Grimwade-Mann, M. | bioRxiv Science

bioRxiv2023

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

BackgroundFusarium Head Blight 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 in 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. 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 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 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 in Fg and 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.

genetics↗

bioRxiv2020

The vesicular trafficking system component MIN7 is required for minimizing Fusarium graminearum infection

Plants have developed intricate defense mechanisms, referred to as innate immunity, to defend themselves against a wide range of pathogens. Plants often respond rapidly to pathogen attack by the synthesis and delivery of various antimicrobial compounds, proteins and small RNA in membrane vesicles to the primary infection sites. Much of the evidence regarding the importance of vesicular trafficking in plant-pathogen interactions comes from the studies involving model plants whereas this process is relatively understudied in crop plants. Here we assessed whether the vesicular trafficking system components previously implicated in immunity in Arabidopsis thaliana play a role in the interaction with Fusarium graminearum, a fungal pathogen notoriously famous for its ability to cause Fusarium head blight (FHB) disease in wheat. Among the analyzed vesicular trafficking mutants, two independent T-DNA insertion mutants in the AtMin7 gene displayed a markedly enhanced susceptibility to F. graminearum. Earlier studies identified this gene, encoding an ARF-GEF protein, as a target for the HopM1 effector of the bacterial pathogen Pseudomonas syringae pv. tomato, which destabilizes AtMIN7 leading to its degradation and weakening host defenses. To test whether this key vesicular trafficking component may also contribute to defense in crop plants, we identified the candidate TaMin7 genes in wheat and knocked-down their expression through Virus induced gene silencing. Wheat plants in which TaMIN7 were silenced displayed significantly more FHB disease. This suggests that disruption of MIN7 function in both model and crop plants compromises the trafficking of innate immunity signals or products resulting in hyper-susceptibility to various pathogens. One sentence summaryDisruption of an ARF-GEF protein encoding gene AtMin7 in Arabidopsis thaliana and silencing of the orthologous gene in wheat result in hyper susceptibility to the fungal pathogen Fusarium graminearum.

plant biology↗