynthesis of arthrobotrisins were also downregulated during vegetative growth. Arthrobotrisins produced by A. oligospora and other NT fungi are involved in the regulation of hyphal growth and trap formation.⁶³^,⁶⁴ These results suggest that Fus3 plays an important regulatory role in secondary metabolism in diverse fungi.

In summary, our results show that Fus3 is an essential component of the MAPK signaling cascade, is indispensable for hyphal fusion, trap morphogenesis, and pathogenicity, and plays an important role in asexual growth and development, conidiation, stress response, DNA damage, autophagy, and secondary metabolism. In addition, the regulatory network analysis of Fus3 suggested that it can impair diverse phenotypic traits by regulating downstream target genes that are involved in protein synthesis and processing, autophagy, oxidative phosphorylation, cell cycle, endocytosis, peroxisome, and proteasome. In summary, our study revealed the conserved roles of Fus3 in different fungi, such as mycelial growth, sexual development, and autolysis, importantly, several unique effects of Fus3 were found in this study including the hyphal fusion, trap morphogenesis, and nematode predation. Our study provides deep insights into the mechanism of MAPK signaling in trap formation and lifestyle transition in NT fungi, and lays a foundation for uncovering the regulatory mechanism of NT fungal cell fusion, asexual reproduction, and pathogenicity.

Limitations of the study

In this study, we constructed the regulatory networks of Fus3 based on the transcriptomic data and STRING analysis. Our results showed that Fus3 can interact with numerous proteins, which are involved in the MAPK signaling pathway and multiple cellular processes, whereas we did not confirm what kind of interaction between them? In addition, several predicted interacting proteins were verified by Y2H, showing that Fus3 interacts with several targets including Ste12, FlbA, Pyp1, and Ptp, however the interaction between Fus3 and targets should be furtherly confirmed by other methods, such as co-immunoprecipitation and pull-down assays.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains

Escherichia coli DH5a	Takara	Cat#9057

Chemicals, peptides, and recombinant proteins

Tryptone	OXOID	Cat#LP0042B
Yeast extract	OXOID	Cat#LP0021B
Molasses	Solarbio	Cat#FA0070
Sorbitol	Solarbio	Cat#S8090
NaCl	Solarbio	Cat#S8210
SDS	Solarbio	Cat#S8010
Congo red	Solarbio	Cat#C8450
Menadione	Sigma	Cat#M5750-25G
Sucrose	Solarbio	Cat#S8271
Calcofluor white (CFW)	Sigma	Cat#910090
4ʹ,6-diamidino-2-phenylindole (DAPI)	Sigma	Cat#D9524
FM4-64	Biotium	Cat#70021

Critical commercial assays

one-step TUNEL apoptosis detection kit	Beyotime	Cat#C1086
PrimeScriptH^RT reagent kit	Takara	Cat#RR037A
LightCycler 480 SYBR Green I Master	Roche	Cat#4887352001

Experimental models: Organisms/strains

Arthrobotrys oligospora	ATCC	ATCC: 24927
Δfus3 mutant strains generated in this study	This paper	N/A
Caenorhabditis elegans	CGMCC	N2

Deposited data

Transcriptomic data	This paper	GSE212172

Oligonucleotides

For all oligonucleotides used for gene manipulation and RT-qPCR	See Table S1	N/A

Software and algorithms

pI/MW tool	Expasy	http://web.expasy.org/compute_pi/
InterProScan		http://www.ebi.ac.uk/Tools/pfa/iprscan/
DNAman	Lynnon Biosoft	https://www.lynnon.com/qa.html
MEGA 5	Tamura et al.⁶⁵	https://www.megasoftware.net/
ImageJ	Schneider et al., 2012	https://imagej.net/Welcome
Prism 5	GraphPad Software	https://www.graphpad.com/
STRING	Hao et al.³⁷	https://cn.string-db.org/
Cytoscape	Franz et al.³⁸	https://cytoscape.org/

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Resource availability

Lead contact

Further information and requests for resources and reagent should be directed to and will be fulfilled by the Lead Contact, Jinkui Yang (jinkui960@ynu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Experimental model and study participant details

Fungal strains

The WT strain A. oligospora (ATCC24927) and two positive transformant (Δfus3-71 and Δfus3-72) strains were routinely incubated on PDA medium at 28°C. Fungal strains were cultured at 28°C on PDA, TG, and TYGA media for mycelial growth analysis as previously described.²⁰^,⁶⁶ The S. cerevisiae FY834 strain (ATCC90845), as a host of recombinant plasmid vector, was cultured with yeast extract–peptone–glucose (YPD) medium.

Bacterial strain

Escherichia coli DH5α strains carrying pRS426 and pCSN44 plasmids (TaKaRa, Japan) were cultured in Luria-Bertani broth agar supplemented with 50 μg/mL kanamycin or 100 μg/mL ampicillin, respectively.

Nematode

The Caenorhabditis elegans used to induce trap formation in the bioassays were incubated on oat medium.⁴²

Method details

Sequence analysis of Fus3

The sequences of Fus3 (GenBank: AOL_s00110g154) were retrieved on the basis of the homologous sequences of the model fungi S. cerevisiae (GenBank: AAA34613) and M. oryzae (GenBank: XP_003712175) using BLAST searches in the NCBI database (https://www.ncbi.nlm.nih.gov/). The theoretical isoelectric point (pI) and molecular mass of Fus3 were analyzed by the pI/MW tool (http://www.expasy.ch/tools/pi_tool.html). Sixteen orthologous Fus3 were retrieved from different fungi in the NCBI database, the structural domains of the homologs of Fus3 were analyzed on the Pfam website (http://pfam.xfam.org/), and the sequence similarity between Fus3 and other orthologs was compared using DNAman software (version 5.2.2). A phylogenetic tree was constructed using MEGA software (version 5) with the neighbor-joining method.⁶⁵

Deletion of fus3

Disruption of the fus3 gene was performed using the homologous recombination method as previously described.⁶⁷ The 5′- and 3′-flanking regions of the fus3 gene (2,064 and 1,927 bp) and the hygromycin resistance cassette (hph) were individually amplified by PCR with paired primers (Table S1). The three DNA fragments were isolated using a PCR product recovery kit (TaKaRa) and then co-transformed into the S. cerevisiae FY834 strain by electroporation⁶⁸^,⁶⁹ with linearized pRS426 (digested with EcoRI and XhoI) to generate the pRS426-fus3-hph recombinant plasmid. Complete disruption sequences were amplified using primers fus3-5f/fus3-3r (Table S1) and transformed into A. oligospora protoplasts as previously described.⁶⁶^,⁶⁹ Then, positive transformants grown on PADS medium were verified by PCR and Southern blotting.⁷⁰ Genomic DNA of the WT and Δfus3 mutant strains were extracted using a plant genomic DNA Kit (TaKaRa) and digested using StuI for Southern blotting analysis.

Analysis of vegetative growth, morphogenesis, and conidiation

To analyze mycelial growth, WT and Δfus3 mutant strains were cultured on PDA, TYGA, and TG agar plates at 28°C for 7 days.²⁰ To determine the spore yield for each strain, they were assayed as previously described after 15 days post inoculation (dpi) in corn meal yeast extract (CMY) medium.⁷¹^,⁷² Freshly harvested hyphae and spores were stained with 20 μg/mL calcofluor white (CFW) (Sigma-Aldrich, USA) to visualize their morphology and hyphal fusion, and 20 μg/mL 4ʹ,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA) to visualize their nuclei. Samples were all observed and photographed with an inverted fluorescence microscope (DS-Ri2, Nikon, Tokyo, Japan).

To observe endocytosis, sterile coverslips were inserted into WT and Δfus3 mutant strains in PDA medium at 28°C for 7 days, and then the coverslips covered with hyphae were stained with the lipophilic dye FM4-64 was diluted 40 times (Biotium, California, USA) for 1 min and 10 min, respectively.⁷³ The ultrastructures of the mycelium, conidia, and traps were observed using TEM and SEM, and the samples were prepared as previously described.³⁰^,⁴² To observe apoptosis, hyphal coverslip inserts were cultured on a CMY plate (9 cm) at 28°C for 7 days; then, the coverslips covered with hyphae stained with one-step terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, 30 μg/mL) assay solution (Beyotime Biotechnology, Haimen, China) at 37°C for 30 min, followed by 20 mg/mL DAPI for 30 min at room temperature.

Stress assays

WT and Δfus3 mutant strains were cultured on TG medium alone (control) or TG medium supplemented with chemical stressors for 7 days at 28°C to measure responses to chemical stressors. The chemical stressors were oxidants (H₂O₂ and menadione), cell wall-perturbing agents (SDS and Congo red), and osmotic agents (NaCl and sorbitol). The diameter of the colony was measured on day 7, and the relative growth inhibition (RGI) values of the fungal colonies under stress were calculated using the equation (Sc − St)/(Sc − d) × 100, where Sc and St denote the areas of the stressed and unstressed (control) colonies, respectively, and d is the constant area of the hyphal mass discs used to initiate colonies.³¹

Trap formation and morphogenesis

Equal amounts of fresh spores of WT and Δfus3 mutants were evenly spread on the water agar WA plate (6 cm) at 28°C for 3 days. Then, approximately 300 C. elegans were added to each plate for trap induction. The numbers of traps and nematodes captured in each plate were then observed and counted under a light microscopy (Olympus, Tokyo, Japan) every 12 h until 48 h. Three replicates were used for each experiment.

Transcriptome sequencing and analysis

WT and Δfus3 strains were grown on cellophane-overlaid PDA plates at 28°C for 5 days. Approximately 600 nematodes were then added to each plate for 0, 12, 24, and 36 h of induction, and three replicates were used for each sample. After collecting the samples, RNA was extracted with the AxyPrep multisource RNA miniprep Kit (Axygen, Jiangsu, China), and then sent to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for transcriptome sequencing. The final cDNA library was sequenced on an Illumina Novaseq 4000 platform. The clean reads were mapped to the A. oligospora (ATCC 24927) genome sequence. The gene expression changes were evaluated and the DEGs were identified with an FDR value of ≤0.05. High-throughput sequencing data were analyzed using the OmicShare online platform (www.majorbio.com).

LC-MS assay

WT and Δfus3 strains were inoculated into PD broth for 7 days at 28°C; then, mycelium and fermentation broth were separated by filtration using a vacuum filter pump. The isolated mycelium was further dried and weighed, and corresponding fermentation broth was prepared according to the same weight of the mycelium. The fermentation broth was added to an equal volume of ethyl acetate and left to stand for extraction for 12 h, and then the crude extract obtained using a rotary evaporator was dissolved in 1 mL of methanol.⁷⁴ Extracts were used for subsequent LC-MS (Thermo Scientific Ultimate 3000, Thermo Fisher Scientific, USA) analysis. Metabolic profiles and untargeted metabolomics of WT and Δfus3 mutant strains were analyzed using Thermo Xcalibur software and Compounds Discoverer 3.0 software (Thermo Fisher Scientific), respectively.

RT-qPCR analysis

WT and Δfus3 strains were cultured on TYGA at 28°C for 3, 5, or 7 days, and total RNA from mycelium was extracted with AxyPrep multisource RNA miniprep kit, and then translated into cDNA a FastQuant RT kit with gDNase (TaKaRa).The cDNA was further used as a template for RT-qPCR using SYBR Premix Ex TaqTM (TaKaRa) and specific primers (Table S1) to detect the transcript levels of candidate genes related to conidiation, cell wall biosynthesis, and oxidative stress response; the β-tubulin-encoding gene was used as an internal standard. The mRNA level was analyzed using the 2^−ΔΔCt method.⁵¹

Y2H screening

The CDS regions of fus3, pyp1, ste12, and flbA were amplified from total cDNA using specific primers (Table S1) and inserted into pGADT7-AD and pGBKT7-BD vectors. The constructed plasmids were sequenced at Tsingke Biotechnology Co., Ltd. (Beijing, China). Then, the constructed vector was co-transformed into S. cerevisiae strain AH109 using the LiAc/SS-DNA/PEG transformation protocol (TaKaRa, Dalian, China), and further cultured on synthetic defined (SD) medium without Trp and Leu at 30°C for 3–5 days. Finally, self-activation or interaction was screened by SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade/+AbA/+X-α-Gal plates.

Analysis of regulatory network of Fus3

Based on the transcriptome analysis between the WT and Δfus3 mutant strains, several genes related to remarkably enriched GO terms were selected, including cell communication-, basal transcription factor-, cell cycle-, translation-, cell response to stress-, nutrient metabolism-, and sporulation-related genes, as well as several genes that have been reported to interact with Fus3, such as Far11-, Cdc24-, and Cdc42-related genes.³^,¹³ The network of Fus3 and its putative target genes were analyzed using STRING³⁷ and then mapped using Cytoscape version 3.9.0.³⁸ String ID and annotation of each target gene in the network are listed in Table S10.

Quantification and statistical analysis

Each experiment was conducted with three parallel replicates, and the data were shown as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was applied in statistical analysis of multiple measurements for all strains, and the significance was determined by Tukey’s honest significance test (Tukey’s HSD). P value < 0.05 was considered significant. Statistical analyses were performed with the software of GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

Acknowledgments

We are grateful to Microbial Library of the Germplasm Bank of Wild Species from Southwest China for preserving and providing experimental strains, and Guo Ying-qi (Kunming Institute of Zoology, Chinese Academy of Sciences) for her help with taking and analyzing TEM images. Funding for this study was provided by National Natural Science Foundation of China (nos. 31960556, 32160665, and U1402265), Special fund of the Yunnan University “double first-class” construction, Yunnan Fundamental Research Projects (nos. 202201BC070004 and 202001BB050004), and General Project of Yunnan Provincial Department of Science and Technology (202101AT070779).

Author contributions

K-Q.Z. and J.Y. conceived and designed the study. M.X. and N.B. performed the experiments. M.X., N.B., X.Y., and Y.L. analyzed the data. M.X. prepared figures and tables. M.X. and J.Y. contributed to manuscript preparation and revision. All authors read and approved the final manuscript.

Declaration of interests

We declare that we have no conflicts of interests.

Published: August 7, 2023

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2023.107404.

Supplemental information

Document S1. Figures S1–S4 and Tables S1–S9

mmc1.pdf^{(516.1KB, pdf)}

Table S10. Annotation of proteins in the Fus3 interaction network in A. oligospora ATCC 24927, related to Figure 9

mmc2.xlsx^{(18.1KB, xlsx)}

Data and code availability

•
Data: All data and methods necessary to reproduce this study are included in the manuscript and Supplemental Information. Sequencing data were deposited to the National Center for Biotechnology Information under accession number GSE212172 and are publicly available.
•
Code: This paper does not report original code.
•
Other items: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S4 and Tables S1–S9

mmc1.pdf^{(516.1KB, pdf)}

Table S10. Annotation of proteins in the Fus3 interaction network in A. oligospora ATCC 24927, related to Figure 9

mmc2.xlsx^{(18.1KB, xlsx)}

Data Availability Statement

•
Data: All data and methods necessary to reproduce this study are included in the manuscript and Supplemental Information. Sequencing data were deposited to the National Center for Biotechnology Information under accession number GSE212172 and are publicly available.
•
Code: This paper does not report original code.
•
Other items: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

Fus3 regulates asexual development and trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora

Meihua Xie

Na Bai

Xuewei Yang

Yankun Liu

Ke-Qin Zhang

Jinkui Yang

Summary

Graphical abstract

Highlights

Introduction

Results

Sequence analysis of Fus3

Fus3 regulates mycelial growth, cell nucleus number, and endocytosis

Figure 2.

Figure 1.

Fus3 regulates conidiophore morphology and conidia yield

Fus3 regulates multiple stress responses

Figure 3.

Fus3 regulates hyphal fusion, trap morphogenesis, and pathogenicity

Figure 4.

Fus3 regulates DNA damage repair and mitophagy

Figure 5.

Transcriptomic insights into the regulatory role of Fus3

Figure 6.

Figure 7.

Fus3 is involved in the regulation of secondary metabolism

Figure 8.

Prediction and analysis of proteins interacting with Fus3

Figure 9.

Discussion

Limitations of the study

STAR★Methods

Key resources table

Resource availability

Lead contact

Materials availability

Experimental model and study participant details

Fungal strains

Bacterial strain

Nematode

Method details

Sequence analysis of Fus3

Deletion of fus3

Analysis of vegetative growth, morphogenesis, and conidiation

Stress assays

Trap formation and morphogenesis

Transcriptome sequencing and analysis

LC-MS assay

RT-qPCR analysis

Y2H screening

Analysis of regulatory network of Fus3

Quantification and statistical analysis

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Supplemental information

Data and code availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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