We have previously described a forward genetic approach for the isolation of C. elegans mutants with enhanced susceptibility to pathogens (Esp mutants), which led to the identification of a requirement for a PMK-1
p38 MAPK pathway in pathogen resistance [1]. More recently, we have reported genome-wide microarray analysis of candidate immune effector genes regulated by the PMK-1 pathway and induced by pathogen infection [2]. Based on these data, we generated a strain carrying the integrated transgene agIs219 that expresses GFP under the control of the promoter for the T24B8.5 gene that is regulated by PMK-1 and induced by pathogen infection. We have used this GFP reporter gene in a forward genetic strategy aided by the COPAS fluorescence-activated worm sorter (Union Biometrica) to isolate C. elegans Esp mutants with diminished PMK-1 activation. Thus far, we have screen ~140,000 haploid genomes and have identified 65 mutant isolates. These isolates comprise at least 6 complementation groups, which represent multiple alleles of known PMK-1 pathway components as well as mutants that do not correspond to previously known pathway components. We anticipate that our genetic strategy will identify upstream inputs into the PMK-1 pathway that may function in pathogen recognition and provide insights into the mechanism of action of the Toll-Interleukin-1 Receptor adaptor protein, TIR-1 [3, 4]. 1.Kim, D.H., et al. Science, 2002. 297(5581): p. 623-6. 2.Troemel, E.R., et al. PLoS Genet, 2006. 2(11): p.
e183. 3.Couillault, C., et al. Nat Immunol, 2004. 5(5): p. 488-94. 4.Liberati, N.T., et al. Proc Natl Acad Sci U S A, 2004. 101(17): p. 6593-8.