CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNPbased cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.

Das Zytosol ist der Hauptort der Proteinbiosynthese. Während viele Proteine im Zytosol bleiben, muss ein Großteil zu unterschiedlichen Kompartimenten der Zelle transportiert werden. Die korrekte Lokalisation der Polypeptide ist essentiell für die Homöostase der Zelle. Werden Proteine fehlgeleitet oder gar nicht transportiert, können diese in der Zelle aggregieren, was zu Stress bis hin zum Zelltod führen kann. Obwohl der Import mitochondrialer Proteine über die verschiedenen Membranen der Mitochondrien sehr gut erforscht ist, war lange unklar, wie diese Proteine zu ihrem Zielorganell transportiert werden. In den letzten Jahren wurde diese Wissenslücke teilweise gefüllt, neue zytosolische Faktoren wurden identifiziert und alternative Transportwege aufgedeckt. Eine solche Entdeckung war der Transportweg namens ER-SURF. Hier werden mitochondriale Proteine an die Membran des endoplasmatischen Retikulums transportiert, wo sie vom Co-Chaperon Djp1 gebunden und zu den Mitochondrien gebracht werden. Im Zuge der Studie zu ER-SURF wurde ein Protein identifiziert, das bisher noch uncharakterisiert war. Dieses Protein nannten wir Ema19 („Efficient Mitochondria Targeting–Associated Protein 19”). Es ist ein Membranprotein des endoplasmatischen Retikulums, das vier Transmembrandomänen besitzt. Ziel dieses Projekts war es, die Funktion von Ema19 für die Zelle zu analysieren. Durch ein Alignment konnte ich feststellen, dass das Protein bis in den Menschen hoch konserviert ist, was auf eine wichtige Rolle für die Zelle schließen ließ. Da Ema19 im Zusammenhang mit dem ER-SURF Transportweg identifiziert wurde, habe ich zunächst eine mögliche Rolle für den Transport und Import mitochondrialer Proteine in unterschiedlichen Experimenten getestet. Im Laufe der Arbeit wurde jedoch deutlich, dass Ema19 keine direkte Rolle beim Import von mitochondrialen Proteinen spielt. Allerdings konnte ich durch mehrere unabhängige Versuche einen Zusammenhang mit der Lokalisation und dem Abbau mitochondrialer Proteine feststellen. Fehlt Ema19 in der Zelle, ist vor allem das mitochondriale Protein Oxa1 mehr am endoplasmatischen Retikulum vorzufinden. Ebenso konnte ich feststellen, dass Oxa1, sowie das Intermembranraumprotein Erv1, langsamer abgebaut werden als in Wildtypzellen. Diese Experimente geben erste Hinweise auf eine mögliche Rolle von Ema19 für den Abbau mitochondrialer Proteine an der ER-Membran. Nichtsdestotrotz bleiben noch viele Fragen offen und weitere Versuche sind nötig, um diese Hypothese weiter zu unterstützen.

The number of sequenced genomes increases rapidly due to the development of faster, better and new technologies. Thus, there is a great interest in automation, and standardization of the subsequent processing and analysis stages of the generated enormous amount of data. In the current work, genomes of clones, strains and species of Streptococcus were compared, which were sequenced, annotated and analysed with several technologies and methods. For sequencing, the 454- and Illumina-technology were used. The assembly of the genomes mainly was performed by the gsAssembler (Newbler) of Roche, the annotation was performed by the annotation pipeline RAST, the transfer tool RATT or manually. Concerning analysis, sets of deduced proteins of several genomes were compared to each other and common components, the so-called core-genome, of the used genomes of one or closely related species determined. Detailed comparative analysis was performed for the genomes of isolates of two clones to gather single nucleotide variants (SNV) within genes. This work focusses on the pathogenic organism Streptococcus pneumoniae. This species is a paradigm for transformability, virulence and pathogenicity as well as resistance mechanisms against antibiotics. Its close relatives S. mitis, S. pseudopneumoniae and S. oralis have no pathogenicity potential as high as S. pneumoniae available and are thus of high interest to understand the evolution of S. pneumoniae. Strains of two S. pneumoniae clones were chosen. One is the ST10523 clone, which is associated with patients with cystic fibrosis and is characterized by long-term persistence. This clone is lacking an active hyaluronidase, which is one of the main virulence factors. The lack of two phage clusters possibly contributed to the long persistence in the human host. The clone ST226 shows a high penicillin resistance but interestingly one strain is sensitive against penicillin. Here it could be seen that the penicillin resistance mainly arose from the presence of mosaic-PBPs, while special alleles of MurM and CiaH - both genes are associated with penicillin-resistance – were present in resistant and sensitive strains as well. Penicillin resistance of S. pneumoniae is the result of horizontal gene transfer, where DNA of closely related species, mainly S. mitis or S. oralis, served as donor. The transfer of DNA from the high-level penicillin-resistant strain S. oralis Uo5 to the sensitive strain S. pneumoniae R6 was intentioned to reveal the amount of transferred DNA and whether it is possible to reach the high resistance level of S. oralis Uo5. Altogether, about 19kb of S. oralis DNA were transferred after three successive transformation steps, about 10-fold less than during transfer from S. mitis, which is more closely related to S. pneumoniae, as donor. MurE was identified as new resistance determinant. Since the resistance level of the donor strain could not be reached, it is assumed, that further unknown factors are present which contribute to penicillin resistance. The comparison of S. pneumoniae and its close relatives was performed using deduced protein sequences. 1.041 homologous proteins are common to the four complete genomes of S. pneumoniae R6, S. pseudopneumoniae IS7493, S. mitis B6 and S. oralis Uo5. Most of the virulence and pathogenicity factors described for S. pneumoniae could also be found in commensal species. These observations were confirmed by further investigations by Kilian et al. (Kilian, et al., 2019). After adding 26 complete S. pneumoniae genomes to the analysis, only 104 gene products could be identified as specific for this species. Investigations of a larger number of related streptococci, which were isolated from human and several primates, confirmed the presence of most of the virulence factors of human pneumococci in S. oralis and S. mitis strains from primates. While NanBC is common among S. pneumoniae and is missing in all S. oralis, all S. oralis contain a ß-N-acetyl-hexosaminidase which vice versa is missing in S. pneumoniae. The occurrence of S. oralis also in free-living chimpanzees suggests the assumption, that this species is part of the commensal flora of these Old-World monkeys unlike S. pneumoniae which has evolved with its human host. Compared to S. pneumoniae, S. oralis shows an amazing variability in factors important for biosynthesis of peptidoglycan and teichoic acid (PBP, MurMN, lic-cluster). Some streptococci contain a second PGP3 homologue. Additional analyses with further isolates, especially of wild animals, are necessary to determine host-specific components.

Cell division and cell elongation are fundamental processes for growth. In contrast to animal cells, plant cells are surrounded by rigid walls and therefore loosening of the wall is required during elongation. On the other hand, vacuole size has been shown to correlate with cell size and inhibition of vacuolar expansion limits cell growth. However, the specific role of the vacuole during cell elongation is still not fully resolved. Especially the question whether the vacuole is the leading unit during cellular growth or just passively expands upon water uptake remains to be answered. Here, we review recent findings about the contribution of the vacuole to cell elongation. In addition, we also discuss the connection between cell wall status and vacuolar morphology. In particular, we focus on the question whether vacuolar size is dictated by cell size or vice versa and share our personnel view about the sequential steps during cell elongation.

Cells depend on the continuous renewal of their proteome composition during the cell cycle and in order to replace aberrant proteins or to react to changing environmental conditions. In higher eukaryotes, protein synthesis is achieved by up to five million ribosomes per cell. With the fast kinetics of translation, the large number of newly made proteins generates a substantial burden for protein homeostasis and requires a highly orchestrated cascade of factors promoting folding, sorting and final maturation. Several of the involved factors directly bind to translating ribosomes for the early processing of emerging nascent polypeptides and the translocation of ribosome nascent chain complexes to target membranes. In plant cells, protein synthesis also occurs in chloroplasts serving the expression of a relatively small set of 60–100 protein-coding genes. However, most of these proteins, together with nucleus-derived subunits, form central complexes majorly involved in the essential processes of photosynthetic light reaction, carbon fixation, metabolism and gene expression. Biogenesis of these heterogenic complexes adds an additional level of complexity for protein biogenesis. In this review, we summarize the current knowledge about co-translationally binding factors in chloroplasts and discuss their role in protein folding and ribosome translocation to thylakoid membranes.