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	Fig 1. TRPA1 regulates defecation in response to the uracil-triggered Duox pathway.A Schematic diagram illustrating the uracil-dependent defecation assay (see Materials and Methods for details). B Dose-dependence of uracil-responsive defecation in wild type flies (wcs) versus TrpA1ins (n = 4–13). Sucrose solution containing uracil at 0.2, 20, 20,000 and 100,000 nM was fed to the indicated genotypes, and the defecation was compared with that from sucrose-only-fed flies. C Requirement of TrpA1 for uracil-induced defecation was confirmed with three independent TrpA1 alleles (n = 6–13). Fold change of defecation was acquired by comparing defecation frequencies from two sibling groups fed with or without 20 μM uracil. Right: The defecation defect in TrpA1ins was rescued by the WT TrpA1 gene inserted in the fly genome. D Ingestion of electrophile N-methyl maleimide (NMM) promotes defecation depending on TrpA1 (n = 4–5), indicating that reactive chemicals in the gut lumen can induce defecation increases through TrpA1. E Co-ingestion of ROS-counteracting dithiothreitol (DTT) with uracil abolishes the defecation increase that is dependent on TrpA1 (n = 6–8). See also S2E–S2G Fig. F Ubiquitous expression of a peroxiredoxin gene Jafrac1 by da-Gal4 readily suppresses uracil-dependent defecation increase (n = 5–10). G Duox is required for uracil-induced defecation, as RNAi knockdown abolishes uracil-dependent defecation (n = 8–11). H The imd pathway is dispensable for TrpA1-dependent defecation, as animals with the strong loss-of-function allele, rele20, of the NF- κB gene retain the uracil-dependent defecation response (n = 6–13). Mean ± SEM is shown with data distribution. *: p<0.05, **: p<0.01, ***: p<0.001, Student’s t- (B) or Tukey tests (C to H). See also S1 Fig for alternative presentation of data and S3A–S3D Fig for mechanical aspects of uracil in relation to the TRPA1 channel activity and feeding behavior.
	Fig 2. TrpA1(A) in a subset of enteroendocrine cells is necessary for uracil-dependent defecation.A Co-immunostaining of TRPA1 and either Prospero or GFP in the anterior midgut from indicated genotypes. Bottom, Arrowheads: TRPA1 and GFP co-immunostaining. B TrpA1 RNAi in TrpA1(A)-Gal4 EECs dramatically suppresses uracil-dependent defecation (n = 5–8). C TrpA1 gene structure illustrating alternative exon usage (Top) and TRPA1 protein organization showing variable domains (Bottom). The primers used in reverse transcription PCR analyses shown in (D and E) are indicated as arrows. Most of primers in these analyses were designed to straddle two separate exons in order to prevent PCR amplification of genomic DNA. D and E The representative results of RT-PCR analyzing alternative exons of TrpA1 transcripts in the gut for the N-terminal (D) and exon10-encoded domains (E). ‘Plasmid’ indicates the PCR reactions with cloned cDNAs of TrpA1 transcripts as DNA polymerization templates, and the bands resulting from the‘plasmid’ reaction were used as size and intensity markers. Proboscis, Head and Gut denote the tissues where total RNA was prepared for reverse transcription. The band of the “Ladder” lane is a 500 bp long DNA fragment of Generuler 1 kb ladder (E). 10a and 10b indicate cDNA templates or PCR products of TrpA1(A)10a and TrpA1(A)10b, respectively, and the PCR products from the template of cloned cDNA (indicated by ‘Plasmid’) serve as size markers for the RT-PCR experiments with total cDNA prepared from the gut. Bar graphs show mean ± SEM with distribution. ***: p<0.001, Tukey test.
	Fig 3. The TRPA1(A)10b isoform is the HOCl receptor essential for uracil-evoked defecation.A Rescue of the defect in uracil-dependent defecation of TrpA1ins animals by expressing TrpA1(A) cDNA with either exon10a (TrpA1(A)10a) or exon10b (TrpA1(A)10b) in TrpA1(A)-Gal4 cells (n = 6–10). Expression of TrpA1(A)10b by EEC-expressing TrpA1(A)-Gal4 but not pan-neuronal c155-Gal4 restored uracil-dependent defecation in the TrpA1ins animal (Lower), while TrpA1(A)10a expression in TrpA1(A)-Gal4 EECs did not (Upper). B Rescue of the defect in NMM-dependent defecation of TrpA1ins mutants by the two alternative TrpA1(A) isoforms (n = 4–5). The NMM-mediated defecation was rescued by expressing TRPA1(A)10a in TrpA1(A)-Gal4 EEC cells, although uracil-dependent defecation was not. C-E Representative dose dependence (Left) and current-voltage relationship (Right) of NaOCl responses in Xenopus oocytes heterologously expressing indicated isoforms. Note that the time scales and dose sequences are set identical for simple comparison of activation kinetics. F Normalized dose dependences of TRPA1 isoforms averaged from the results at -60 mV in C-E panels (n = 4–10). TRPA1(A)10b show more sensitive response to NaOCl than TRPA1(A)10a or TRPA1(B). Current amplitudes at varying doses of NaOCl was normalized with respect to that at 100 ppm. The data were fitted to Hill equation to determine EC50s. G Time to 70% of the steady state current amplitude induced at each NaOCl concentration was measured for indicated TRPA1 isoforms for activation kinetics comparison of macroscopic NaOCl currents (n = 5–9, -60 mV). Mean ± SEM shown. *: p<0.05, **: p<0.01, ***: p<0.001, Tukey test. ###: p<0.001, Student’s t-test. See also S9 Fig for further functional characterization data of fly and human TRPA1s in oocytes, and for HOCl- or NMM-elicited action potential responses in sugar cells ectopically expressing fly TRPA1 isoforms.
	Fig 4. TrpA1-dependent defecation is required for bacteria expulsion from the gut.A Defecation of indicated Drosophila lines fed with either Ecc15 pyrE or Ecc15 wild-type (WT) was presented as output/input denoting the fecal spot number normalized by ingestion volume (n = 5–17). B Quantitative representation of (A) as defecation fold difference of indicated genotypes. Left, The wcs flies fed with ECC15 WT at OD600 of 10 defecate ~1.5 fold more than those fed with ECC15 pyrE, while TrpA1ins did not show higher defecation with ECC15 WT. Right, rele20 lacking the imd pathway show higher defecation frequency with ECC15 WT over pyrE. C Defecation comparison between pyrE ingestions with and without supplementation of 1 mM uracil (n = 5–8). Defecation of ECC15 pyrE-ingested flies is increased by supplementing uracil, and the increase requires TrpA1. #: p<0.05, Student’s t-test between wcs and rele20 fed with Ecc15 pyrE (A). *: p<0.05, **: p<0.01, Student’s t- or Tukey tests.
	Fig 5. Defecation deficit in TrpA1 mutants is associated with longer ECC15 gut persistence and rel-dependent increased mortality.A Gut persistence presented as relative colony-forming units (CFUs) at 2-, 5- and 8-hr time points of the experiments (n = 6–12). The time point indicates the time after start of ECC15 feeding. After ingestion of ECC15-GFP for indicated time, the dissected intestine was homogenized and CFUs of intestinal ECC15-GFP were determined to estimate bacterial growth in the gut. CFUs at 5 and 8 hr time points were divided by that at 2 hr time point to minimize experimental variation. B Defecation in the assay time windows of 0–5 hrs and 5–8 hrs is displayed as output/input (n = 5–17). Consistent with (A), TrpA1-dependent defecation is more prominent between 0 and 5 hr time points. C Loss of TrpA1 increases ECC15 gut persistence in the gut deficient for the imd pathway. D Ingestion of ECC15 pyrE does not differentiate wcs and TrpA1ins in gut persistence (Left). Supplementation with uracil allows ECC15 pyrE to proliferate in the guts of TrpA1ins more than wcs albeit at a delayed time point of 8 hrs (Right). E Germ-free flies show similar difference in ECC15 gut persistence among the wcs, TrpA1ins and genomic rescue strains, indicating that the persistence phenotypes resulted from genetic manipulation in flies not commensal bacteria which may differ in the fly strains. F and G Ingestion of ECC15 shortens life span of immune-compromised flies. When daily ingesting ECC15 WT (F), the rele20 mutant defective in the imd innate immune pathway shows reduced life span, which was further exacerbated by additional loss of TrpA1, indicating uracil-dependent defecation contributes to ECC15 resistance. Ingestion of ECC15 pyrE did not differentiate rele20 and TrpA1ins, rele20. Left, Kaplan-Meier LogRank survival analysis of indicated genotypes ingesting ECC15 WT (F) or pyrE (G) (p<0.001). Right, Life span distribution of the animals tested in the panel of Left. All pairwise multiple comparison by Holm-Sidak method. Letters indicate significantly distinct groups (p<0.001). §: p<0.02, §§: p<0.01, ANOVA Repeated Measures Holm-Sidak method (A). Mean ± SEM. *: p<0.05, **: p<0.01, Student’s t- or Tukey tests.

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