Figure 1.
Life cycle of the nematode-trapping fungus M. haptotylum.
(A) Saprophytic growth with hyphae and knob; (B) Newly captured nematode; (C) Knobs (black arrows) adhering to immobilized nematode (∼1 hour after adding the nematode); (D) Fungal degradation of the nematode; (E) Emergence of new hyphae from the nematode; (F) Trap formation after degrading nematode. The whole infection process from adhesion to new trap formation after degrading the nematode lasts ∼36 hours. Scale bar, 50 µm. The infected nematode shown is C. briggsae.
Table 1.
Main features of the M. haptotylum and A. oligospora genomes.
Figure 2.
Gene family expansions, contractions and lineage-specific proteins of the nematode-trapping fungi M. haptotylum and A. oligospora.
(A) Rooted maximum likelihood tree constructed from 602 single copy orthologous proteins using the Dayhoff amino acid substitution model showing evolutionary relationships of 16 fungal species. Branches labeled with letters show the taxonomic classification O: Orbiliomycetes, P: Pezizomycotina, S: Saccharomycotina, T: Taphrinomycotina. The bootstrap support values were 100 on all branches. Pie charts based on CAFE analysis of 13,402 orthoMCL gene families indicate expanded and contracted gene families for each branch in the phylogenetic tree. The size of each pie chart is proportional to the total number of gene families that have either expanded or contracted (Figure S3). (B) Venn diagram of the predicted proteins in M. haptotylum and A. oligospora versus those of 14 other fungal species. The slight difference in number of genes between M. haptotylum and A. oligospora in each category is due to different gene copy numbers.
Figure 3.
Comparison of gene families in filamentous ascomycetes.
(A) Principal component analysis of eight filamentous pathogens based on gene counts from all gene families (13,402) identified by OrthoMCL clustering. Scale was log2(gene counts+1) and variance filtering was set to 0.2. The colors indicate the host infected by each species. Filamentous pathogens infecting plants are green (M. oryzae, formerly M. grisea, Fusarium graminearum), insects are red (M. anisopliae, M. acridum) and other animals (humans) are orange (Arthroderma benhamiae, A. fumigatus). (B) Heat map of expanded gene families in common between nematode-trapping fungi and other filamentous pathogens infecting plants (M. grisea, F. graminearum), insects (M. anisopliae, M. acridum) and other animals (A. benhamiae, A. fumigatus). The gene families where clustered using hierarchical clustering of log2(gene counts+1). Only 39 gene families passing the F-test with variance filtering of 0.2 and q-value<0.05 (false discovery rate, adjusted for multiple testing) are shown. Pfam domains present in proteins belonging to each gene family are shown to the right. Scale is log2(gene counts+1) .
Figure 4.
Expanded gene families in M. haptotylum.
The number of proteins in 25 Pfam families that were found to be significantly (P<0.001) enriched in the genome of M. haptotylum are compared with their sizes in 20 other fungal genomes. The symbol ‘*’ indicates that the family were also found among the lineage-specific families (Figure 2B). The symbol ‘Φ’ indicates that the Pfam family contains M. haptotylum proteins that match proteins (BLASTP, threshold value <1E -10) in the pathogen–host interaction (PHI-base) database [29]. Species and group abbreviations: NEM, the nematode-trapping fungi M. haptotylum (MH) and Arthrobotrys oligospora (AO); E, the entomopathogenic fungus Metarhizium anisopliae (MAA); APA, animal pathogenic fungi including Arthroderma benhamiae (AB), Aspergillus fumigatus (AF), Coccidioides immitis (CI), Cryptococcus neoformans var neoformans (CN), Candida albicans (CA) and Malassezia globosa (MGL); PPA, plant pathogenic fungi including Magnaporthe oryzae (formerly M. grisea) (MGR), Stagonospora nodorum (SN), Fusarium graminearum (FG) and Ashbya gossypii (AG); SAP, the saprotrophic fungi including Neurospora crassa (NC), Aspergillus niger (AN), Trichoderma reesei (TR), Emericella nidulans (WN), Podospora anserina (PA), Saccharomyces cerevisiae (SC) and Schizosaccharomyces pombe (SP); SYM, the symbiotic fungus Tuber melanosporum (TM).
Figure 5.
Features of core, lineage-specific (LS) and species-specific (SS) protein-coding genes in M. haptotylum and A. oligospora.
The proportions of proteins among the core, LS and SS categories that have homologs in the NCBI nr database, the EuKaryotic Orthologous Groups (KOG) database and the pathogen–host interaction protein database (PHI-base) are shown. “Pfam” indicates the fraction of proteins with Pfam domains; “Expanded Pfam” indicates proteins with Pfam domains of expanded gene families (see Figure 4); “Secreted proteins (SPs)” indicates proteins with predicted signal peptide; SSPs indicates SPs with a length <300 amino acids; “Expressed” indicates the fraction of proteins supported by expression (RNASeq) data. The total number of genes in the core, LS and SS categories are shown in parentheses.
Figure 6.
Gene expression by M. haptotylum during the infection of the nematode C. briggsae.
(A) Shown is the proportion of protein-coding genes that were upregulated or down-regulated at 2-fold or 10-fold levels when comparing the expression levels in penetrating mycelium (4 hours of infection) and knobs. The genes were classified into core, lineage-specific (LS) and species-specific (SS) genes. (B) Features of the highly upregulated (>10 fold) and expressed (>10%) genes. “*” indicates the categories of genes that were significantly (P<0.001) enriched among the upregulated genes (left) as compared with their distribution in the genome (right). “Combined” includes core + LS + SS.
Table 2.
Number of highly upregulated or highly expressed transcripts in expanded Pfam domain families of M. haptotylum and A. oligospora during nematode infection.
Figure 7.
Comparison of gene expression in M. haptotylum and A. oligospora during infection of the nematode C. briggsae.
(A) Log10 scatter plot of gene expression pattern between orthologs of M. haptotylum (mean of two replicates, 4 hours of infection) and A. oligospora (mean of samples from 6 and 10 hours of infection as replicates). The red circles indicate 2,599 significantly differentially expressed genes (q-value <0.01). (B) Features of M. haptotylum genes that are >10 fold upregulated in comparison with A. oligospora. (C) Features of A. oligospora genes that are >10 fold upregulated in comparison with M. haptotylum. “*” indicates the categories of genes that were significantly (P<0.001) enriched among the upregulated genes as compared with their distribution in the genome (right). “Combined” includes core + LS + SS.
Figure 8.
Gene cluster of secreted proteins in M. haptotylum that were highly expressed during nematode infection.
The gene cluster (id 74) contains five of the 117 genes that were most expressed and upregulated during nematode infection (see Figure 5). All genes in the cluster contain a secretion signal; SSPs and Pfam domains are indicated below each gene. The bar chart shows the gene expression level (fold change) of each of the five genes in the cluster and is ordered in which they are found in the genome.
Figure 9.
Sequence homology clustering of secreted proteins in M. haptotylum.
Clusters of secreted proteins (nodes) containing at least three protein members are shown and Pfam domains in the clusters are indicated. The two rings indicate a cluster containing two groups of proteins with different Pfam domains and joined by only one protein. Small secreted proteins, SSPs (<300 amino acids) are colored: Blue nodes indicates proteins belonging to the core set; green are lineage-specific proteins and red nodes are species-specific proteins. Grey nodes are secreted proteins with a length of 300 amino acids or longer. Five red stars indicate SSPs that are more than 10 fold upregulated during infection versus knobs and belong to the 10% most highly expressed genes during infection. O indicates orphan sequences lacking matches to the Pfam database and other species in NCBI nr database. Blue Δ indicates core genes and green Δ indicates lineage-specific genes with 10 fold differential gene expression pattern between orthologs of M. haptotylum and A. oligospora during infection. Cluster 1 contains the following Pfam domains: Abhydrolase_2, Adeno_E1B_55K, Alpha_GJ, ArabFuran-catal, ASF1_hist_chap, BBE, CBM_1, Cellulase, Cutinase, CVNH, DUF1023, DUF1191, DUF1680, DUF291, End_N_terminal, Esterase, Esterase_phd, FAD_binding_4, Glyco_hydro_6, 7, 10, 11, 12, 16, 18, 26, 28, 30, 43, 45, 61, 62, 72, GMC_oxred_C, GMC_oxred_N, HC2, Herpes_gE, Kei1, Lipase_GDSL, LysM, Melibiase, Muc_lac_enz, Mucin, OmpW, Pec_lyase_C, Pectate_lyase, Pectinesterase, Peptidase_C8, Podoplanin, Pyr_redox, Pyr_redox_2, RE_AccI, Ricin_B_lectin, Sec10, SKN1, Stig1, Syndecan, Tyrosinase, X8 and Yip1.