Fig 1.
Mutations in nhr-66 promote Caenorhabditis elegans escape from the predatory fungus A. oligospora.
(A) Schematic of the forward genetic screen for trap-resistant C. elegans. (B) Quantification of nematode escaping rates in strains obtained from forward genetic screens (yph406-408, 410, and 412), CRISPR STOP-IN (yph413), and fosmid-rescued strains (yphXXX alleles correspond to nhr-66 variants shown in Fig 1D) (mean ± SEM; n is shown below the x-axis; two-tailed unpaired Student t test) (See S1 Video for more). (C) SNP mapping of two of the screen mutants. Trap-resistant mutants were crossed with Hawaiian strain CB4856, and the F2 progeny from each mutant line which exhibited trap resistance were analyzed for eight SNP markers on each chromosome. Regions containing only N2 SNPs (indicated by the red rectangle) were assumed to contain the causative mutation (See S1 Data for more). (D) Genetic mapping and whole-genome sequencing of trap-resistant mutants reveal causative mutations (marked by asterisks) in the gene nhr-66. Boxes and lines represent exons and introns, respectively. The data underlying this figure can be found in S1 Data.
Fig 2.
NHR-66 promotes adhesion through cuticle-secreting tissues without altering cuticle structure.
(A) Quantification of nematode escaping rates in wild-type Caenorhabditis elegans (N2), the nhr-66(yph413) mutant, and tissue-specific rescue lines expressing nhr-66 cDNA under various promoters (mean ± SEM; n is shown below the x-axis; two-tailed unpaired Student t test). (B, C) Atomic Force Microscopy (AFM) topography images (top), quantification and their line profiles (bottom) of adult cuticle annuli, furrows (B) and alae (C) for both wildtype and nhr-66(yph413) mutant. Data are presented as Mean ± SEM (n is shown below the x-axis; two-tailed unpaired Student t test). (D) TEM images and quantification of cuticle (top) and strut width (bottom) from wild-type (N2) and nhr-66(yph413) animals (scale bar: white, 0.2 µm; black, 2 µm). Data are presented as Mean ± SEM (n is shown below the x-axis; two-tailed unpaired Student t test). The data underlying this figure can be found in S2 Data.
Fig 3.
NHR-66 regulates the expression of the collagen gene family in Caenorhabditis elegans.
(A) Principal component analysis of transcript expression from wildtype (N2) and nhr-66(yph413) mutants across three biological replicates. (B) Heatmap comparison of differentially expressed genes (≥ 4-fold change) between N2 and nhr-66(yph413) mutants. The numerical labels beneath the heatmap indicate the number of differentially expressed genes between N2 and the nhr-66(yph413) mutant. (See S3 Data for more). (C) Gene ontology enrichment analysis results for genes down-regulated more than 4-fold in the nhr-66(yph413) mutant. (See S3 Data for more). (D) Quantification of nematode escaping rates in wild-type C. elegans (N2), the nhr-66(yph413) mutant, and collagen mutants (mean ± SEM, n is shown below the x-axis, two-tailed unpaired Student t test). (E) Protein structure of collagens used in rescue experiments. Collagens are classified into clusters based on interruptions in their main collagenous domain (blue boxes), with numbers in the boxes indicating the count of Gly-X-Y repeats. All cuticular collagens typically contain cysteine-rich regions (yellow), an N-terminal helical Gly-X-Y repeat (red box), a transmembrane region (pink) or predicted signal peptides (green). (F) Quantification of nematode escaping rates in collagen-rescued nhr-66(yph413) mutants (mean ± SEM, n is shown below the x-axis, two-tailed unpaired Student t test). (G) Fluorescence images and quantification of Pcol-14::GFP reporter expression in N2, the nhr-66(yph413) mutant, and rescued strains (scale bar, 100 µm). Data are presented as the Mean ± SEM (n is shown below the x-axis, two-tailed unpaired Student t test). The data underlying this figure can be found in S3 Data.
Fig 4.
The nhr-66 mutant is resistant to multiple species of nematode-trapping fungi of the genus Arthrobotrys.
(A) Nematode escape rates of nhr-66 mutants exposed to various species of nematode-trapping fungi (NTF phylogeny shown on the left). The phylogeny is based on single-copy orthologs. Data are presented as Mean ± SEM (n is shown on the left the y-axis, two-tailed unpaired Student t test, P-values comparing WT and nhr-66 mutant against each NTF strain are < 0.001 with the exact p-values provided in the source data). The data underlying this figure can be found in S4 Data.
Fig 5.
nhr-66 is critical for the fitness and survival of Caenorhabditis elegans in natural environments.
(A) A pie chart showing the number of wild isolates bearing nhr-66 missense variants out of the total number of isolates characterized on CaeNDR. (See S4 Table for more). (B) Nematode escaping rates in wildtype, nhr-66(yph413) mutants, and nhr-66 natural variants (mean ± SEM, n is shown below the x-axis; two-tailed unpaired Student t test). (C) Percentage of nematodes that exploded under hypoosmotic stress (mean ± SEM, n is shown in the figure; two-tailed unpaired Student t test). Right, images of nematodes exposure to deionized water for 15 min (scale bar, 500 μm) (See S2 Video for more). (D) Occupancy rates of wildtype and the nhr-66(yph413) mutant in a soil competition assay at 20 °C (n is shown below the x-axis, two-tailed unpaired Student t test). The data underlying this figure can be found in S5 Data.