Fig 1.
pnp-1 mutants have increased expression of IPR genes.
A) Gene structure of the two isoforms of pnp-1 with exons indicated as black boxes. 5’ and 3’ untranslated regions are not shown. B-D) pals-5p::gfp IPR reporter expression in wild-type animals, pnp-1(jy90) and pnp-1(jy121) mutants. myo-2p::mCherry is a pharyngeal marker for the presence of the IPR reporter transgene. Scale bar is 100 μm. E) qRT-PCR of a subset of IPR genes in pnp-1(jy90) and pnp-1(jy121) mutants. Fold change in gene expression is shown relative to wild-type animals. Graph shows the mean fold change of three independent experiments. Error bars are standard deviation (SD). Mixed stage populations of animals were used. **** indicates p < 0.0001 by one-tailed t-test. F) Quantification of inosine and hypoxanthine levels in pnp-1 and pals-22 mutants from metabolomics analysis. Graph shows the mean levels of metabolites from six independent experiments for pnp-1(jy121), pnp-1(jy90) and pals-22(jy1) mutants, and five independent experiments for wild-type animals. Error bars are standard error of the mean (SEM). ** indicates p < 0.01 by the Kruskal-Wallis test. E, F) Red dots indicate values from individual experiments. See materials and methods for more information.
Fig 2.
pnp-1 regulates intracellular pathogen resistance.
A) qRT-PCR for Orsay viral load in wild-type animals, pnp-1(jy90), pnp-1(jy121) and pals-22(jy1) mutants. Fold change in gene expression is shown relative to wild-type animals. Graph shows results of three independent experiments. Mean is shown with error bars as SD. Red dots indicate values from individual experiments. Synchronized fourth larval stage (L4) animals were used. **** indicates p < 0.0001 by a one-tailed t-test. B) Quantification of N. parisii sporoplasm number in wild-type animals, pnp-1(jy90), pnp-1(jy121) and pals-22(jy1) first larval stage (L1) mutants at 3 hpi. n = 225 animals per genotype. Box represents 50% of the data closest to the median while whiskers span the values outside the box. C) Quantification of N. parisii pathogen load in wild-type animals, pnp-1(jy90), pnp-1(jy121) and pals-22(jy1) L1 mutants at 30 hpi. n = 300 animals per genotype. N. parisii load per animal was quantified with the COPAS Biosort machine and normalized to time-of-flight as proxy for the length of the animal. Each dot represents an individual animal. Mean is shown with error bars as SD. B, C) N. parisii was visualized using an N. parisii rRNA specific probe. Each graph shows the combined results of three independent experiments. D) Survival of wild-type, pnp-1(jy90) and pnp-1(jy121) mutants after infection with N. parisii. n = 120 per genotype. One experiment of three independent experiments is shown (see S4 Fig for additional two experiments). **** indicates p < 0.0001 by the Log-rank (Mantel-Cox) test. E) Quantification of fluorescent bead accumulation in wild-type animals, pnp-1(jy90), pnp-1(jy121) and eat-2(ad465) mutants. n = 150 animals per genotype. The corrected total fluorescence per worm was calculated and normalized to worm area. Each dot represents an individual animal. B, C and E) **** indicates p < 0.0001 by the Kruskal-Wallis test. Graphs show combined results of three independent experiments.
Fig 3.
pnp-1 functions in the intestine to regulate the IPR.
A) Expression of PNP-1::EGFP::3XFLAG under control of the wild-type pnp-1 genomic locus. Asterisks indicate intestines and arrows indicate neurons. Scale bar is 100 μm. B) Quantification of N. parisii sporoplasm number at 3 hpi in pnp-1 mutants containing the rescuing pnp-1::egfp::3Xflag genomic locus in L1 animals (indicated as "+WT pnp-1"), as well as their non-transgenic siblings (indicated as "-WT pnp-1"). n = 255 animals per genotype. C) Quantification of N. parisii sporoplasm number at 3 hpi in pnp-1 mutants containing wild-type pnp-1 cDNA under the control the vha-6 (intestinal) or unc-119 (neuronal) promoters in L1 animals. n = 150 per genotype. B, C) N. parisii was visualized using an N. parisii rRNA specific probe. Each graph shows the combined results of three independent experiments. In the graphs, the box represents the 50% of the data closest to the median while the whiskers span the values outside the box. **** indicates p < 0.0001 by the Kruskal-Wallis test. D) qRT-PCR of a subset of IPR genes in adult pnp-1 mutants containing wild-type pnp-1 cDNA under the control the vha-6 or unc-119 promoter. Fold change in gene expression is shown relative to control. Graphs show the combined results of three independent experiments. Red dots indicate values from individual experiments. **** indicates p < 0.0001 by a one-tailed t-test.
Fig 4.
Lifespan, thermotolerance and P. aeruginosa resistance phenotypes of pnp-1 mutants.
A) Lifespan of wild-type animals, pnp-1(jy90) and pnp-1(jy121) mutants. n = 90 animals per genotype. Results from one representative experiment of four independent experiments is shown (see S6 Fig for additional three experiments). Survival of each mutant population was compared to that of the wild-type population with the Log-rank (Mantel-Cox) test. B) Survival of wild-type animals, pnp-1(jy90) and pnp-1(jy121) mutants 24 hours after 2 hours 37°C heat shock. C) Survival of wild-type animals, pnp-1(jy90), pals-22(jy1) and pals-22(jy1); pnp-1(jy90) mutants 24 hours after a 2 hour 37°C heat shock. B, C) For one experiment, three plates per genotype with 30 worms per plate were tested. One dot represents the survival from one plate. The graphs show the mean survival of three independent experiments. Error bars are SD. D) Quantification of PA14-dsRED pathogen load in L4 stage wild-type animals, pnp-1(jy90) and pnp-1(jy121) mutants at 16 hpi. E) Quantification of PA14-dsRED pathogen load in L4 stage wild-type animals, pnp-1(jy90), pals-22(jy1), pmk-1(km25), pnp-1(jy90) pmk-1(km25) and pals-22(jy1); pmk-1(km25) mutants at 16 hpi. D, E) PA14-dsRED red fluorescence per animal was quantified with the COPAS Biosort machine and normalized to time-of-flight as proxy for the length of the animal. n = 300 animals per genotype. Graph shows the combined results of three independent experiments. Each dot represents an individual animal. Mean is shown with error bars as SD. B-E) **** indicates p < 0.0001 by the Kruskal-Wallis test.
Fig 5.
RNA-seq analysis demonstrates that pnp-1 represses expression of many IPR genes.
A) Venn diagram of differentially expressed genes in pnp-1(jy90) and pnp-1(jy121) mutants as compared to wild-type animals. Both up (rf = 31.5; p < 0.000e+00) and down (rf = 239.6; p < 3.489e-58) regulated genes have significant overlap between the two mutant alleles. B) Venn diagram of upregulated genes in pnp-1(jy90), pnp-1(jy121) and pals-22(jy3) mutants as compared to wild-type animals. Upregulated genes in both pnp-1(jy90) (rf = 2.9; p < 4.566e-62) and pnp-1(jy121) (rf = 2.8; p < 1.591e-52) mutants have significant overlap with those upregulated in pals-22(jy3) mutants from a previous study [17]. C) Venn diagram of upregulated genes in pnp-1(jy90), pnp-1(jy121) and the IPR genes (defined in [17]). Upregulated genes in both pnp-1(jy90) (rf = 28.8; p < 1.242e-88) and pnp-1(jy121) (rf = 30.9; p < 2.567e-87) mutants have significant with the IPR genes [17]. A-C) rf is the ratio of actual overlap to expected overlap where rf > 1 indicates overrepresentation and rf < 1 indicates underrepresentation (see S7 Table for more detail) D) Log2 fold-change of a subset of pals genes in pnp-1(jy90) and pnp-1(jy121) mutants normalized to wild-type. E) Log2 fold change of a subset of non-pals genes in pnp-1(jy90) and pnp-1(jy121) mutants normalized to wild-type. F) Correlation between genes differentially expressed by various known IPR activators and those differentially expressed in pnp-1(jy90) or pnp-1(jy121) mutants. G) Wormcat analysis for significantly enriched categories in differentially expressed gene sets of pnp-1(jy90) and pnp-1(jy121) mutants. Size of the circles indicates the number of the genes and color indicates value of significant over representation in each Wormcat category. H) Correlation of genes differentially expressed by bacterial pathogens, immune regulators, and various stressors to those differentially expressed in pnp-1(jy90) or pnp-1(jy121) mutants. F, H) Analysis was performed using GSEA 3.0 software, and correlations of genes sets (S5 Table) were quantified as a Normalized Enrichment Score (NES) (S6 Table). NES’s presented in a heat map. Blue indicates significant correlation of downregulated genes in a pnp-1 mutant with the tested gene set, yellow indicates significant correlation of upregulated genes in a pnp-1 mutant with the tested gene set, while black indicates no significant correlation (p > 0.05 or False Discovery Rate < 0.25).
Fig 6.
Model for pnp-1 regulation of immune responses.
A) pnp-1 negatively regulates mRNA expression of IPR genes induced by infection with the intracellular pathogens the Orsay virus and N. parisii (microsporidia). Loss of pnp-1 or pals-22 results in constitutive expression of IPR genes and resistance to the Orsay virus and N. parisii. Epistasis analysis indicates that pnp-1 acts in parallel to pals-22/pals-25. pmk-1 (p38 MAPK) mutants display increased susceptibility to N. parisii as compared to wild-type animals. Because IPR genes are distinct from pmk-1-regulated genes, we favor a model where pmk-1 acts in parallel to the IPR. B) pnp-1 negatively regulates genes that are induced by various extracellular pathogens and pnp-1 mutants are resistant to infection by the extracellular Gram-negative pathogen P. aeruginosa. Resistance to P. aeruginosa in pnp-1 mutants requires pmk-1. In addition, upregulation of genes induced by wild-type pmk-1 in pnp-1 mutants requires functional pmk-1, suggesting that here, pnp-1 functions upstream of pmk-1.