C. elegans were grown on standard NGM plates with E. coli OP50 [37] (link) unless otherwise noted. The previously published C. elegans strains used in this study were: N2 Bristol [37] (link), pmk-1(km25)[5] (link), AY101 [acIs101[pDB09.1(pF35E12.5::GFP); pRF4(rol-6(su1006))] [10] (link), XA7702 mdt-15(tm2182)[19] (link), [21] (link), CF512 fer-15(b26);fem-1(hc17)[38] (link), and AU0133 [agIs17(pirg-1::GFP; pmyo-2::mCherry)] [14] (link). The C. elegans strains created for this study were: AU0307 [agIs44(pF08G5.6::GFP::unc-54-3′UTR; pmyo-2::mCherry)], AU0316 [mdt-15(tm2182); agIs44], AU0325 [mdt-15(tm2182); agEx116 (mdt-15;pmyo-3::mCherry)], AU0326 [mdt-15(tm2182); agEx117 (mdt-15;pmyo-3::mCherry)], AU0327 [mdt-15(tm2182); agEx118 (mdt-15;pmyo-3::mCherry)] and AU0323 [mdt-15(tm2182); agIs44; agEx114 (mdt-15;pmyo-3::mCherry)].
The strain carrying agIs44 was constructed by PCR amplification from N2 genomic DNA of an 851 bp region upstream of the start codon of the F08G5.6 gene (primers GACTTGTCAAATGAACAATTTTATCAAATCTCA and CGCCTAGGTGTCAATTGATAATGAATA) and ligated to the GFP coding region and unc-54-3′UTR sequences amplified from pPD95.75 using published primers, and a previously described protocol [39] (link). The agIs44 construct was transformed into N2 animals with the co-injection marker pmyo-2::mCherry using established methods [40] (link). A strain carrying the pF08G5.6::GFP::unc-54-3′UTR and pmyo-2::mCherry transgenes in an extrachromosomal array was irradiated, and strains carrying the integrated array agIs44 were isolated. AU0307 was backcrossed to N2 five times.
The mdt-15 rescuing arrays agEx116, agEx117 and agEx118 contain a 4.8 kb mdt-15 genomic fragment, which includes 707 bp upstream and 1075 bp downstream of the mdt-15 coding region, amplified from N2 genomic DNA (primers GGAGTATCAGAAGCTCACGATGCTC and CCAAATAATACTAACCACCACATATCTTCCATT). This mdt-15 genomic fragment was transformed into N2 animals or AU0316 with the co-injection marker pmyo-3::mCherry using established methods.
RNAi clones presented in this study were from the Ahringer [41] (link) or Vidal [42] (link) RNAi libraries unless otherwise stated. The atf-7[12] (link) and the pmk-1[5] (link) RNAi clones have been previously reported. All RNAi clones presented in this study have been confirmed by sequencing. The P. aeruginosa strain PA14 were used for all studies, unless otherwise indicated. The P. aeruginosa strains used in Figure S1 have been previously described [13] (link) and were (in order of descending virulence toward C. elegans): CF18, PA14, MSH10, S54485, PA01, PAK, 19660, and E2. The P. aeruginosa PA14 phenazine null mutant (Δphz) lacks both the phzA1-G1 and phzA2-G2 operons and has been previously described [43] (link). The BL21 E. coli strain that expresses the bacterial toxin Exotoxin A (ToxA) has been previously described [15] (link).
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