Figure 1.
Summary of C. elegans genes up-regulated after D. coniospora infection.
A–B Venn diagrams showing the number of genes identified as robustly up-regulated by D. coniospora infection after 12 h and/or 24 h using cDNA (A) and oligo (B) microarrays. C List, with brief annotation, of 14 genes identified as up-regulated at 24 h using statistical methods, (supporting evidence: 1, cDNA microarray; 2, oligo microarray; 3, statistical significance according to both MAANOVA and BRB-Array tool; 4, BRB-Array tools only).
Figure 2.
Phylogenetic analysis of nlp genes.
A The nlp-29 cluster in C. elegans together with syntenic regions from C. brigssae (Cbr) and C. remanei (Cre). a, B0213.7 (str-83); b, K09D9.9; c, ortholog of B0213.16; d, ortholog of K09D9.10 (srx-62); only the 3′ extremities of a and b are shown. B Phylogenetic tree for C. elegans CNC and selected NLP proteins, inferred from protein sequences by maximum likelihood (ML). Branches are drawn in proportion to the estimated number of substitutions per site. The results of bootstrapping (200 replicates) are indicated next to the branches. A phylogenetic tree with all the NLP proteins is provided in Figure S2A. The corresponding genes that were found to be up- or down-regulated by D. coniospora infection, are represented in red and blue, respectively; those not represented on either array are marked with asterisks. C Unrooted tree for nlp genes. Trees were inferred from aligned DNA sequences using ML. Branch-lengths are drawn in proportion to the estimated number of substitutions per site. ML analysis of NLP peptide sequences yielded the same tree with the exception of the exact relationships among nlp-28 to nlp-31. Bootstrap support (500 replicates) is given next to the branches for the peptide (before slashes) and DNA (after slashes) analysis. Branches for which there is support for adaptive sequence evolution are indicated in red; see also Figure S3 and Table S2.
Figure 3.
Induction and regulation of nlp gene expression.
A–C Quantification of the expression of the genes in the nlp-29 locus by qRT-PCR after 24 h of infection by D. coniospora (A), 2 h after needle wounding (B) and 6 h of osmotic stress in liquid (C). + is wild type; – pmk-1(km25). The columns show the average expression level (arbitrary units, +/− SEM) for at least 3 experiments. The level of nlp-27 expression in control animals is set at 1024 (see Materials and Methods). Constitutive expression of the nlp genes can vary across experiment due to differences in the exact age of the worms or conditions (solid or liquid culture). Within a single experiment, age-matched worms were used. D–F Quantification with the Biosort of the normalized fluorescent ratio (green/red) of worms carrying the integrated frIs7 transgene that contains pnlp-29::GFP and pcol-12::DsRed reporters [19] after 6 h in liquid culture in the presence of increasing concentrations of NaCl (D) and following a osmotic stress in 300 mM NaCl (E & F; see Materials and Methods). The fluorescent ratio in different backgrounds, sek-1(ag1), sek-1(km4), nsy-1(ky397), pmk-1(km25) or tir-1(tm3036), for worms after osmotic stress (purple) is compared to control worms (blue). As explained more fully elsewhere [19], due to the nature of the distribution, standard deviations are not an informative parameter and are not shown on this or subsequent figures using the Biosort. The number of worms used in each test is shown in parenthesis. The results shown are representative of at least 3 independent experiments.
Figure 4.
Osmotic stress resistant mutants constitutively express pnlp-29::GFP and resist fungal infection.
A–D Osmotic resistant mutants such as osm-11(n1604) (B) or dpy-9(e12) (D) constitutively express pnlp-29::GFP while other Dpy mutants including dpy-13(e184) (C) behave like wild-type (A) and do not. E Quantification with the Biosort of the normalized fluorescent ratio (green/red) of the integrated frIs7 transgene in different mutant backgrounds. The number of worms used in each test is shown in parenthesis. F & G dpy-9 and osm-11 mutants resist D. coniospora infection significantly better than wild-type worms and dpy-17 mutants (F, p<0.001, one-side log rank test), but do not have a significant different life span in the absence of pathogen (G). Both survival experiments were done on heat killed OP50. H Quantification with the Biosort of the normalized fluorescent ratio (green/red) of worms carrying the integrated frIs7 transgene in different mutant backgrounds, osm-11(n1604), pmk-1(km25) and osm-11(n1604);pmk-1(km25). The number of worms used in each test is shown in parenthesis. The results shown are representative of at least 3 independent experiments. I Acute osmotic stress resistance in wild type worms osm-11(n1604), pmk-1(km25) and osm-11(n1604);pmk-1(km25) mutants.
Figure 5.
Overexpression of the nlp-29 locus.
A Transgenic worms carrying extra copies of the nlp-29 locus have elevated levels of gene expression. The level of gene expression for the 6 genes of the nlp-29 locus was compared between transgenic worms carrying a DNA fragment specifically encompassing the locus (+) and non-transgenic worms (−), either following D. coniospora infection (yellow) or not (blue). B Overexpression of the nlp-29 locus is associated with increased resistance to infection. Transgenic worms carrying the nlp-29 locus (in light blue) are more resistant to D. coniospora infection than their non-transgenic sibling (in red). The difference in survival is highly significant (p<0.001, one-side log rank test).
Figure 6.
The GATA factor ELT-3 is required for gene induction in the epidermis.
A Quantification with the Biosort of the relative fluorescent ratio following infection (yellow) or osmotic stress (300 mM NaCl in liquid) compared to control worms (blue) in wt or an elt-3 background. The number of worms used in each test is shown in parenthesis. Fluorescence images of a pgpdh-1::GFP reporter gene in wt (B,D) or an elt-3 (C,E) background after overnight exposure to 200 mM NaCl. The tail region is detailed in D&E. In wt, pgpdh-1::GFP is induced in both the epidermis (arrowhead) and intestine, as compared to elt-3 mutant where pgpdh-1::GFP is only present in the intestine (arrow). F & G elt-3 nlp-29 tir-1 mutants are significantly more susceptible to D. coniospora infection better than wild-type worms (F, p<0.001, one-side log rank test), but do not have a significant different life span in the absence of pathogen. (G). Both survival experiments were done on heat killed OP50.