Supplemental Material for Chen et al., 2020
Identification and phylogenic analysis of MAPKs in Arthrobotrys oligospora.
(A) Hypothetical model of MAPK signalings during trap morphogenesis in A. oligospora. Targeted deletion strains of genes highlighted in red were acquired in the A. oligospora TWF154 background. (B) Phylogeny of MAPKs from A. oligospora and orthologs from other model fungi. Sce: Saccharomyces cerevisiae. Mor: Magnaporthe oryzae. Fox: Fusarium oxysporum. Ncr: Neurospora crassa. Cne: Cryptococcus neoformans. Ao: Arthrobotrys oligospora. (C) Phylogenetic tree of A. oligospora and other fungal model organisms based on nucleotide and protein sequence databases of INSDC and NCBI.
Targeted gene deletions and Southern blot confirmations of mutants in A. oligospora
(A) Overview of gene deletion using CRISPR-Cas9 ribonucleoproteins (RNPs) coupled with repair template in A. oligospora. Two sgRNAs targeting the start and end of the SLT2 ORF were mixed with Cas9 protein to generate functional RNPs. Repair template and RNPs were co-transformed into protoplasts of A. oligospora to generate gene deletion mutants. (B) Southern blot confirmation of CRISPR-Cas9 mediated SLT2 deletion. The wild type TWF154 strain displays a predicted band of size 2.7 kb, whereas the slt2 TWF1029 mutant has a predicted band of 5.7 kb together with multiple ectopic integrations. (C-F) Southern blot confirmations for homologous recombination-based gene deletions of FUS3, STE7, BCK1, and STE12 in A. oligospora.
Characterization of the cell wall integrity MAPK pathway in the nematode-trapping fungus A. oligospora.
(A) Colony morphologies of WT, bck1, slt2, and a slt2 SLT2 complemented strain after culturing for 4 days at 25 °C on PDA plates (5-cm diameter). (B) Trap morphologies of WT, mutant, and complemented strains after 24 hours of exposure to 30 C. elegans nematodes for fungal cultures grown on LNM plates (2.5 cm). The arrows indicate traps produced by A. oligospora. (C) Trap morphologies after 24 hours of induction by ascr#18 (10 µM, 1 µl). The arrows indicate traps produced by A. oligospora. (D) Conidiation of WT, mutant, and complemented strains after culturing for 4 days at 25 °C on PDA plates. (E) Trap morphologies of slt2 mutant after 72 hours of exposure to ~1000 C. elegans on LNM plates.
Transcriptomic analysis of the ku70 and ku70 ste12 lines in response to nematode induction.
(A) PCA analysis of the transcriptomes of ku70 and ku70 ste12 under control and nematode-induced conditions. At least two replicates are presented for each condition. (B) Gene ontology enrichment analysis of the Ste12-dependent regulon. The bar chart displays the percentage of sequences in the test and reference sets for each GO term. The top ten GO terms with the highest number of genes in the test set are shown. (C) Summary of Ste12-dependent regulon orthologs exclusively in NTF or conserved in fungi other than NTF. Y-axis displays the percentage of transcripts for the described categories.
Table S1. Strains used in this study
Table S2. Protein sequences of MAPKs used for phylogenetic analysis
Table S3. Primers used in this study
Table S4. Ste12-dependent regulon