Aufklärung des Weizen Fusarium-Resistenz-Locus Fhb1

Fusarium head blight (FHB) is a highly destructive fungal diseases in wheat and Fhb1 the most effective and deployed resistance quantitative trait locus (QTL). Fhb1 confers resistance to fungal spread and to the major mycotoxin deoxynivalenol (DON) associated with conjugation into the non-toxic DON-3-O-glucoside (D3G). While four conflicting studies report the identification of the gene controlling fungal spread at the locus, the regulation of the concomitant DON detoxification is completely unknown. We revealed one candidate Fhb1 region affecting both FHB-related traits using forward genetics and an ethyl methane sulfonate (EMS)-treated population of the Fhb1-carrier CM82036. Yet, the target interval comprises a gene with ambiguous annotation and stretches a large physical distance of 20 kb. To resolve this resistance locus we aim to employ CRISPR/Cas-induced mutagenesis and evaluate the effect of defined alterations in the candidate region for their functional causality for resistance and DON glycosylation. Transcriptomics will establish the complete gene model and identify splicing variants instrumental for fungal spreading and DON detoxification. Joint analysis of the triggered CRISPR/Cas and the already available EMS-induced alterations leading to susceptibility and reduced D3G contents will determine the ‘hub’ of Fhb1-mediated resistance and unravel the underlying mechanism. In an unbiased approach CRISPR/Cas-induced genome editing will generate an allelic series for this locus. Additionally, prime editing will test a specific hypothesis of DON detoxification, through mutating cysteine residues in the N-terminus of the protein which are putatively involved in covalent DON binding. Edited CM82036 individuals will be phenotyped for DON/FHB resistance and DON/D3G ratios. Illumina and Pacific Biosciences RNA sequencing and quantitative PCR of toxin and pathogen-challenged spike tissue will elucidate the complex gene model and identify those isoforms causative for Fhb1-mediated resistance. Genetic mutants are an extremely valuable resource for gene isolation and understanding gene function. CRISPR/Cas genome-editing enables targeted candidate gene screens with high specificity. The direct infiltration of DON into wheat florets allows unambiguous and fast identification of loss-of-function mutants; unique plant material the detection of splicing variants controlling the resistance phenotype.