Divergent C. elegans toxin alleles are suppressed by distinct mechanisms
- Department of Human Genetics, University of California, Los Angeles, United States
- Department of Biological Chemistry, University of California, Los Angeles, United States
- Howard Hughes Medical Institute, United States
- Department of Biology, Johns Hopkins University, United States
Figures
Figure 1 with 1 supplement
Discovery and characterization of a novel toxin-antidote (TA).
(A) The gray line represents the frequency of XZ1516 alleles across the genome after four generations of intercrossing with QX1211. Each panel corresponds to a C. elegans chromosome and each x-axis tick indicates 5 Mb. The dotted blue line represents the expected allele frequency for each chromosome with no selection. The region highlighted in red on the right side of chromosome V shows the greatest allele frequency deviation from expectation. (B) Crosses between XZ1516 (purple) and QX1211 (yellow) establish the inheritance pattern of the TA element. Bar plots show the fraction of dead L1s observed in each cross. Error bars indicate 95% binomial confidence intervals calculated using the normal approximation method. Crosses from left to right: selfing of XZ1516/QX1211 heterozygous hermaphrodites; XZ1516/QX1211 heterozygous hermaphrodites crossed to QX1211 males; XZ1516/QX1211 heterozygous males crossed to QX1211 hermaphrodites. The observed fraction of dead L1s was not significantly different from the expected fractions for a maternally inherited TA element, exact binomial test. (C) Model of the TA inheritance. Punnett square shows the lethality pattern expected in progeny from selfing of XZ1516/QX1211 heterozygous hermaphrodites. A maternally deposited toxin (black square) is present in all progeny and causes L1 lethality unless a zygotically expressed antidote (white circle) is also present.
Figure 1—figure supplement 1
DL238 is also susceptible to the XZ1516 toxin-antidote (TA).
(A) Estimates of the XZ1516 allele frequency at each position in the genome after four (gray) or ten generations of intercrossing with QX1211 (black, gray) or DL238 (red). Each panel corresponds to a C. elegans chromosome and each x-axis tick represents 5 Mb. The dotted blue line represents the expected allele frequency for each chromosome. (B) Top three panels are representative images of rod-like lethality phenotype caused by the tmrl-1 toxin. The bottom panel is a rod worm next to a normally developing worm derived from the same cross. (C) Crosses between XZ1516 (purple) and DL238 (yellow) that establish the inheritance pattern of the TA element. The y-axis represents the fraction of dead L1s observed for each cross depicted on the x-axis. Crosses from left to right: selfing of XZ1516/DL238 heterozygous; XZ1516/DL238 heterozygous hermaphrodites crossed to DL238 males; XZ1516/DL238 heterozygous males crossed to DL238 hermaphrodites. The observed fraction of dead L1s was not significantly different from the expected fractions for a maternally inherited TA element, exact binomial test.
Figure 2 with 2 supplements
Identification of the toxin-antidote (TA) components.
(A) Localization of the TA element genes in XZ1516. Top panel: Strain genotypes of near-isogenic lines (NILs) are displayed as colored rectangles (XZ1516 in purple; QX1211 in yellow; Cas9-induced deletion in red) for chromosome V. The fraction of L1 lethality after selfing of the NIL/QX1211 hermaphrodites is shown to the right of each NIL. The bottom panel depicts a summary of QX1211 sequencing reads aligned to the XZ1516 genome corresponding to the mapped TA element. Gray bars denote short-read sequencing depth in 200 bp windows, and red dots denote the number of variants detected between QX1211 and XZ1516 in each window. The XZ1516 and QX1211 genomes are so diverged that short reads derived from QX1211 don’t align to the XZ1516 genome in the 200 bp windows with no corresponding read depth, as indicated by a lack of a gray bar. The toxin and antidote genes are highlighted in green and light blue, respectively. (B) Knockout and transgenic rescue experiments define the TA components. Bar plots denote the fraction of dead L1s derived from selfing F1 heterozygous individuals. Error bars indicate 95% binomial confidence intervals calculated using the normal approximation method. Blue and green boxes with ‘A’ and ‘T’ indicate intact antidote and toxin genes, respectively; white boxes indicate deletions of these genes. XZ1516 genotypes are depicted in purple and QX1211 genotypes are depicted in yellow. Panels from top to bottom: XZ1516/QX1211 control cross, the observed lethality is not significantly different from the expected 25%; toxin knockout cross to QX1211, the observed lethality is significantly different from the expected 25% p=1.38e-31; antidote transgenic rescue cross the observed lethality is significantly different from the expected 25% p=1.22e-53; toxin and antidote double knockout cross to XZ1516, the observed lethality is not significantly different from the expected 25%. An exact binomial test was used to determine significance.
Figure 2—figure supplement 1
Isoforms of the tmrl-1 toxin.
Long-read RNA sequencing reads of the different tmrl-1 isoforms aligned to the XZ1516 genome visualized in the Integrative Genomics Viewer (IGV). Light purple reads represent isoform B, the dark purple reads represent isoform A, and gray reads represent the 5’ UTR of tmrl-1. The functional gene model of tmrl-1 is depicted in green above the computationally predicted ‘FUN’ gene models.
Figure 2—figure supplement 2
The tmrl-1 transcript persists until the L1 stage.
Representative tmrl-1 FISH images in XZ1516. Yellow corresponds to tmrl-1 transcript puncta, and nuclei staining with DAPI is in blue. (A) Max projection of one- and two-cell embryos. (B) Post-gastrulation embryos showing tmrl-1 localization to the Z2/Z3 cells. (C) L1 larvae showing tmrl-1 localization to the Z2/Z3 cells.
Figure 3 with 1 supplement
Demographics of the tmrl-1/amrl-1 toxin-antidote (TA).
(A) A dendrogram showing the relatedness of 550 wild C. elegans strains at the TA locus. Branches are colored to represent the three distinct clades, where purple denotes the XZ1516-like clade, yellow denotes the N2-like clade, and pink denotes the NIC195-like clade. (B) Isolation location of strains collected in Hawaii. Pie charts show the number of isolates from each clade when multiple strains were collected at one location, with colors as in A. (C) Bar plots show the fraction of dead L1s in crosses between XZ1516 and NIC195 (left) and between XZ1516 and NIC195 with its antidote allele knocked out (right), indicating that this antidote is active against the XZ1516 toxin. Error bars indicate 95% binomial confidence intervals calculated using the normal approximation method. The observed lethality in the NIC195 × XZ1516 cross is significantly different from the expected 25% (p=6.14e-19, exact binomial test), while the antidote knockout difference is not significantly different. (D) Synteny plot of the TA region between the XZ1516 (top) and N2 (bottom) genomes. The TA components tmrl-1 and amrl-1 are colored green and blue, respectively. (E) Percent amino acid identity of ~5500 one-to-one orthologs identified between the XZ1516 and N2 genomes. Amino acid identity for tmrl-1 is indicated with a red line.
Figure 3—figure supplement 1
Phenotypes induced by the two tmrl-1 alleles.
The percent of healthy (blue) or arrested or delayed (red) individuals is shown on the y-axis. The first two bars represent technical replicates of tetracycline-induced tmrl-1 in the XZ1516 genetic background. The remaining three bars correspond to tetracycline-induced B0250.8 in XZ1516, DL238, and N2, respectively.
Figure 4 with 2 supplements
The N2 tmrl-1 allele contributes to larval arrest in the absence of MUT-16.
(A) Density plots showing the distribution of animal lengths on the x-axis for the Δtmrl-1, Δmut-16, and the Δtmrl-1; ∆mut-16 double knockout lines. The distribution of animal lengths is significantly different for all comparisons (Kruskal-Wallis test; p=1.56e-133 for the ∆mut-16 to double knockout comparison, p=7.51e-67 for the ∆tmrl-1 to double knockout comparison, and p ≈ 0 for the ∆mut-16 to ∆tmrl-1 comparison). (B) Animal length data from (A) were binned to approximate larval stages as described in the methods. Stacked bar charts of the fraction of animals for each developmental stage for the Δtmrl-1, ∆mut-16, and the ∆tmrl-1; ∆mut-16 double knockout lines are shown. The fraction of the population is shown on the y-axis for each developmental stage—yellow: L1, green: L2/L3, and blue: L4. The fraction of adults is omitted for clarity, but corresponds to the fraction that brings the total to 1 for each genotype.
Figure 4—figure supplement 1
Small RNAs (sRNAs) are involved in the suppression of the N2 tmrl-1 allele.
The (A) mRNA and (B) sRNA abundance is shown for Δmut-16 on the x-axis and WT on the y-axis (Reed et al., 2020). The N2 tmrl-1 (B0250.8) allele is represented as a yellow dot. (C) sRNA abundance of WAGO-1-associated sRNAs for two independent replicates (Seroussi et al., 2023).
Figure 4—figure supplement 2
SVM bubble detection for COPAS biosort.
The fraction of individual worms that developed to adults is shown on the y-axis for increasingly strict SVM cutoffs that differentiate worms from bubbles on the x-axis. The most strict cutoff of 0.9999999 was used to generate Figure 4.
Additional files
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Supplementary file 1
Strains.
- https://cdn.elifesciences.org/articles/106269/elife-106269-supp1-v1.csv
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Supplementary file 2
Plasmids.
- https://cdn.elifesciences.org/articles/106269/elife-106269-supp2-v1.csv
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Supplementary file 3
Plasmid construction.
- https://cdn.elifesciences.org/articles/106269/elife-106269-supp3-v1.csv
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Supplementary file 4
Crosses.
- https://cdn.elifesciences.org/articles/106269/elife-106269-supp4-v1.csv
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Supplementary file 5
Oligos.
- https://cdn.elifesciences.org/articles/106269/elife-106269-supp5-v1.csv
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Supplementary file 6
Guide RNAs (gRNAs) and repair.
- https://cdn.elifesciences.org/articles/106269/elife-106269-supp6-v1.csv
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Supplementary file 7
Inducible construct phenotypes.
- https://cdn.elifesciences.org/articles/106269/elife-106269-supp7-v1.csv
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Supplementary file 8
COPAS biosort data.
- https://cdn.elifesciences.org/articles/106269/elife-106269-supp8-v1.csv
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MDAR checklist
- https://cdn.elifesciences.org/articles/106269/elife-106269-mdarchecklist1-v1.pdf
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Divergent C. elegans toxin alleles are suppressed by distinct mechanisms
eLife 14:RP106269.
https://doi.org/10.7554/eLife.106269.3
