Supplementary Information

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1 Supplementary Information Supplementary figures % Occupancy Wt tol-1(nr2033) Figure S1. Avoidance behavior to S. enterica was not observed in wild-type or tol-1(nr2033) mutant nematodes. The behavioral avoidance assay was performed as described (Pradel et al., 2007). Twenty 1 day old adult nematodes were placed on a small spot of S. enterica in a 6 cm plate and monitored at 16 hr post-exposure for presence or absence on the lawn. Experiments were performed in duplicate S. marcescens S. enterica E. coli

2 Figure S2. Pharyngeal infection of tol-1(nr2033) mutants with S. enterica expressing GFP. Confocal images of the terminal bulb of the tol-1(nr2033) pharynx infected with S. enterica expressing GFP for 48 hr. Intact bacteria are observed in all images. The confocal plane of column A shows the intact lumen of the pharynx (arrows) and the grinder (arrow heads). Column B shows a different confocal plane and column C shows higher magnification images of the same animal.

3 Ave. pumping/15 sec min 24 hr Wt tol-1(nr2033) Figure S3. Wild-type and tol-1(nr2033) mutant nematodes exhibit similar pharyngeal pumping rates on S. enterica. Rates of pharyngeal pumping of wild type and the tol-1(nr2033) mutant were similar. Pumping rates using one day old adult hermaphrodites challenged with S. enterica were determined as described by (Chow et al., 2006) with minor modifications. Briefly, the number of contractions of the terminal bulb over 15 seconds was counted. A contraction was defined as the backward movement of the grinder in the terminal bulb of the pharynx. The pumping rates for ten nematodes were determined at 30 min and 24 hr postinfection.

4 100 Wt tol-1(nr2033) Figure S4. The tol-1(nr2033) mutant nematodes survive longer than wild-type nematodes on Streptococcus pneumoniae. Thirty-six one day old adult wild-type N2 and tol- 1(nr2033) nematodes were challenged with S. pneumoniae and monitored for survival. tol- 1(nr2033) nematodes were more resistant to S. pneumoniae (P<0.0001). The graph is representative of at least three independent experiments Hours

5 Supplementary experimental procedures C. elegans killing assay For all strains except E. faecalis OG1RF, individual bacterial colonies were inoculated into LB and grown for 8 hours on a rotary wheel at 37 C. Ten microliters of culture were plated onto a 3.5 cm plate containing modified NGM (3.5 % peptone instead of 2.5 %). E. faecalis OG1RF was grown in 2 ml BHI broth at 37 C and 20 µl of culture were plated onto a 6.0 cm plate of BHI medium containing 50 µg/ml gentamicin. All plates were incubated overnight at 37 C. One day old adult hermaphroditic nematodes were transferred to lawns of the various bacteria and transferred daily to a fresh lawn until progeny was no longer detected. All experiments were performed at 25 C. Animals were scored at the indicated times and considered dead upon failure to respond to touch. Animals missing from the agar plate were censored on day of loss. Lifespan assay Synchronized L4 larvae were transferred to a lawn of heat-killed E. coli OP50 on a plate of modified NGM containing 100 µg/ml 5-fluorodeoxyuridine (FUdR) and 50 µg/ml ampicillin. FUdR is an inhibitor of DNA synthesis that blocks the development of progeny. The assay was performed at 25 C. Animals were scored at the indicated times and considered dead upon failure to respond to touch. Animals missing from the agar plate were censored on day of loss. Expression of tol-1 in tol-1(nr2033), ikb-1(nr2027), and trf-1(2014) animals tol-1(nr2033), ikb-1(nr2027), and trf-1(2014) mutants were microinjected the cosmid C07F11 containing approximately forty-one kilobases of sequence which includes C07F11.2 and the

6 promoter region and full-length sequence of C07F11.1 (tol-1). Animals were co-injected with prf4 which contains the rol-6(su1006) allele as the marker. Pharyngeal invasion assay and invasion-mortality assay Fifty 1 day old adult hermaphroditic nematodes were placed on lawns of S. enterica or P. aeruginosa expressing GFP. Nematodes were monitored at 48 hours for infected pharynxes using fluorescence microscopy. An infected pharynx was defined as the presence of GFP in the terminal bulb excluding the pharyngeal lumen. For the pharyngeal invasion-mortality assay, twenty 1 day old adult hermaphroditic nematodes were placed individually on a lawn of S. enterica or P. aeruginosa expressing GFP. Nematodes were monitored daily by fluorescence microscopy for infected terminal bulbs of the pharynx and mortality. All experiments were performed at 25 C. Confocal microscopy Seventy-five microliters of S. enterica or P. aeruginosa expressing GFP grown in 3 ml LB with Kan 50 µg/ml were plated onto a 10 cm plate of modified NGM medium containing 50 µg/ml Kan. The plates were incubated at 37 C overnight. One day old adult nematodes were transferred to a lawn of bacteria expressing GFP and incubated for two days with daily transfers to a new lawn of pathogen. After 2 days, the nematodes were moved to a lawn of E. coli OP50. This enables rapid identification of nematodes with infected pharynxes and reduces the fluorescence from the background lawns. Twenty nematodes were anesthetized in 10% sodium azide on an agar pad (2% agarose) and examined using a Leica TCS SL confocal microscope with Leica

7 Confocal software version 2.61 Build 1537 (Leica Microsystems Heidelberg GmbH). The confocal images were imported into Adobe Photoshop for processing including size adjustments, layering, and brightness-contrast. qrt-pcr Gravid wild-type and tol-1(nr2033) nematodes were lysed using a solution of sodium hydroxide and bleach, washed, and the eggs were synchronized overnight in S basal liquid medium at room temperature. Synchronized L1 animals were placed onto NGM plates seeded with E. coli OP50 and grown until L4. The L4 animals were exposed to S. enterica for 24 hours at 25 C and then harvested. The animals were collected by washing the plates with M9 buffer, and RNA extracted using Trizol reagent. Genomic DNA was removed by treating the RNA samples with DNase using the DNA-free kit according to manufacturer s instruction (Ambion). qrt-pcr was conducted using the Applied Biosystems Taqman One-Step Real-time PCR protocol using SYBR Green fluorescence (Applied Biosystems) on an Applied Biosystems 7900HT real-time PCR machine in 96 well plate format. Fifty nanograms of RNA were used for real-time PCR. Twenty-five microliter reactions were set-up and performed as outlined by the manufacturer (Applied Biosystems). Genes tested include the control, act-1 (act-1 F 5 gtgtgacgacgaggttgccgctcttgttgtagac-3 and act-1 R 5 -ggtaaggatcttcatgaggtaatcagtaagatcac-3 ), abf-2 (F 5 -gccaccatcgtggctgccg-3, abf-2 R 5 cctctcttaataagagcac-3 ), and hsp (hsp F 5 - aatttttccgataatattggggag-3, and hsp R 5 - ttctggtttgaaatgagagacatc-3 ). Gene expression in tol-1(nr2033) was compared to wild type using the comparative Ct method and normalization to act-1 (actin) was used.

8 Statistical analyses Animal survival was plotted as a non-linear regression curve using the PRISM (version 4.00) computer program. Survival curves are considered significantly different than the control when P values are <0.05. Prism uses the product limit or Kaplan-Meier method to calculate survival fractions and the logrank test, which is equivalent to the Mantel-Heanszel test, to compare survival curves. The time for 50% of the nematodes to die (time to death 50, TD 50 ) was calculated using Prism software (version 4.01) using a non-linear regression analysis of survival proportions utilizing the equation: Y=Bottom + (Top-Bottom)/( (LogEC 50 -X)*Hill Slope) ), where Top is set at 100, Bottom is set at 0, X is the time in days and Y is the percentage of nematodes alive at time X. In this instance, TD 50 is equivalent to EC 50. Correlation between pharyngeal infection and mortality was determined using linear regression analysis. Mann-Whitney test and Student s exact t test were used to analyze the pharyngeal invasion and qrt-pcr results, respectively. Supplementary References Chow, D.K., Glenn, C.F., Johnston, J.L., Goldberg, I.G. and Wolkow, C.A. (2006) Sarcopenia in the Caenorhabditis elegans pharynx correlates with muscle contraction rate over lifespan. Exp Gerontol, 41, Pradel, E., Zhang, Y., Pujol, N., Matsuyama, T., Bargmann, C.I. and Ewbank, J.J. (2007) Detection and avoidance of a natural product from the pathogenic bacterium Serratia marcescens by Caenorhabditis elegans. Proc Natl Acad Sci U S A, 104,