The insertion of organellar membrane proteins with the correct topology requires the following: First, the proteins must contain topogenic signals for translocation across and insertion into the membrane. Second, proteinaceous complexes in the cytoplasm, membrane, and lumen of organelles are required to drive this process. Many complexes required for the intracellular distribution of membrane proteins have been described, but the signals and components required for the insertion of plastidic β-barrel-type proteins into the outer membrane are largely unknown. The discovery of common principles is difficult, as only a few plastidic β-barrel proteins exist. Here, we provide evidence that the plastidic outer envelope β-barrel proteins OEP21, OEP24, and OEP37 from pea (Pisum sativum) and Arabidopsis thaliana contain information defining the topology of the protein. The information required for the translocation of pea proteins across the outer envelope membrane is present within the six N-terminal β-strands. This process requires the action of translocon of the outer chloroplast (TOC) membrane. After translocation into the intermembrane space, β-barrel proteins interact with TOC75-V, as exemplified by OEP37 and P39, and are integrated into the membrane. The membrane insertion of plastidic β-barrel proteins is affected by mutation of the last β-strand, suggesting that this strand contributes to the insertion signal. These findings shed light on the elements and complexes involved in plastidic β-barrel protein import.

Arabidopsis thaliana polyamine oxidase 5 gene (AtPAO5) functions as a thermospermine (T-Spm) oxidase. Aerial growth of its knock-out mutant (Atpao5-2) was significantly repressed by low dose(s) of T-Spm but not by other polyamines. To figure out the underlying mechanism, massive analysis of 3’-cDNA ends was performed. Low dose of T-Spm treatment modulates more than two fold expression 1,398 genes in WT compared to 3186 genes in Atpao5-2. Cell wall, lipid and secondary metabolisms were dramatically affected in low dose T-Spm-treated Atpao5-2, in comparison to other pathways such as TCA cycle-, amino acid- metabolisms and photosynthesis. The cell wall pectin metabolism, cell wall proteins and degradation process were highly modulated. Intriguingly Fe-deficiency responsive genes and drought stress-induced genes were also up-regulated, suggesting the importance of thermospermi’ne flux on regulation of gene network. Histological observation showed that the vascular system of the joint part between stem and leaves was structurally dissociated, indicating its involvement in vascular maintenance. Endogenous increase in T-Spm and reduction in H2O2 contents were found in mutant grown in T-Spm containing media. The results indicate that T-Spm homeostasis by a fine tuned balance of its synthesis and catabolism is important for maintaining gene regulation network and the vascular system in plants.

Proper and size selective blood filtration in the kidney depends on an intact morphology of podocyte foot processes. Effacement of interdigitating podocyte foot processes in the glomeruli causes a leaky filtration barrier resulting in proteinuria followed by the development of chronic kidney diseases. Since the function of the filtration barrier is depending on a proper actin cytoskeleton, we studied the role of the important actin-binding protein palladin for podocyte morphology. Podocyte-specific palladin knockout mice on a C57BL/6 genetic background (PodoPalldBL/6-/-) were back crossed to a 129 genetic background (PodoPalld129-/-) which is known to be more sensitive to kidney damage. Then we analyzed the morphological changes of glomeruli and podocytes as well as the expression of the palladin-binding partners Pdlim2, Lasp-1, Amotl1, ezrin and VASP in 6 and 12 months old mice. PodoPalld129-/- mice in 6 and 12 months showed a marked dilatation of the glomerular tuft and a reduced expression of the mesangial marker protein integrin α8 compared to controls of the same age. Furthermore, ultrastructural analysis showed significantly more podocytes with morphological deviations like an enlarged sub-podocyte space and regions with close contact to parietal epithelial cells. Moreover, PodoPalld129-/- of both age showed a severe effacement of podocyte foot processes, a significantly reduced expression of pLasp-1 and Pdlim2, and significantly reduced mRNA expression of Pdlim2 and VASP, three palladin-interacting proteins. Taken together, the results show that palladin is essential for proper podocyte morphology in mice with a 129 background.

Multi-drug resistant (MDR), gram-negative Enterobacteriaceae, such as Escherichia coli (E. coli) limit therapeutic options and increase morbidity, mortality, and treatment costs worldwide. They pose a serious burden on healthcare systems, especially in developing countries like Rwanda. Several studies have shown the effects caused by the global spread of extended-spectrum beta-lactamase (ESBL)-producing E. coli. However, limited data is available on transmission dynamics of these pathogens and the mobile elements they carry in the context of clinical and community locations in Sub-Saharan Africa. Here, we examined 120 ESBL-producing E. coli strains from patients hospitalized in the University Teaching Hospital of Butare (Rwanda), their attending caregivers as well as associated community members and livestock. Based on whole-genome analysis, the genetic diversification and phylogenetics were assessed. Moreover, the content of carried plasmids was characterized and investigated for putative transmission among strains, and for their potential role as drivers for the spread of antibiotic resistance. We show that among the 30 different sequence types (ST) detected were the pandemic clonal lineages ST131, ST648 and ST410, which combine high-level antimicrobial resistance with virulence. In addition to the frequently found resistance genes bla CTX-M-15 , tet(34), and aph(6)-Id, we identified csg genes, which are required for curli fiber synthesis and thus biofilm formation. Numerous strains harbored multiple virulence-associated genes (VAGs) including pap (P fimbriae adhesion cluster), fim (type I fimbriae) and chu (Chu heme uptake system). Furthermore, we found phylogenetic relationships among strains from patients and their caregivers or related community members and animals, which indicates transmission of pathogens. Also, we demonstrated the presence and potential transfer of identical/similar ESBL-plasmids in different strains from the Rwandan setting and when compared to an external plasmid. This study highlights the circulation of clinically relevant, pathogenic ESBL-producing E. coli among patients, caregivers and the community in Rwanda. Combining antimicrobial resistance with virulence in addition to the putative exchange of mobile genetic elements among bacterial pathogens poses a significant risk around the world.

Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.

Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification.