New Nipi alleles.

1<p>WormBase Release WS228.</p

<i>nipi-4</i> encodes a pseudokinase required for the induction of <i>nlp-29</i>.

<p>(A) SNP mapping with WGS. The positions of SNP loci on Chromosome V for the <i>fr106</i> allele are depicted as a XY scatter plot, where the ratio ‘Hawaiian/total number of reads’ for each SNP is represented, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033887#pone.0033887-Doitsidou1" target="_blank">[35]</a>. The region without Hawaiian SNPs contains the mutation (red arrow). (B) Exon-intron structure of <i>nipi-4</i>, adapted from WormBase (WS220), with the positions of the <i>fr68</i>, <i>fr71</i>, <i>fr99</i> and <i>fr106</i> mutations indicated. Also shown is the structure of the p<i>nipi-4</i>::GFP & p<i>nipi-4</i>::NIPI-4 constructs. (C) Biosort quantification of the normalized fluorescence ratio in wild type, <i>sta-2(ok1860)</i> and the 4 <i>nipi-4</i> alleles <i>fr68</i>, <i>fr71</i>, <i>fr99</i> and <i>fr106</i> carrying <i>frIs7</i> following infection. For this and subsequent figures, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033887#s4" target="_blank">Materials and Methods</a> for details of the data processing and the number of worms analyzed. The results are representative of 3 independent experiments. (D) Biosort quantification of the normalized fluorescence ratio in wild type, <i>nipi-4(fr106)</i> and <i>nipi-4(fr106)</i> with a rescuing transgene p<i>nipi-4</i>::NIPI-4, carrying <i>frIs7</i> following infection.</p

The <i>nipi-4</i> gene acts cell autonomously in the epidermis.

<p>(A–E) Expression of <i>nipi-4</i> is seen throughout the epidermis (A & B)), in larvae (C) and adults (A,B,D&E), from head (D) to tail (E), in vulval cells (arrow in B), in rectal cells (arrow in E), but not in the seam cells (arrowhead in A), scale bar 10 µm. (F–G) <i>nipi-4</i>(<i>fr71</i>) and <i>nipi-4</i>(<i>fr71</i>);<i>frEx496</i> (P<i>col-19</i>::NIPI-4) worms strains carrying an integrated P<i>nlp-29</i>::GFP reporter (<i>frIs7</i>) following infection. The expression of <i>nipi-4</i> in epidermal cells in the adult rescues the <i>nipi-4</i> phenotype. Green and red fluorescence is visualized simultaneously with a GFP long pass filter.</p

New Nipi alleles isolated in a large scale screen.

<p>(A) Biosort quantification of the fluorescence in wild type and different mutants strains carrying an integrated P<i>nlp-29</i>::GFP reporter (<i>frIs7</i>) following infection including <i>sta-2(ok1860)</i>, <i>nipi-3(fr4)</i>, <i>tpa-1(k530)</i> and 38 new alleles, 11 of which have been determined to define 6 new independent complementation groups. The average fold induction for each strain is represented after standardization across different independent experiments by normalizing to 10 the fold induction between the wild type strain infected versus non infected. (B) Genetic map of Nipi loci identified from screens or from candidate gene approaches. The map has been scaled to the genome sequence, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033887#pone.0033887-Yook1" target="_blank">[48]</a>. The Nipi genes identified in the present mutagenesis and in previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033887#pone.0033887-Couillault1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033887#pone.0033887-Pujol3" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033887#pone.0033887-Ziegler1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033887#pone.0033887-Dierking1" target="_blank">[21]</a> (Couillault <i>et al.</i> submitted) are represented in red and black respectively.</p

The <i>nipi-4</i> gene is required for the response to infection and wounding.

<p>(A) <i>nipi-4</i> mutants do not block the induction of <i>nlp-29</i> expression upon osmotic stress. Biosort quantification of the normalized fluorescence ratio in wild type, <i>sta-2(ok1860)</i> and <i>nipi-4(fr71)</i> worms carrying <i>frIs7</i> following infection by <i>D. coniospora</i>, wounding, PMA treatment and osmotic stress. (B) Quantitative RT-PCR analysis of gene expression levels in non- infected and infected wild type, <i>sta-2(ok1860)</i> and <i>nipi-4(fr106)</i> worms. The columns show the average expression level (arbitrary units) and SEM from 4 experiments. The level of <i>nlp-34</i> expression in control animals is set at 1024 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033887#s4" target="_blank">Materials and Methods</a>).</p