(A) Mitochondrial proteostatic stress was triggered by overexpression of a misfolding mitochondrial protein (ΔOTC) or knockdown of the complex I subunit ND75, as previously reported. The ubiquitous DaGS::GeneSwitch driver was activated for 24 hr at day 7 of adulthood, and female thoracic tissue collected for RNAseq analysis. Venn and bar diagrams show genes upregulated 3-fold/downregulated 2-fold with both treatments relative to controls, and with control FKPM > 10. Full raw data found in Supplementary File 1. (B) Gene Ontology analysis of the shared stress response; upregulated genes include chaperones, stress pathways, and antimicrobial peptides/innate immune genes. Redundant GO terms were trimmed using REVIGO. (C) Mitochondrial proteostatic stress response genes from RNAseq analysis verify by qPCR and are not activated by heat shock or induction of the ER-UPR through RNAi of Hrd1. p-values for ΔOTC ± RU486 are 0.109, 0.130, 0.255 and 0.008. (D) Knocking down the transcription factors FoxO or Relish, or the JNK kinase, during ΔOTC expression blocks induction of response genes. The kinase ASK1 and the mitochondrial membrane protein PGAM5 are also required for the mitochondrial proteostatic stress response. See supplementary for verification of additional UPRmt genes. p-values relative to ΔOTC + RU are (left to right)<0.0001,<0.0001,<0.0001,<0.0001,<0.0001, 0.0185 and 0.9888; 0.002,<0.0001,<0.0001, and <0.0001. (E) Proposed signaling pathway for immune activation by mitochondrial proteostatic stress. All error bars are SEM of 3+ independent experiments. ANOVA with Dunnett’s multiple comparisons test; *p<0.05, **p<0.01, then each *=0.1 x.. 200 µM RU486 was used in all experiments.