Identification and functional characterisation of a locus for target site integration inFusarium graminearum

Abstract

BackgroundFusarium Head Blight is a destructive floral disease of different cereal crops. The Ascomycete fungusFusarium graminearum(Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence ofFggenes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies whereFggenes functions were characterised that integration of expression cassettes occurred randomly. Random insertion of a cassette may disrupt gene expression and/or protein functions and hence the overall conclusion of the study. Target site integration (TSI) is an approach that consists in identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI inFgwould avert the potential risks of ectopic integration.ResultsHere, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region the TSI locus 1. We developed an efficient cloning vector system based on the Golden Gate method to clone different expression cassettes for use in combination with TSI locus 1. We present evidence that integrations in the TSI locus 1 affects neither fungal virulence nor fungal growth under different stress conditions. Integrations at the TSI locus 1 resulted in the expression of different gene fusions. In addition, the activities ofFgnative promoters were not altered by integration into the TSI locus 1. We have developed a bespoke bioinformatic pipeline to analyse the existence of ectopic integrations and tandem insertions of the cassette that may occurred during the transformation process. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus 1.ConclusionThe TSI locus 1 can be used inFgand potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work can be an alternative to southern blotting, the gold standard technique to identify ectopic integration and tandem insertions in fungal transformation.

The trichothecene mycotoxin deoxynivalenol facilitates cell‐to‐cell invasion during wheat‐tissue colonization by Fusarium graminearum

“…Inoculations occurred through the placement of a cut pipette tip with a filter paper insert soaked with 5 × 10 5 spores/mL solution, with distilled water used as a negative control. The coleoptiles were left in the dark for 3 days to aid infection, after which inoculation tips were subsequently removed, and coleoptiles were left to grow under normal growth conditions for a further 4 days (Darino et al., 2024). Disease phenotypes on the coleoptiles were assessed at 7 days post‐inoculation by imaging lesions on an M205 FA stereomicroscope (Leica Microsystems).…”

The trichothecene mycotoxin deoxynivalenol facilitates cell-to-cell invasion during wheat-tissue colonisation byFusarium graminearum

“…Inoculations occurred through the placement of a cut pipette tip with a filter paper insert soaked with 5x10 5 spores/ml solution, with dH 2 O used as a negative control. The coleoptiles were left in the dark for 3 days to aid infection, after which inoculation tips were subsequently removed, and coleoptiles were left to grow under normal growth conditions for a further 4 days [39]. Disease phenotypes on the coleoptiles were assessed at 7 days post inoculation by imaging lesions on a Leica M205 FA Stereomicroscope (Leica Microsystems, UK).…”