Biology subjects

Mosbach, A.

Publications and source records attributed to Mosbach, A..

4 recordsLinked to original sources

bioRxiv2024

SATAY-Based Chemogenomic Screening uncovers Antifungal Resistance Mechanisms and Key Determinants of ATI-2307 and Chitosan Sensitivity

Multidrug-resistant fungal pathogens are a serious threat to public health and global food security. Mitigation requires the discovery of antifungal compounds with novel modes of action, along with a comprehensive understanding of the molecular mechanisms governing antifungal resistance. Here, we apply SAturated Transposon Analysis in Yeast (SATAY), a powerful transposon sequencing method in Saccharomyces cerevisiae, to uncover loss- and gain-of-function mutations conferring resistance to 20 different antifungal compounds. These screens identify a diverse array of novel resistance mechanisms and multiple modes of action. SATAY is performed in drug-sensitive strains to generate chemogenomic profiles for compounds that lack activity against conventional laboratory strains. This study therefore provides a significant resource for exploring cellular responses to chemical stresses. We discover that the natural antifungal Chitosan electrostatically interacts with cell wall mannosylphosphate, and that the transporter Hol1 concentrates the novel antifungal ATI-2307 within yeast. This latter finding presents an avenue for drug design initiatives, but also unveils a straightforward evolutionary path to ATI-2307 resistance with minimal fitness cost.

microbiology↗

bioRxiv2020

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

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 transiently stable telomeres containing vectors, which allowed temporary selection and convenient screening of marker-free editing. We demonstrate that this approach provides vastly superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. The high performance of telomere vector-mediated coediting 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 RNP-based cotransformation is expected to greatly facilitate molecular research in B. cinerea and other fungi.

genetics↗

bioRxiv2019

Exploiting homologous recombination increases SATAY efficiency for loss- and gain-of-function screening

The analysis of large-scale transposon mutant libraries is becoming a method of choice for functional genomics in bacteria and fungi. We previously established SAturated Transposon Analysis in Yeast (SATAY) to uncover genes necessary for growth in any condition in S. cerevisiae (Michel et al., 2017). We present an improved version leveraging homologous recombination to increase transposition efficiency by a factor 10, allowing a single experimenter to rapidly perform several parallel screens. We demonstrate its potential by presenting (1) a comparison of the essential gene sets between two yeast laboratory backgrounds, (2) a comprehensive description of essential genes displaying phenotypic delays - we highlight their common features and propose plausible explanations for this phenomenon -, (3) a genome-wide analysis of loss- and gain-of-function mutations conferring sensitivity or resistance to a compendium of 9 anti-fungal compounds. This study highlights the power of this improved SATAY protocol for yeast functional- and pharmaco-genomics.

cell biology↗

bioRxiv2019

A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici

Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used for the control of a broad range of fungal diseases. This has been the most rapidly expanding fungicide group in terms of new molecules discovered and introduced for agricultural use over the past fifteen years. A particular pattern of differential sensitivity (resistance) to a subclass of chemically-related SDHIs (SHA-SDHIs) was observed in naive Zymoseptoria tritici populations. Class specific SHA-SDHI resistance was confirmed at the enzyme level but did not correlate with the genotypes of the succinate dehydrogenase (SDH) encoding genes. Mapping and characterization of the genetic factor responsible for standing SHA-SDHI resistance in natural field isolates identified a gene (alt-SDHC) encoding a paralog of the C subunit of succinate dehydrogenase. This paralog was not present within our sensitive reference isolates and found at variable frequencies within Z. tritici populations. Using reverse genetics, we showed that alt-SDHC associates with the three other SDH subunits leading to a fully functional enzyme and that a unique Qp-site residue within the alt-SDHC protein confers SHA-SDHI resistance. Enzymatic assays, computational modelling and docking simulations for the two types of SQR enzymes (alt-SDHC, SDHC) enabled us to describe protein-inhibitor interactions at an atomistic level and to propose rational explanations for differential potency and resistance across SHA-SDHIs. European Z. tritici populations displayed a presence (20-30%) / absence polymorphism of alt-SDHC, as well as differences in alt-SDHC expression levels and splicing efficiency. These polymorphisms have a strong impact on SHA-SDHI resistance phenotypes. Characterization of the alt-SDHC promoter in European Z. tritici populations suggest that transposon insertions are associated with the strongest resistance phenotypes. These results establish that a dispensable paralogous gene determines SHA-SDHIs fungicide resistance in natural populations of Z. tritici. This study paves the way to an increased awareness of the role of fungicidal target paralogs in resistance to fungicides and demonstrates the paramount importance of population genomics in fungicide discovery.\n\nAuthor SummaryZymoseptoria tritici is the causal agent of Septoria tritici leaf blotch (STB) of wheat, the most devastating disease for cereal production in Europe. Multiple succinate dehydrogenase inhibitor (SDHI) fungicides have been developed and introduced for the control of STB. We report the discovery and detailed characterization of a paralog of the C subunit of the SDH enzyme conferring standing resistance towards a particular chemical subclass of the SDHIs. The resistance gene is characterized by its presence/absence, expression and splicing polymorphisms which in turn affect resistance levels. The identified mechanism influenced the chemical optimization phase which led to the discovery of pydiflumetofen, exemplifying the importance of population genomics for discovery and rational design of the most adapted solutions.

microbiology↗