The transcription factor DAF-12 is activated in <i>irg-7(zc6)</i> mutants.

<p>(A-B) The <i>irg-7(zc6)</i> mutation is sufficient to increase the expression of the <i>Pcdr-6</i>::<i>gfp</i> reporter (Student's t-test, p<0.0001) in a <i>daf-12(+)</i> background but not in a <i>daf-12(-)</i> background (Student's t-test, p = 0.075). Asterisks mark Student's t-test values of P<0.001 compared to the fluorescence in wild-type animals. 30–35 animals analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) The <i>irg-7(zc6)</i> mutation is not sufficient to increase the expression of the DAF-16 reporter <i>Psod-3</i>::<i>gfp</i> (Student's t-test, P = 0.067). (D) The <i>irg-7(zc6)</i> mutation is not sufficient for the accumulation of DAF-16::GFP translational reporter in the intestine.</p

<i>irg-7(zc6)</i> is a gain of function mutation in F40F4.6.

<p>(A) <i>F40F4</i>.<i>6</i> RNAi significantly shortened the lifespan of <i>irg-7(zc6)</i> mutants (Mantel-Cox, P<0.0001) but did not affect the lifespan of otherwise wild-type animals (Mantel-Cox,P = 0.9). Mean lifespans are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s004" target="_blank">S2 Table</a></b> for more lifespan data. (B) Representative fluorescence micrographs (100-fold magnification) of day-1 adults harboring an integrated <i>Phsp-4</i>::<i>gfp</i> transgene. <i>F40F4</i>.<i>6</i> RNAi significantly reduced the expression of the <i>Phsp-4</i>::<i>gfp</i> in <i>zc6</i> mutants. Asterisks mark Student's T-test values of P<0.001 compared to fluorescence on control RNAi. 40 animals were analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) Expression of fosmid WRM0619aB08 (which includes F40F4.6) significantly extended the lifespan of wild-type animals (Mantel-Cox, P<0.001), whereas expression of the partially overlapping fosmid WRM0635bF05 (which does not include F40F4.6) did not extend the lifespan of wild-type animals (Mantel-Cox, P = 0.4). Mean lifespans are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s004" target="_blank">S2 Table</a></b> for more lifespan data. (D) Expression of an NLS-RFP reporter fused to the putative promoter upstream of the F40F4.6 gene drives expression in the posterior cells of the intestine. Fluorescence of this reporter increased upon exposure of L4 animals to Photorhabdus luminescens subsp Hb bacteria (HB) or Enterococcus faecalis bacteria (EF). Asterisks mark Student's T-test values of P<0.001 compared to wild-type fluorescence on OP50 bacteria. (E) Schematic representation of the major domains in the F40F4.6 protein. The region deleted by the <i>zc6</i> mutation is indicated by a dashed line. This region includes an EGF domain. Note that the deletion preserves the ORF of the original gene. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s002" target="_blank">S2 Fig</a> for sequence data.</p

Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7

<div><p>In <i>C</i>. <i>elegans</i>, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named <i>irg-7</i>, as a novel mediator of longevity in germlineless animals. We consider <i>irg-7</i> to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, <i>irg-7</i> activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks.</p></div

<i>irg-7(zc6)</i> mutation extends lifespan by increasing pathogen resistance.

<p>(A) Feeding animals with dead OP50 bacteria extended the lifespan of wild-type animals (Mantel-Cox, P<0.001), but did not extend the lifespan of <i>irg-7(zc6)</i> mutants (Mantel-Cox, P = 0.53). (B) The <i>irg-7(zc6) gof</i> mutation improved the survival of animals fed with pathogenic HB bacteria (Mantel-Cox, P<0.0001). (C-D) <i>irg-7</i> inactivation by pre-treatment with F40F4.6 RNAi from eggs to early adulthood hindered survival of animals fed henceforth with pathogenic HB bacteria (C) (Mantel-Cox, P<0.0001), but did not affect the survival of animals fed henceforth with OP50 bacteria (D) (Mantel-Cox, P = 0.7). (E) <i>irg-7</i> inactivation by pre-treatment with F40F4.6 RNAi from eggs to early adulthood did not affect the survival of <i>daf-16(mu86)</i> mutants fed henceforth with HB bacteria (Mantel-Cox, P = 0.43). Mean lifespan are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s005" target="_blank">S3 Table</a></b> for additional lifespan data.</p

The reproductive innate immunity pathway model.

<p>Pathogens are one of the factors that limit lifespan in <i>C</i>. <i>elegans</i> and in higher organisms. Our data implies that exposure to pathogens and/or their toxins can directly or indirectly modulate germline proliferation and survival resulting in a reproductive system with less germ cells. Germline depletion induces the transcription of <i>irg-7</i>, an innate immunity-related secreted protein whose induction promotes the activation of a somatic defense response. On one hand, <i>irg-7</i> enables the activation of bona-fida longevity-associated transcription factors, which support the induction of a longevity-promoting transcriptome. At the same time, <i>irg-7</i> enables the activation of <i>atf-7</i>, a dedicated innate immunity-related transcription factor. In this way, exposure to pathogens can be sensed by perturbations in germline homeostasis. Consequently, the reproductive system can serve as a signaling center to divert key metabolic resources towards defending against the putative pathogen attack by activating the innate immune response.</p

<i>irg-7</i> is part of the germline longevity pathway.

<p><i>F40F4</i>.<i>6</i> RNAi significantly shortened the lifespan of <i>glp-1</i> germlineless animals, but did not shorten the long lifespan of insulin/IGF1 signaling mutants (<i>daf-2</i>), dietary restricted mutants (<i>eat-2)</i>, mutants in mitochondrial respiration mutants (<i>clk-1</i>) or wild-type animals. Mean lifespans and Mantel-Cox P values are indicated within each graph. Mantel Cox P-values for control RNAi vs. <i>irg-7</i> RNAi-treated wild-type animals are in gray. Mantel Cox P-values for control RNAi vs. <i>irg-7</i> RNAi-treated long-lived mutants are in blue. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s007" target="_blank">S5 Table</a></b> for more lifespan data.</p

Sensitivity of germline homeostasis to pathogenic HB bacteria improves the animals' survival.

<p>(A) Treatment with pathogenic HB bacteria induced germline apoptosis in an <i>egl-1</i> dependent manner. Asterisks mark Mann-Whitney P values of P<0.001 of HB-treated animals (gray) compared to OP50 treated animals (black) of the same genotype. 45–50 animals were analyzed per genotype. Error bars represent SD. (B) Amount of mitotic germ cells was determined in wild-type animals and in animals with activated <i>irg-7</i> upon exposure to OP50 (black) or HB bacteria (gray). Asterisks mark Mann-Whitney values of P<0.001 of HB-treated animals (gray) compared to OP50 treated animals (black) of the same genotype. Additional Mann-Whitney P values are indicated in the graph. Note that treatment with pathogenic HB bacteria reduced the amount of mitotic germ cells in the gonad of wild-type animals but not in animals with activated <i>irg-7</i>. Also note that activation of <i>irg-7</i> did not reduce, and even increased, the amount of mitotic germ cells in the animals. 25–30 DAPI-stained gonads were analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) In the absence of pathogen-induced germline apoptosis, animals are more sensitive to the pathogenic bacteria. Mean survival and Mantel-Cox P-values are indicated within the graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s009" target="_blank">S7 Table</a></b> for additional survival data.</p

<i>irg-7</i> is required for DAF-12 and DAF-16 activation in germline-less animals.

<p>(A) Expression of an NLS-RFP reporter fused to the promoter of upstream of the <i>F40F4</i>.<i>6</i> gene is induced in <i>irg-7(zc6)</i> mutants and upon germ cell removal (<i>glp-1</i>). Asterisks mark Student's T-test values of P<0.005 compared to the fluorescence in wild-type animals. 30 animals were analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (B) F40F4.6 RNAi reduced the expression of the DAF-16 and DAF-12 reporters <i>Psod-3</i>::<i>gfp</i> and <i>Pcdr-6</i>::<i>gfp</i> in germline-less animals, but did not affect the expression of the PHA-4 and NHR-80 reporters <i>Plgg-1</i>::<i>gfp</i> and <i>Pfat-6</i>::<i>gfp</i>. Asterisks mark Mann-Whitney P values <0.0001 compared to the fluorescence in control RNAi-treated animals. 30 animals were analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) <i>F40F4</i>.<i>6</i> RNAi reduced DAF-16::GFP nuclear translocation in germline-less animals (left panels) but not in <i>daf-2(e1370)</i> mutants (right panels). </p

<i>irg-7(zc6)</i> mutation extends lifespan independently of UPR genes.

<p>(A) Representative fluorescence micrographs (100-fold magnification) of day-1 adults harboring an integrated <i>Phsp-4</i>::<i>gfp</i> transgene. <i>irg-7(zc6)</i> mutants expressed higher levels of the <i>Phsp-4</i>::<i>gfp</i> than did wild type animals, predominantly in the posterior end of the intestine. Asterisks mark Student's T-test values of P<0.001 compared to wild-type fluorescence. 50 animals were analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (B) The <i>irg-7(zc6)</i> mutation significantly extended the lifespan of otherwise wild-type, <i>pek-1(ok275)</i>, <i>atf-6(ok551)</i> or <i>ire-1(RNAi)</i> animals. Mantel Cox P-value for the wild type vs. <i>irg-7(zc6)</i> single mutant comparison is in green. Mantel Cox P-value for the mutant vs. the mutant; <i>irg-7(zc6)</i> double mutant comparison is in black. Mean lifespan and P-values are indicated within each graph. <b>See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s003" target="_blank">S1 Table</a></b> for additional lifespan data.</p

The innate immunity-related transcription factor ATF-7 promotes longevity and pathogen resistance in <i>irg-7(zc6)</i> mutants.

<p>(A) The <i>irg-7(zc6)</i> mutation increases the expression of the <i>T24B8</i>.<i>5</i>::<i>gfp atf-7</i> target gene reporter (Mann-Whitney, P<0.0001). This increased expression by <i>irg-7(zc6)</i> is similar to that induced by exposure to HB or EF bacteria (Mann-Whitney, P = 1.00 and P = 0.98 respectively). Asterisks mark Mann-Whitney values <0.001 compared to the fluorescence in wild-type animals on OP50 bacteria. 30 animals were analyzed per genotype. Error bars represent SE of 3–5 independent biological replicates. (B) The <i>irg-7(zc6)</i> mutation did not extend the lifespan of animals treated with <i>atf-7</i> RNAi (Mantel-Cox, P = 0.07). Mean survival is indicated within the graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s006" target="_blank">S4 Table</a></b> for additional lifespan data. (C) <i>atf-7(gk715); irg-7(zc6)</i> double mutants animals did not survive on HB bacteria better than <i>atf-7(gk715)</i> single mutants (Mantel-Cox, P = 0.9). Mean survival is indicated within the graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s006" target="_blank">S4 Table</a></b> for additional survival data. (D) Representative fluorescence micrographs (100-fold magnification) of day-1 adults harboring an integrated <i>Phsp-4</i>::<i>gfp</i> transgene. <i>atf-7</i> and <i>pmk-1</i> RNAi significantly reduced the expression of the <i>Phsp-4</i>::<i>gfp</i> in <i>irg-7(zc6)</i> mutants. Asterisks mark Student's T-test values of P<0.002 compared to fluorescence on control RNAi. 30 animals were analyzed per genotype. Error bars represent SE of 3 independent biological replicates.</p