Immunohistochemistry of <i>ogt-1(fosmid)</i> line, with all channels shown.

Representative immunohistochemistry images of dissected males, as shown in Fig 1D, including O-GlcNAc (RL2 antibody, in green), tubulin (anti-TBB-2 antibody, in red), DAPI (in blue), and all three fluorescent channels merged with differential interference contrast (DIC, greyscale). All worms are in the him-5 background. (TIF)</p

Data from analysis of mating videos, data underlying S6 Fig.

(XLSX)</p

Uncropped western blot with <i>ogt-1</i> point mutant lines from Fig 4B.

Left: O-GlcNAc (RL2 antibody), right: actin antibody. Genotypes of strains given above with regard to the ogt-1 and oga-1 genes. gfp = ogt-1(dr84), Δ = deletion (ogt-1(jah01) or oga-1(av82)), H612A = ogt-1(dr91), K957M = ogt-1(dr89). All strains are in the him-5 background. (TIF)</p

Additional behavioral tests of catalytic-dead and hypodermal-rescue <i>ogt-1</i> lines.

(A) Repeat of the mate-response assay, including ogt-1(K957M) and dpy-7p::ogt-1, compared with wild-type and with ogt-1(jah01) by pairwise Fisher’s exact test. Results include five independent replications of the experiment. (B) Scoring of turn quality in videos of males mating with anesthetized fem-1 animals, shown as percent of total turns assessed for each genotype. Good = successful turn with no loss of contact, sloppy = successful turn with loss of contact, missed = failed turn where male loses contact with mate. The experimenter was blinded to the genotype of the males while scoring turns. Statistical comparisons to WT and ogt-1 were performed with pairwise Chi-square analyses. * = pogt-1(K957M) = ogt-1(dr89). All strains are in the him-5 background, and dpy-7p::ogt-1 is additionally in the ogt-1 background. (TIF)</p

Data underlying Fig 4.

(XLSX)</p

One <i>ogt-1</i> allele showed increased incidence of ray development defects.

(A) Percentage of males with normal V6 ray morphology, with total number of observed normal and total males of each genotype. Defects include ray fusions and missing rays. Ratio of normal to deviant males was compared to wild-type with Fisher’s exact test and is shown above. ** = pogt-1(ok1474) line. All males are in the him-5 background. (TIF)</p

<i>ogt-1</i> mutations reduce male fertility and can be rescued by expression in the hypodermis.

(A) 24-hour brood count from self-sterile fem-1 worms mated with males of each specified genotype, shown as individual values overlaid on violin plots with quartiles. Statistical comparisons to him-5 by one-way ANOVA are shown above. Below, a schematic of the three ogt-1 deletion alleles in the context of the OGT-1 protein structure, with the tetracopeptide repeat (TPR) domain in blue and the catalytic domain in bright green. (B) 24-hour brood by various ogt-1 rescue males mating with fem-1. All extrachromosomal array rescue lines are in the deletion background ogt-1(jah01). Statistical comparisons by one-way ANOVA to WT and ogt-1(jah01) are shown above. (C) Western blot of worm lysates demonstrates rescue of anti-O-GlcNAc antibody (RL2) signal in fosmid-rescued line, with anti-actin antibody signal shown for comparison. (D) Representative immunohistochemistry images of dissected males shows O-GlcNAc staining is restored in somatic tissues (intestine, dashed orange line) of the fosmid-rescued line, but not in the germline (gonad, dotted yellow line). (E) 24-hour brood count of hlh-3p::ogt-1 rescue lines by mating with fem-1 with statistical comparisons to WT and ogt-1 by one-way ANOVA shown above. All males in panels B-E are in the him-5 background. * = p<0.05, ** = p<0.01, **** = p<0.0001, ns = not significant.</p

Data underlying Fig 2.

(XLSX)</p

OGT-1 is required for the <i>C. elegans</i> response to <i>S. aureus</i>.

<p><i>O-</i>GlcNAc cycling null nematodes are similarly susceptible to <i>P. aeruginosa</i> exposure as N2 animals: (A) <i>ogt-1(ok1474)</i>, (B) <i>oga-1(tm3642)</i>, (C) <i>ogt-1(ok430)</i>, and (D) <i>oga-1(ok1207)</i>. (A–H) <i>pmk-1(km25)</i> animals are hypersensitive to <i>P. aeruginosa</i> and <i>S. aureus</i>. (A, C) <i>ogt-1; pmk-1</i> and (B, D) <i>oga-1; pmk-1</i> are equally susceptible to <i>P. aeruginosa</i> as <i>pmk-1(km25)</i> single mutants. (E, G) <i>ogt-1</i> animals are hypersensitive to <i>S. aureus</i> while (F, H) <i>oga-1</i> mutants maintain survival similar to N2. (E, G) <i>ogt-1; pmk-1</i> are more susceptible to <i>S. aureus</i> than either <i>ogt-1</i> or <i>pmk-1</i> single mutants and (F, H) <i>pmk-1; oga-1</i> are similarly susceptible to <i>S. aureus</i> as <i>pmk-1</i> single mutants. Results are representative of at least two independent assays and are represented by comprehensive plots. <i>n</i>≧162. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231.s008" target="_blank">Table S1</a> for individual assay statistical analysis.</p

Conserved Nutrient Sensor O-GlcNAc Transferase Is Integral to <i>C. elegans</i> Pathogen-Specific Immunity

<div><p>Discriminating pathogenic bacteria from bacteria used as a food source is key to <i>Caenorhabidits elegans</i> immunity. Using mutants defective in the enzymes of O-linked N-acetylglucosamine (O-GlcNAc) cycling, we examined the role of this nutrient-sensing pathway in the <i>C. elegans</i> innate immune response. Genetic analysis showed that deletion of O-GlcNAc transferase (<i>ogt-1</i>) yielded animals hypersensitive to the human pathogen <i>S. aureus</i> but not to <i>P. aeruginosa</i>. Genetic interaction studies revealed that nutrient-responsive OGT-1 acts through the conserved β-catenin (BAR-1) pathway and in concert with p38 MAPK (PMK-1) to modulate the immune response to <i>S. aureus</i>. Moreover, whole genome transcriptional profiling revealed that O-GlcNAc cycling mutants exhibited deregulation of unique stress- and immune-responsive genes. The participation of nutrient sensor OGT-1 in an immunity module evolutionarily conserved from <i>C. elegans</i> to humans reveals an unexplored nexus between nutrient availability and a pathogen-specific immune response.</p></div