Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated induced pluripotent stem cells (iPSCs) from people carrying insulin (INS) mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation into mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunits expression were all reduced in INS-mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development.

Cuticular defects trigger a battery of reactions including enhanced reactive oxygen species (ROS) production and resistance to necrotrophic pathogens. However, the source of ROS generated by such impaired cuticles has remained elusive. Here, we report the characterization of Arabidopsis thaliana ohy1 mutant, a Peroxidase 57 (PER57) – overexpressing line that demonstrates enhanced defense responses that result both from increased accumulation of ROS and permeability of the leaf cuticle. The ohy1 mutant was identified in a screen of A. thaliana seedlings for oligogalacturonides (OGs) insensitive/hypersensitive mutants that exhibit altered growth retardation in response to exogenous OGs. Mutants impaired in OG sensitivity were analyzed for disease resistance/susceptibility to the necrotrophic phytopathogens Botrytis cinerea and Pectobacterium carotovorum. In the ohy1 line, the hypersensitivity to OGs was associated with resistance to the tested pathogens. This PER57 overexpressing line exhibited a significantly more permeable leaf cuticle than wild-type plants and this phenotype could be recapitulated by overexpressing other class III peroxidases. Such peroxidase overexpression was accompanied by the suppressed expression of cutin biosynthesis genes and the enhanced expression of genes associated with OG-signaling. Application of ABA completely removed ROS, restored the expression of genes associated with cuticle biosynthesis and led to decreased permeability of the leaf cuticle, and finally, abolished immunity to B. cinerea. Our work demonstrates that increased peroxidase activity increases permeability of the leaf cuticle. The loss of cuticle integrity primes plant defenses to necrotrophic pathogens via the activation of DAMP-responses.

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EFR is a plasma-membrane resident receptor responsible for recognition of microbial elongation factor Tu (EF-Tu) and thus triggering plant innate immunity to fend off phytopathogens. Functional EFR must be subject to the endoplasmic reticulum quality control (ERQC) machinery for the correct folding and proper assembly in order to reach its final destination. Genetic studies have demonstrated that ERD2b, a counterpart of the yeast or mammalian HDEL receptor ERD2 for retaining proteins in the endoplasmic reticulum (ER) lumen, is required for EFR function in plants (Li et al., 2009). In this study, we characterized the Arabidopsis glucosidase II beta-subunit via the HDEL motif against the non-redundant protein database. Data mining also revealed that the glucosidase II beta-subunit gene has a highly similar expression pattern to ERD2b and the other known ERQC components involved in EFR biogenesis. Importantly, the T-DNA insertion lines of the glucosidase II beta-subunit gene showed that EFR-controlled responses were substantially reduced or completely blocked in these mutants. The responses include seedling growth inhibition, induction of marker genes, MAP kinase activation, and callose deposition, triggered by peptide elf18, a full mimic of EF-Tu. Taken together, our data indicate a requirement of the glucosidase II beta-subunit for EFR function.