EXPERIMENTAL PROCEDURES

Strains. Wild-type N2 worms and the conditional sterile mutant fer-15(b26ts) LGII strain were maintained at 15, 20 or 25 °C on NGM (nematode growth medium) agar with E. coli OP50 bacteria. The S. marcescens strains Db11 and Db1140 were grown as previously described .

Microarray and northern analyses. Eggs from fer-15(b26ts) worms cultivated at 15 °C were placed at 25 °C and allowed to hatch in the absence of food. The thus synchronised larvae were transferred after 16-20 h to OP50 and cultivated at 25 °C until the L4 stage. Worms were then transferred to fresh plates seeded either with OP50 or Db11, and harvested 24 h or 48 h later. Total RNA was extracted using Trizol (Gibco/BRL). Microarray analyses, using cDNA arrays were carried out as previously described . Northern analysis was performed as previously described , using the rpp-1 gene (Y37E3.7; transcript size 0.4 kb) as a loading control. The blots were quantified using a phosphoimager, ensuring that the signals quantified were within the linear range.

Generation of lys-1::GFP and control transgenic worms. To construct a lys-1::GFP reporter, we inserted a PstI/BamHI-digested PCR amplicon (generated from genomic DNA with primers AA CTGCAG TGG ACA TGG GTT AAG AAA AGA GA and CG GGATCC ACA TGC CAA TAC CAG AGA AGA AC and corresponding to the entire coding sequence of the lys-1 gene together with 2 kb of upstream sequence) into the vector pPD95.75 (the kind gift of A. Fire). Reporter DNA was injected at 50 ng/µl into N2 worms using the rol-6(su1006) plasmid pRF4 at 100 ng/µl as a co-injection marker. Among the several transgenic lines obtained, IG36 gave the strongest GFP signal. The strain IG66 obtained upon injection of pRF4 alone at 100 ng/µl into N2 worms was used as a control.

RNAi. The insert from yk557g2 was cloned as an EcoRI/XhoI fragment into pPD129.36 (the kind gift of A. Fire) to give pNP102. RNAi was performed by feeding as previously described . Briefly, L4 IG36 worms were placed on plates spread with HT115 bacteria transformed either with pPD129.36 or with pNP102, allowed to grow for 72 h at 15 °C and then transferred to 25 °C. After 48 h L4 worms of the F1 generation were put on fresh plates seeded with OP50, Db11 or Db1140. The survival of the worms was followed as described below. In parallel, worms were harvested for western analysis.

Western blotting. 50 IG36 worms for each condition were picked directly into electrophoresis sample buffer and boiled for 5 minutes. The samples were separated on 12% SDS-PAGE gels and transferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA). Membranes were blocked in TBS overnight at 4 °C. Primary and secondary antibodies (JL-8 anti-GFP, Clontech and goat anti-mouse peroxydase, Sigma, respectively) were added before revelation with the enhanced chemoluminescence system (ECL, Amersham, UK). A single band of the expected size (60 KDa) was obtained.

Survival tests. Assays for S. marcescens killing were conducted as described previously . 10 L4 stage hermaphrodite worms from the test strains were placed on each plate. Plates were incubated at 25 °C and scored for live and dead worms every 24 hours. During the first few days, worms were transferred to fresh plates daily. Animals were considered dead when they ceased moving and responding to prodding. One-sided rank log tests (within Prism; www.graphpad.com) were used to assess the similarity between two groups.

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