Correction: Surveillance-Activated Defenses Block the ROS-Induced Mitochondrial Unfolded Protein Response

Abstract

[This corrects the article DOI: 10.1371/journal.pgen.1003346.].

Fig 6. Activities of rpl-36 and atfs-1 are required for the hsp-6 response to paraquat.

Representative micrographs (A) and quantification of GFP fluorescence intensity (B) of two screening positives (rpl-36, atfs-1) show a block of the paraquat triggered induction of the hsp-6 reporter (Phsp-6::gfp) after their RNAi. Worms were raised on respective RNAi plates from L1 larval stage and exposed to 0.5 mM paraquat at early L3 stage. GFP fluorescence was analyzed after two days. Columns represent pooled normalized values of three independent experiments plus standard error of the mean (SEM). Numbers in or on columns indicate the number of analyzed animals (n_total = 485). ***: p<0.001; Kruskal-Wallis test plus Dunn's Multiple Comparison Test; Mann Whitney test. Equal optical settings, scale bar 200 μm. (i): RNAi; L4440: empty vector control.

Fig 7. Knockdown of rpl-36 and atfs-1 suppresses the isp-1(qm150) mediated induction of the hsp-6 reporter.

The isp-1(qm150) mutant has increased levels of mitochondrial superoxide [17] and constitutively activated the Phsp-6 reporter (Phsp-6::gfp). RNAi of rpl-36 and atfs-1 suppressed (p<0.001) the constitutive hsp-6 reporter gene induction. Representative micrographs (A) and quantification of GFP fluorescence intensity (B). hsp-6 reporter worms carrying the qm150 allele were analyzed for GFP expression after one week on the respective RNAi plates. Columns represent pooled values of three independent experiments plus standard error of the mean (SEM). Numbers in columns indicate the number of analyzed animals (n_total = 244). ***: p<0.001; Kruskal-Wallis test plus Dunn's Multiple Comparison Test. Equal optical settings per row, scale bar 100 μm. (i): RNAi; L4440: empty vector control.

Fig 8. The downregulation of rpl-36 and atfs-1 increases paraquat sensitivity.

L1 staged N2 worms were placed on the respective RNAi plates containing 0.4 mM or no paraquat, development was analyzed five days later. Downregulation of both genes enhanced sensitivity towards paraquat, indicated by delayed development. Columns represent pooled values of three independent experiments in percent. Numbers on columns indicate the number of animals analyzed (n_total = 2004). (i): RNAi; L4440: empty vector control.

Fig 9. The knockdown of rpl-36 and atfs-1 does not prevent non-mitochondrial stress responses.

Worms were grown from L1 larval stage on the respective RNAi plates before being exposed to the respective stress and analyzed four days after L1. A. A reporter strain for the SKN-1 dependent phase II response (Pgst-4::gfp) was exposed to 2.1 mM acrylamide starting at early L3 stage. RNAi of rpl-36 and atfs-1 did not prevent reporter gene induction as compared to vector control. Columns represent pooled normalized values of four independent experiments plus standard error of the mean (SEM). Numbers in columns indicate the number of analyzed animals (n_total = 613). ***: p<0.001; Kruskal-Wallis test plus Dunn's Multiple Comparison Test; Mann Whitney test (comparison of vector with and without acrylamide). Equal optical settings, scale bar 200 μm. B. Cytosolic UPR (heat shock) reporter worms (Phsp-16.2::gfp) were exposed to 34°C for 4h at L4. The knockdown of rpl-36 and atfs-1 significantly decreased heat stress induced reporter expression (p<0.001). Columns represent pooled normalized values of two independent experiments plus standard error of the mean (SEM). Numbers in or on columns indicate the number of analyzed animals (n_total = 266). ***: p<0.001; Kruskal-Wallis test plus Dunn's Multiple Comparison Test; Mann Whitney test (comparison of vector with and without heat shock). Equal optical settings, scale bar 100 μm. C. The UPR^ER reporter strain (Phsp-4::gfp) was raised from L1 stage RNAi plates (with 7.2 μg/ml tunicamycin). UPR^ER induction was not blocked by any RNAi tested here, but rpl-36 (RNAi) strongly impaired its induction (p<0.001). Columns represent pooled normalized values of four independent experiments plus standard error of the mean (SEM). Numbers in or on columns indicate the number of analyzed animals (n_total = 363). ***: p<0.001; Kruskal-Wallis test plus Dunn's Multiple Comparison Test; Mann Whitney test (comparison of vector with and without tunicamycin). Equal optical settings, scale bar 100 μm. (i): RNAi; L4440: empty vector control.

Fig 10. cSADDs inhibits paraquat mediated signaling to hsp-6 through KGB-1.

A-B. In kgb-1(um3) mutant animals, which are cSADDs deficient, paraquat induced hsp-6 induction is not blocked by elt-2 RNAi. Thus, ROS induced UPR^mt is activated in the absence of functional cSADDs. In contrast, kgb-1(um3) does not prevent inhibition of hsp-6 induction by atfs-1 knockdown, suggesting that it functions downstream of kgb-1 and the cSADDs. Columns represent normalized values plus standard error of the mean (SEM). Numbers in or on columns indicate the number of analyzed animals (n_total = 181). ***: p<0.001, *: p<0.05; Kruskal-Wallis test plus Dunn's Multiple Comparison Test (A). Equal optical settings, scale bar 400 μm. (i): RNAi; L4440: empty vector control; +: wild type allele (B). C. Model: Genes activating the cSADDs (cellular surveillance system) inhibit the paraquat-triggered induction of the UPR^mt.