Biology subjects

Scalliet, G.

Publications and source records attributed to Scalliet, G..

10 recordsLinked to original sources

bioRxiv2026

Mapping ortholog-restricted ligandable cysteines in the wheat pathogen Zymoseptoria tritici

Zymoseptoria (Z.) tritici is the fungal phytopathogen responsible for Septoria tritici leaf blotch (STB), the main foliar disease of wheat. Fungicides mitigate crop loss caused by STB, but multidrug-resistant Z. tritici strains pose a major threat to the global food supply and underscore the need to identify new fungicidal targets. Here, we use activity-based protein profiling to generate a covalent ligandability map of Z. tritici, leading to the discovery of cysteines in a diverse set of essential proteins that are targeted by stereochemically defined, electrophilic small molecules (stereoprobes). We identify stereoprobes that display nucleotide-dependent reactivity with an ortholog-restricted cysteine in the GTPase SAR1, leading to accumulation of this protein and other COPII components at apparent ER exit sites and corresponding impairments in Z. tritici growth. Our findings thus expand the scope of ligandable, essential proteins in Z. tritici and illuminate opportunities to selectively target this destructive plant pathogen.

biochemistry↗

bioRxiv2025

A pangenomics-enabled platform for the high-throughput discovery of antifungal resistance factors in crop pathogens

The rise of antifungal resistance is a global challenge for both human health and food security, because resistance emergence easily outpaces the antifungal development pipeline. Furthermore, resistance arises often in parallel and through alternative mechanisms creating challenges to predict emergence. In agriculture, where vast areas are sprayed by diverse cocktails, antifungal resistance gains are particularly complex. Despite broad efforts, knowledge of resistance mechanisms is often limited to model genotypes and empirical evidence from the field is lacking. Here, we define and validate a high- throughput pipeline for antifungal resistance discovery informed by emerging resistance gains at continental scale. We analyzed a thousand-genome European diversity panel of the major wheat pathogen Zymoseptoria tritici and assessed resistance levels against over 29 fungicides covering all major classes. We optimized high-throughput phenotyping assays to comprehensively capture emerging resistance phenotypes. Pangenome-informed genotyping techniques revealed a total of 2192 genes associated with antifungal resistance. This expands by an order of magnitude the current knowledge and establishes a refined atlas of resistance mutations. We generated mutants to recapitulate several of the discovered resistance factors. Hence, our approach captures in-field resistance gains across Europe for all major fungicide classes and can define exact molecular targets. Broad knowledge of resistance gains will guide more sustainable fungicide development pipelines.

microbiology↗

bioRxiv2025

A large European diversity panel reveals complex azole fungicide resistance gains of a major wheat pathogen

Fungicide resistance in crop pathogens poses severe challenges to sustainable agriculture. Demethylation inhibitors (DMIs) are critical for controlling crop diseases but face rapid resistance gains in the field. Even though the main molecular basis of resistance is well established, field surveys have repeatedly revealed alternative resistance mechanisms. The European continent in particular has seen rapid and heterogeneous gains in azole resistance in the past decades. Here, we establish a large genome panel to dissect the genetic architecture of emerging resistance in the major wheat pathogen Zymoseptoria tritici. The European diversity panel spans 15 sampling years and 27 countries for a total of 1394 sequenced and phenotyped strains. Using two complementary assays to quantify resistance levels of each strain, we captured fine-grained shifts in DMI resistance over space and time. We conducted genome-wide association studies based on a comprehensive set of genotyping approaches for six DMIs. We mapped a total of 21,220 genetic variants and 158 genes linked to resistance. The substantial scope in genetic mechanisms underpinning DMI resistance significantly expands our mechanistic understanding how continent-wide resistance arises in fungal pathogens over time. Diversification of the Cyp51 coding sequence was particularly striking with new resistant haplotypes emerging with complex configurations and geographic patterns. This study provides expansive new insights into fungicide resistance gains of crop pathogens relevant for future resistance management strategies.

microbiology↗

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↗

bioRxiv2023

Improved gene annotation of the fungal wheat pathogen Zymoseptoria tritici based on combined Iso-Seq and RNA-Seq evidence

Despite large omics datasets, the establishment of a reliable gene annotation is still challenging for eukaryotic genomes. Here, we used the reference genome of the major fungal wheat pathogen Zymoseptoria tritici (isolate IPO323) as a case study to develop methods to improve eukaryotic gene prediction. Four previous IPO323 annotations identified 10,933 to 13,260 gene models, but only one third of these coding sequences (CDS) have identical structures. To resolve these discrepancies and improve gene models, we generated full-length transcripts using long-read sequencing. This dataset was used together with other evidence (RNA-Seq transcripts and protein sequences) to generate novel ab initio gene models. The selection of the best structure among novel and existing gene models was performed according to transcript and protein evidence using InGenAnnot, a novel bioinformatics suite. Overall, 13,414 re-annotated gene models (RGMs) were predicted, including 671 new genes among which 53 encoded effector candidates. This process corrected many of the errors (15%) observed in previous gene models (coding sequence fusions, false introns, missing exons). While fungal genomes have poor annotations of untranslated regions (UTRs), our Iso-Seq long-read sequences outlined 5 and 3UTRs for 73% of the RGMs. Alternative transcripts were identified for 13% of RGMs, mostly due to intron retention (75%), likely corresponding to unprocessed pre-mRNAs. A total of 353 genes displayed alternative transcripts with combinations of previously predicted or novel exons. Long non-coding transcripts (lncRNAs) and double-stranded RNAs from two fungal viruses were also identified. Most lncRNAs corresponded to antisense transcripts of genes (52%). lncRNAs that were up or down regulated during infection were enriched in antisense transcripts (70%), suggesting their involvement in the control of gene expression. Our results showed that combining different ab initio gene predictions and evidence-driven curation using InGenAnnot improved the quality of gene annotations of a compact eukaryotic genome. Our analysis also provided new insights into the transcriptional landscape of Z. tritici, helping develop an increasingly complex picture of its biology.

genomics↗

bioRxiv2022

A thousand-genome panel retraces the global spread and climatic adaptation of a major crop pathogen

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.

genomics↗

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↗

bioRxiv2018

Analysis of small RNA silencing in Zymoseptoria tritici - wheat interactions

Cross-kingdom small RNA (sRNA) silencing has recently emerged as a mechanism facilitating fungal colonization and disease development. Here we characterized RNAi pathways in Zymoseptoria tritici, a major fungal pathogen of wheat, and assessed their contribution to pathogenesis. Computational analysis of fungal sRNA and host mRNA sequencing datasets was used to define the global sRNA populations in Z. tritici and predict their mRNA targets in wheat. 389 in planta-induced sRNA loci were identified. sRNAs generated from some of these loci were predicted to target wheat mRNAs including those potentially involved in pathogen defense. However, molecular approaches failed to validate targeting of selected wheat mRNAs by fungal sRNAs. Mutant strains of Z. tritici carrying deletions of genes encoding key components of RNAi such as Dicer-like (DCL) and Argounate (AGO) proteins were generated, and virulence bioassays suggested that these are dispensable for full infection of wheat. Nonetheless, our results did suggest the existence of non-canonical DCL-independent pathway(s) for sRNA biogenesis in Z. tritici. dsRNA targeting essential fungal genes applied in vitro or generated from an RNA virus vector in planta in a procedure known as HIGS (Host-Induced Gene Silencing) was ineffective in preventing Z. tritici growth or disease. We also demonstrated that Z. tritici is incapable of dsRNA uptake. Collectively, our data suggest that RNAi approaches for gene function analyses in this fungal species and potentially also as a control measure may not be as effective as has been demonstrated for some other plant pathogenic fungi.

plant biology↗