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Additional file 1: Figure S1. B12 supplementation alleviates DTT toxicity in a mmcm-1 mutant but not metr-1 mutant. Embryos of (A-C) wild type (N2), (D-F) metr-1(ok521), (G-I) mmcm-1(ok1637), and (J-L) rips-1(ij109) were added to plates supplemented with 0 (top row) or 5 mM DTT (middle and bottom rows), in the presence (bottom row) or absence (top and middle rows) of 64 nM vitamin B12. Development to adult stage was assessed 4 days later and representative images are shown in panels (A-L). Scale bars denote 1 mm. The number of animals used in this experiment are as follows: (A) (n = 118), (B) (n = 136), (C) (n = 73); (D) (n = 197), (E) (n = 147), (F) (n = 197); (G) (n = 59), (H) (n = 93), (I) (n = 56); (J) (n = 109), (K) (n = 62), (L) (n = 94). (M) Plotted development to adult stage in percentage under the above treatments. p-values were determined from Fisher’s exact test. NS not significant, *** p < 0.001. For all panels, purple significance marks indicate comparison of mutant worm strains to N2 wild type for each treatment group and blue significance marks indicate comparison of treatment groups (i.e., DTT or DTT+B12) to no DTT groups for each worm strain. Figure S2. DTT resistance mutants map to a single SAM methyltransferase gene. (A-B) HA mapping output from DTT resistance screen for (A) rips-1 allele ij109 (strain TP193) and (B) rips-1 allele ka14 (strain TP251). Clear peak visible on Chromosome V. (C) Protein sequence of RIPS-1 SAM methyltransferase highlighting location of mutation generated via EMS DTT resistance screen. Underlined residues exon/exon junctions and position and nature of mutation highlighted in colour and allele designation in brackets. Location of mutation relative to methyltransferase domain highlighted in cartoon (Pfam (PF13847) Methyltransf_31 residues 176-285; InterPro domain (IPR025714) Methyltranfer_dom residues 176-285. Figure S3. The loss of rips-1 causes DTT resistance phenotype. (A) High degree of identity between rips-1 (R08E5.3) and its closest homologue R08E5.1 that will account for potential RNAi cross-reaction. (B) N2 wild type [a], rips-1(ij109) mutant [TP193] [b], or wild type worms fed on E. coli expressing RNAi targeting rips-1 (R08E5.3) [c] or its homologues (R08E5.1 [d], R08F11.4 [e], or K12D9.1 [f]) was treated with 5 mM DTT for confirmation of causative gene for DTT resistance phenotype. Worms harbouring rips-1(ij109) allele or fed on rips-1 RNAi or R08E5.1 RNAi survived and reached adult stage on DTT while wild type (N2) or worms fed on R08F11.4 RNAi or K12D9.1 RNAi arrested development on DTT. Scale bars denote 1 mm. (C) The rips-1 paralogue R08E5.1 is induced by 5 mM DTT in wild type (N2) as shown via quantitative PCR. Red points with lines denote the mean and SEM. The level of R08E5.1 in wild type increases 4-fold upon exposure to 5 mM DTT (mean 4.527, Standard deviation 1.389). Figure S4. Media composition and bacterial food source do not influence DTT resistance of rips-1 mutant strain. (A-B) Counts for additional alleles of rips-1: (A) TP276 [rips-1(ka23)] and (B) TP251 [rips-1(ka14)]. Survival to adulthood after 4 days on 5 mM DTT treatment was compared to wild type N2. Red points with lines denote the mean and SEM. p-values were determined from Student’s t-test. *** p < 0.001. Significance marks indicate comparison of rips-1 mutants to wild type. (C) rips-1(ij109) embryos were added onto (top row) animal-based peptone NGM or (bottom row) soy plant-based peptone NGM with either (half left) B12-poor E. coli strain OP50 or (half right) B12-rich E. coli strain HT115, with or without 5 mM DTT. Worms were then viewed as adults after 4 days at 22°C. All growth conditions resulted in development to healthy adult populations. Scale bars denote 1 mm. Figure S5. R08E5.3 is a methyltransferase conserved in diverse species. (A) Top BLAST hits from R08E5.3 amino acid sequence (Fig. S2C) as representative sequences: archaea [Nitrosopumilus maritimus, WP_012215840.1], mycobacterium [Mycobacterium mantenii, WP_083099804.1], bacterium [Desulfovibrio brasiliensis, WP_054652021.1], non-nematode multi-cellular eukaryote [Branchiostoma belcheri, XP_019632822.1], Caenorhabditis elegans rips-1 [NP_504045.1], non-Caenorhabditis nematode [Angiostrongylus cantonensis, KAE9416730.1], and fungi [Arthrobotrys oligospora, KAF3112035.1]. (B) Conservation of amino acids between rips-1 and its orthologues: rips-1 [NP_504045.1], R08E5.1 [NP_504044.3], R08F11.4 [NP_504052.1] and K12D9.1 [NP_503823.2]. In (A-B), conserved mutations found in rips-1 DDT resistance mutants are shown in red above the alignments. (C) Genomic location and genes surrounding rips-1 (R08E5.3). Figure S6. RIPS-1::GFP reporter transgene shows subcellular induction following 5 mM DTT exposure. RIPS-1::GFP transgenic strain (A-C) TP313 and (D-F) TP315 show (A, D) weak gut induction in the absence of DTT (white asterisk in (A) denotes the transgenic pharyngeal marker), but (B-C, E-F) strong induction in gut (arrowed) and the hypodermis (arrowhead) following 5 mM DTT exposure. Scale bars denote 0.1 mm. Figure S7. RIPS-1::GFP reporter is induced by DTT in the presence of heat-killed OP50 and in the complete absence of bacteria. Transgenic RIPS-1::GFP (TP313) nematodes were picked to unseeded plates for 1 hour then transferred to plates in the (A) absence or (B-C) presence of 5 mM DTT, with no bacteria (A-B) or (C) heat-killed bacteria (60°C for 30 min), and grown for 24 hours. Insets represent bright field image. Scale bars denote 0.5 mm. (D) GFP quantification of (A-C). Red points with lines denote the mean and SEM. p-values were determined from one-way ANOVA, followed by Tukey’s test. *** p < 0.001. Significance marks indicate comparison of DTT-treated groups to untreated control. Figure S8. RNAi of hypoxia pathway genes induce RIPS-1::GFP reporter expression in TP315. An independent RIPS-1::GFP reporter strain (TP315) was used to carry out the same experiment depicted in Fig. 5. RIPS-1::GFP reporter strain was reared on E. coli expressing (A) Control (L4440) RNAi, (B) rhy-1 RNAi, (C) egl-9 RNAi, (D) vhl-1 RNAi, (E) mxl-3 RNAi, or (F) clk-1 RNAi. Knockdowns of rhy-1 and egl-9 induce RIPS-1::GFP expression in the gut and hypodermis, vhl-1 RNAi induces RIPS-1::GFP expression only in the gut, while control, mxl-3, and clk-1 RNAi do not cause any induction. Insets represent bright field image. Scale bars denote 0.5 mm. (G) Quantification of RIPS-1::GFP expression in panels (A-F). Red points with lines denote the mean and SEM. p-values were determined from one-way ANOVA, followed by Dunnett’s test. *** p < 0.001. Significance marks indicate comparison of DTT-treated groups to untreated control. Figure S9. Hypoxia induction factor HIF-1 controls RIPS-1 activation on DTT exposure. An independent RIPS-1::GFP reporter strain (TP315) was used to carry out the same experiment depicted in Fig. 5. (A-B) Control, (C-D) hif-1, and (E-F) vhl-1 RNAi feeding were carried out on RIPS-1::GFP reporter strain for 3 days. L4 animals with positive myo-2 transgenic marker (red pharynx) were then picked onto the corresponding RNAi plates that (B, D, F) were supplemented with 5 mM DTT or (A, C, E) with no DTT for 24 hours prior to imaging. (A-B) Worms reared on control RNAi only showed strong RIPS-1::GFP induction upon treatment with 5 mM DTT, while (C) hif-1 RNAi alone or (D) followed by 5 mM DTT treatment failed to induce RIPS-1::GFP. (E-F) RNAi of vhl-1 induced RIPS-1::GFP expression in the gut tissues that persisted following DTT exposure. Insets represent bright field image. Scale bars denote 0.5 mm. Quantification of GFP signals for panels (A-F) is depicted in (G), where red points with lines denote the mean and SEM. p-values were determined from one-way ANOVA, followed by Tukey’s post-hoc test. NS not significant, * p < 0.05, *** p < 0.001. In panel (G), blue significance marks indicate comparison of groups treated with 5 mM DTT to untreated control, black and red significance marks indicate comparison of RNAi groups to control (L4440) RNAi groups within no DTT or DTT-treated conditions, respectively. |
