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

A - Papers appearing in refereed journals

Darino, M., Urban, M., Kaur, N., Wood, A. K. M., Smith, D., Grimwade-Mann, M., Beacham, A. and Hammond-Kosack, K. E. 2024. Identification and functional characterisation of a locus for target site integration in Fusarium graminearum. Fungal Biology and Biotechnology. 11 (2), pp. 1-21. https://doi.org/10.1186/s40694-024-00171-8

AuthorsDarino, M., Urban, M., Kaur, N., Wood, A. K. M., Smith, D., Grimwade-Mann, M., Beacham, A. and Hammond-Kosack, K. E.
Abstract

Background
Fusarium Head Blight (FHB) is a destructive floral disease of different cereal crops. The Ascomycete fungus Fusarium graminearum (Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence of Fg genes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies where Fg genes 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 of identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI in Fg would avert the potential risks of ectopic integration.

Results
Here, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region 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 of Fg native 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, cassette truncations 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.

Conclusion
The TSI locus 1 can be used in Fg and potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, protein secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work together with PCR amplification of the insert could be an alternative to Southern blotting, the gold standard technique used to identify ectopic integrations, cassette truncations and tandem insertions in fungal transformation.

KeywordsFusarium graminearum; Fusarium Head Blight; Fungal transformation; Target site integration; Complementation; Secretion; Coleoptiles; Wheat; Confocal microscopy; Genome sequence
Year of Publication2024
JournalFungal Biology and Biotechnology
Journal citation11 (2), pp. 1-21
Digital Object Identifier (DOI)https://doi.org/10.1186/s40694-024-00171-8
Web address (URL)https://rdcu.be/dzHNQ
Open accessPublished as ‘gold’ (paid) open access
FunderBiotechnology and Biological Sciences Research Council
Funder project or codeDFW - Designing Future Wheat - Work package 2 (WP2) - Added value and resilience
Delivering Sustainable Wheat
Delivering Sustainable Wheat (WP2): Delivering Resilience to Biotic Stress
Designing Future Wheat (DFW) [ISPG]
BB/CCG2280/1
Defining the signalling network linking pathogen infection and asparagine accumulation in wheat grain
Bilateral BBSRC-Embrapa: Using disease risk forecasting, NGS and HIGS to explore and control Fusarium Head Blight disease in wheat fields
BBSRC CASE Quota Studentship: Identification and characterisation of resistance to the take-all fungus in wheat
Publisher's version
Copyright license
CC BY 4.0
Accepted author manuscript
Copyright license
CC BY
Supplemental file
Output statusPublished
Publication dates
Online26 Feb 2024
PublisherSpringer

Permalink - https://repository.rothamsted.ac.uk/item/98z75/identification-and-functional-characterisation-of-a-locus-for-target-site-integration-in-fusarium-graminearum

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