The pseudokinase NIPI-4 is a novel regulator of antimicrobial peptide gene expression

Abstract

Hosts have developed diverse mechanisms to counter the pathogens they face in their natural environment. Throughout the plant and animal kingdoms, the up-regulation of antimicrobial peptides is a common response to infection. In C. elegans, infection with the natural pathogen Drechmeria coniospora leads to rapid induction of antimicrobial peptide gene expression in the epidermis. Through a large genetic screen we have isolated many new mutants that are incapable of upregulating the antimicrobial peptide nlp-29 in response to infection (i.e. with a Nipi or 'no induction of peptide after infection' phenotype). More than half of the newly isolated Nipi mutants do not correspond to genes previously associated with the regulation of antimicrobial peptides. One of these, nipi-4, encodes a member of a nematode-specific kinase family. NIPI-4 is predicted to be catalytically inactive, thus to be a pseudokinase. It acts in the epidermis downstream of the PKC∂ TPA-1, as a positive regulator of nlp antimicrobial peptide gene expression after infection. It also controls the constitutive expression of antimicrobial peptide genes of the cnc family that are targets of TGFß regulation. Our results open the way for a more detailed understanding of how host defense pathways can be molded by environmental pathogens.

Figure 1. New Nipi alleles isolated in a large scale screen.

(A) Biosort quantification of the fluorescence in wild type and different mutants strains carrying an integrated Pnlp-29::GFP reporter (frIs7) following infection including sta-2(ok1860), nipi-3(fr4), tpa-1(k530) 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 . The Nipi genes identified in the present mutagenesis and in previous studies , , , (Couillault et al. submitted) are represented in red and black respectively.

Figure 2. nipi-4 encodes a pseudokinase required for the induction of nlp-29.

(A) SNP mapping with WGS. The positions of SNP loci on Chromosome V for the fr106 allele are depicted as a XY scatter plot, where the ratio ‘Hawaiian/total number of reads’ for each SNP is represented, as in . The region without Hawaiian SNPs contains the mutation (red arrow). (B) Exon-intron structure of nipi-4, adapted from WormBase (WS220), with the positions of the fr68, fr71, fr99 and fr106 mutations indicated. Also shown is the structure of the pnipi-4::GFP & pnipi-4::NIPI-4 constructs. (C) Biosort quantification of the normalized fluorescence ratio in wild type, sta-2(ok1860) and the 4 nipi-4 alleles fr68, fr71, fr99 and fr106 carrying frIs7 following infection. For this and subsequent figures, see Materials and Methods 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, nipi-4(fr106) and nipi-4(fr106) with a rescuing transgene pnipi-4::NIPI-4, carrying frIs7 following infection.

Figure 3. The nipi-4 gene acts cell autonomously in the epidermis.

(A–E) Expression of nipi-4 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) nipi-4(fr71) and nipi-4(fr71);frEx496 (Pcol-19::NIPI-4) worms strains carrying an integrated Pnlp-29::GFP reporter (frIs7) following infection. The expression of nipi-4 in epidermal cells in the adult rescues the nipi-4 phenotype. Green and red fluorescence is visualized simultaneously with a GFP long pass filter.

Figure 4. The nipi-4 gene is required for the response to infection and wounding.

(A) nipi-4 mutants do not block the induction of nlp-29 expression upon osmotic stress. Biosort quantification of the normalized fluorescence ratio in wild type, sta-2(ok1860) and nipi-4(fr71) worms carrying frIs7 following infection by D. coniospora, wounding, PMA treatment and osmotic stress. (B) Quantitative RT-PCR analysis of gene expression levels in non- infected and infected wild type, sta-2(ok1860) and nipi-4(fr106) worms. The columns show the average expression level (arbitrary units) and SEM from 4 experiments. The level of nlp-34 expression in control animals is set at 1024 (see Materials and Methods).

Figure 5. nipi-4 genetically interacts with the G-protein/PKCδ/p38 MAPK cascade.

(A) The G-protein/PKCδ/p38 MAPK cascade regulates the expression of nlp-29 after infection and wounding. Biosort quantification of the normalized fluorescence ratio in wild type, sta-2(ok1860) and nipi-4(fr106) mutant worms carrying an integrated Pnlp-29::GFP reporter, with or without a transgene carrying an activated form of GPA-12 under the control of an epidermis promoter (Pcol-19::GPA-12*). (B) Images of the wild type strain carrying frIs7 with (+GPA-12*) or without (−GPA-12*) Pcol-19::GPA-12* in control animal (−Dc) or worm infected by D. coniospora (+Dc). Green and red fluorescence is visualized simultaneously. (C) Quantitative RT-PCR analysis of gene expression levels in wild type and nipi-4(fr106) worms with or without Pcol-19::GPA-12*. The columns show the average expression level (arbitrary units) and SEM from 3 experiments. The level of nlp-34 expression in control animals is set at 1024 (see Materials and Methods).