Figure 1:Calcineurin knockdown enhances C. elegans susceptibility to Pseudomonas aeruginosa(A) Representative survival plots of N2 and tax-6(p675) animals on P. aeruginosa PA14 at 25°C. The animals were grown on E. coli OP50 at 20°C until 1-day-old adults before transferring to P. aeruginosa PA14 at 25°C. p<0.001(B) Representative survival plots of N2 animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control and tax-6 RNAi. p<0.001.(C) Representative survival plots of tax-6(p675) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control and tax-6 RNAi. n.s., nonsignificant.(D) Representative survival plots of N2 animals grown on bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. p<0.001.(E) Representative survival plots of fer-1(b232) animals on P. aeruginosa PA14 at 25°C after treatment with the EV control and tax-6 RNAi at 25°C. p<0.001.(F) Representative survival plots of fer-1(b232) animals grown on bacteria for RNAi against tax-6 along with the EV control at 25°C. The animals were developed at 25°C. Day 0 represents young adults. p<0.001.For all panels, n = 3 biological replicates; animals per condition per replicate > 90.Figure 2:Calcineurin is required for the C. elegans defecation motor program (DMP)(A) Representative fluorescence (top) and the corresponding brightfield (bottom) images of N2 animals incubated on P. aeruginosa-GFP for 6 hours at 25°C after growth on the empty vector (EV) control and tax-6 RNAi bacteria.(B) Colony-forming units (CFU) per animal of N2 worms incubated on P. aeruginosa-GFP for 6 hours at 25°C after growth on the EV control and tax-6 RNAi bacteria. **p < 0.01 via the t-test (n = 3 biological replicates).(C) Representative fluorescence (top) and the corresponding brightfield (bottom) images of N2 and tax-6(p675) animals incubated on P. aeruginosa-GFP for 6 hours at 25°C after growth on E. coli OP50 at 20°C.(D) CFU per animal of N2 and tax-6(p675) worms incubated on P. aeruginosa-GFP for 6 hours at 25°C after growth on E. coli OP50 at 20°C. *p < 0.05 via the t-test (n = 3 biological replicates).(E) Representative photomicrographs of N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. Arrows point to the border of the intestinal lumen.(F) Quantification of the diameter of the intestinal lumen of N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. ***p<0.001 via the t-test (n = 21 worms each).(G) The number of expulsion events observed in 20 minutes in 1-day-old adult N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C. ***p<0.001 via the t-test (n = 6 worms each).(H) Percent of 1-day-old adult worms having irregular DMP. ***p<0.001 via the t-test (n = 4 biological replicates).Figure 3:Calcineurin knockdown enhances lifespan via DMP defects-mediated calorie restriction(A) Representative photomicrographs of oil-red-O (ORO) stained 1-day-old adult N2 animals grown on the empty vector (EV) control and tax-6 RNAi bacteria at 20°C. Scale bar = 200 µm.(B) Quantification of ORO intensity per animal of 1-day-old adult N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C. ***p<0.001 via the t-test (n = 10 worms each).(C) Quantitative reverse transcription-PCR (qRT-PCR) for pha-4 mRNA levels in N2 animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. ***p < 0.001 (n = 3 biological replicates).(D) Representative survival plots of eat-2(ad465) animals grown on the bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. n.s., nonsignificant (n = 3 biological replicates; animals per condition per replicate > 90).(E-I) Representative survival plots of aak-2(ok524) (E), raga-1(ok386) (F), rsks-1(ok1255) (G), daf-16(mu86) (H), and nhr-49(nr2041) (I) animals grown on the bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. p<0.001 for all the plots (n = 3 biological replicates; animals per condition per replicate > 80).Figure 4:Calcineurin inhibition enhances lifespan via HLH-30 and NHR-8(A) Representative survival plots of hlh-30(tm1978) animals grown on the bacteria for RNAi against tax-6 along with the empty vector (EV) control at 20°C. Day 0 represents young adults. n.s., nonsignificant (n = 3 biological replicates; animals per condition per replicate > 90).(B) Representative photomicrographs of oil-red-O (ORO) stained 1-day-old adult hlh-30(tm1978) animals grown on the EV control and tax-6 RNAi bacteria at 20°C. Scale bar = 200 µm.(C) Quantification of ORO intensity per animal of 1-day-old adult hlh-30(tm1978) animals grown on the EV control and tax-6 RNAi bacteria at 20°C. n.s., nonsignificant via the t-test (n = 10 worms each).(D) Representative survival plots of nhr-8(ok186) animals grown on the bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. n.s., nonsignificant (n = 3 biological replicates; animals per condition per replicate > 90).(E) Representative survival plots of daf-9(rh50) animals grown on the bacteria for RNAi against tax-6 along with the EV control at 20°C. Day 0 represents young adults. p<0.001 (n = 3 biological replicates; animals per condition per replicate > 80).(F) Representative photomicrographs of ORO stained 1-day-old adult nhr-8(ok186) animals grown on the EV control and tax-6 RNAi bacteria at 20°C. Scale bar = 200 µm.(G) Quantification of ORO intensity per animal of 1-day-old adult nhr-8(ok186) animals grown on the EV control and tax-6 RNAi bacteria at 20°C. ***p<0.001 via the t-test (n = 10 worms each).(H) Quantitative reverse transcription-PCR (qRT-PCR) for nhr-8 mRNA levels in N2 animals grown on the EV control, tax-6, flr-1, nhx-2, and pbo-1 RNAi bacteria at 20°C until 1-day-old adults. ***p < 0.001 via the t-test (n = 3 biological replicates).(I) Model depicting the mechanism of increased lifespan and enhanced susceptibility to pathogen upon inhibition of calcineurin via the defects in the defecation motor program (DMP).Figure S1:Calcineurin knockdown enhances C. elegans susceptibility to Pseudomonas aeruginosaRepresentative survival plots of N2 animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control, tax-6, crtc-1, and crh-1 RNAi.. p<0.001 for tax-6 and p<0.01 for crtc-1 and crh-1 compared to EV (n = 3 biological replicates; animals per condition per replicate > 90).Figure S2:Calcineurin inhibition enhances susceptibility to P. aeruginosa independent of known immunity pathways(A-E) Representative survival plots of pmk-1(km25) (A), kgb-1(km21) (B), dbl-1(nk3) (C), zip-2(ok3730) (D), and daf-16(mu86) (E) animals on P. aeruginosa PA14 at 25°C after treatment with the empty vector (EV) control and tax-6 RNAi. p<0.001 for all the plots (n = 3 biological replicates; animals per condition per replicate > 90).Figure S3:Calcineurin inhibition leads to defects in the defecation motor program (DMP) without affecting the pharyngeal pumping rate(A) Pharyngeal pumps per 30 seconds of 1-day-old adult N2 animals grown on the empty vector (EV) control and tax-6 RNAi bacteria at 20°C. n.s., nonsignificant via the t-test (n = 20 worms each).(B) The number of expulsion events observed in 20 minutes in 1-day-old adult N2 and regular and irregular tax-6(p675) animals grown on E. coli OP50 at 20°C. ***p<0.001, n.s., nonsignificant via the t-test (n = 6 worms each).Figure S4:Calcineurin inhibition leads to defects in the defecation motor program (DMP) in hlh-30(tm1978) animals(A) The number of expulsion events observed in 20 minutes in 1-day-old adult hlh-30(tm1978) animals grown on the empty vector (EV) control and tax-6 RNAi bacteria at 20°C. ***p<0.001 via the t-test (n = 5 worms each).(B) Representative photomicrographs of hlh-30(tm1978) animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. Arrows point to the border of the intestinal lumen.(C) Quantification of the diameter of the intestinal lumen of hlh-30(tm1978) animals grown on the EV control and tax-6 RNAi bacteria at 20°C until 1-day-old adults. ***p<0.001 via the t-test (n = 13 worms each).Table S1:Primers used in the study