Transcriptomic Analysis Reveals That Rho GTPases Regulate Trap Development and Lifestyle Transition of the Nematode-Trapping Fungus Arthrobotrys oligospora

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


Fig. S2 Verification of the AS events and transcriptional levels of Rho GTPases. (A)
Transcriptome alignment identified 3058 to 4488 splicing events in 15 sample libraries, respectively. (B) Verification of the AS events in Aorho2 using RT-PCR; a. The diagrammatic representation of the Aorho2 gene. b. The AS events of gene Aorho2 were verified using RT-PCR. The target cDNA was amplified using AoRho2-P1 and AoRho2-P2 primers, and the AS events were verified using AoRho2-P2/AoRho2-P3 and AoRho2-P4/AoRho2-P5 primers (Table S9). M1K represents the 1000 DNA marker, M500 represents the 500 DNA marker. (C) Verification of the AS events in Aorac using RT-PCR; a. The diagrammatic representation of the Aorac gene. b. The AS events of gene Aorac were verified using RT-PCR. AoRac-P1 and AoRac-P2 primers were used to amplify the target cDNA, and AoRac-P1and AoRac-P3 primers (Table S9) were used to verify the AS events. M1K represents the 1000 DNA marker, M500 represents the 500 DNA marker. (D) Relative transcript levels (RTLs) of Aorho2, Aocdc42, and Aorac genes in the WT strain were monitored during the trap formation and nematode predation on water agar medium at different time points. The red line indicates the standard (with RTL=1) for statistical analysis of the RTL of each gene in the WT strain under a given condition. Error bars in (D) show SD. Asterisk indicates a significant difference (n = 3 for each gene; Tukey's HSD, P<0.05).

Fig. S3 Multiple sequence alignment of Rho GTPases.
The amino acid sequences of Rho GTPases from different fungi are aligned by DNAman software. Areas shaded in black represent conserved regions (100% similarity), areas shaded in red represent high degree similarity (more than 75% similarity), and those shaded in yellow show middle degree similarity (more than 50% similarity), while unshaded areas are regions of variability between the Rho2, Cdc42, and Rac.

Fig. S4 Three-dimensional structure models of Rho GTPases and phylogenetic analyses of Rho GTPases. (A)
The three-dimensional structures of Rho GTPases in A. oligosporaare predicted by Iterative Threading Assembly Refinement (I-TASSER). (B) Phylogenetic relationship among orthologous Rho2, Cdc42, and Rac proteins from A. oligospora and other fungi. The orthologs of Rho2 (AoRho2), Cdc42 (AoCdc42), and Rac (AoRac) in A. oligospora are marked in blue color.

Fig. S5 Verification of the knockdown of Rho GTPase genes in A. oligospora. (A)
Verification of the Aorho2 gene knockout using PCR and Southern blot analysis. a. The diagrammatic representation of homologous recombination of the Aorho2 gene. Primers AoRho2-5f/AoRho2-5r and AoRho2-3f/AoRho2-3r were used for the amplification of homologous flanks of the target gene, and the primers AoRho2-Yf/AoRho2-Yr (Table S9) were used for the verification of transformants. Probe (P) indicates the site of the Southern blot probe, and XhoI was the restriction enzyme used for Southern blot analysis. b. The transformants of gene Aorho2 were verified using PCR with the primers AoRho2-Yf/AoRho2-Yr. Line 3, 4, and 5 suggest positive transformants, line 1 suggests the wild-type (WT) strain, whereas line 2 suggests heterozygotic transformant with a WT gene copy and hph-replaced copy. M represents the DNA marker. c. Southern blot analysis of the WT and ΔAorho2 mutants. MT represents three independent mutants. (B) Verification of the Aocdc42 gene knockout using PCR and Southern blot analysis. a. The diagrammatic representation of homologous recombination of the Aocdc42 gene. Primers AoCdc42-5f/AoCdc42-5r and AoCdc42-3f/AoCdc42-3r were used for the amplification of homologous flanks of the target gene, and the primers AoCdc42-Yf/ AoCdc42-Yr (Table S9) were used for the verification of transformants. Pindicates the site of the Southern blot probe, and BstEII was the restriction enzyme used for Southern blot analysis. b. The transformants of gene Aocdc42 were verified using PCR with the primers AoCdc42-Yf/AoCdc42-Yr. Line 3, 4, and 5 suggest positive transformants, line 1 suggests the wild-type (WT) strain, whereas line 2 suggests heterozygotic transformant with a WT gene copy and hph-replaced copy. M represents the DNA marker. c. Southern blot analysis of the WT and ΔAocdc42 mutants. MT represents three independent mutants. (C) Verification of the Aorac gene knockout using PCR and Southern blot analysis. a. The diagrammatic representation of homologous recombination of the Aorac gene. Primers AoRac-5f/AoRac-5r and AoRac-3f/ AoRac-3r were used for the amplification of homologous flanks of the target gene, and the primers AoRac-Yf/AoRac-Yr (Table S9) were used for the verification of transformants. P indicates the site of the Southern blot probe, and HindIII was the restriction enzyme used for Southern blot analysis. b. The transformants of gene AorheB were verified using PCR with the primers AoRac-Yf/AoRac-Yr. Line 3, 4, and 5 suggest positive transformants, line 1 suggests the wild-type (WT) strain, whereas line 2 suggests heterozygotic transformant with a WT gene copy and hph-replaced copy. M represents the DNA marker. c. Southern blot analysis of the WT and ΔAorac mutants. MT represents three independent mutants.  represented as mean ± SD. The asterisk in (B and C) indicates a significant difference between the mutants and the WT strain (n = 3 for the WT strain (B and C), n = 9 for each mutant strain (B and C); Tukey's HSD, P<0.05).

Fig. S8
Comparison of trap formation, nematocidal activity, extracellular proteolytic activity, and ROS production. (A) Trap formation in the WT and ΔAorac mutant strains induced by nematodes at different time points. The red arrows show the traps produced by the WT strain and mutants. Bar = 50 μm. (B) The numbers of traps produced by the WT and ΔAorac mutant strains. Error bars: Data are represented as mean ± SD. The asterisk indicates a significant difference between the mutants and the WT strain (n = 3 for the WT strain, n = 9 for the mutant strain; Tukey's HSD, P<0.05).(C) Comparison of the extracellular proteolytic activities on casein plates. (D) Light micrographs of DHE-stained hyphae observed under DIC. The red arrows point to mycelia that do not produce ROS. Bar = 100 μm.