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

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.

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 to the primary infection sites of various antimicrobial compounds, proteins, and small RNA in membrane vesicles. Much of the evidence regarding the importance of vesicular trafficking in plant-pathogen interactions comes from 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 play a role in the interaction with Fusarium graminearum, a fungal pathogen well-known for its ability to cause Fusarium head blight disease in wheat. Among the analysed 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 MIN7 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 genes were silenced displayed significantly more Fusarium head blight 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 hypersusceptibility to various pathogens.

Genome Sequence of Fusarium graminearum Strain CML3066, Isolated from a Wheat Spike in Southern Brazil.

Fusarium graminearum is a global fungal pathogen of wheat and other small grains, causing Fusarium head blight (FHB) disease, also known as wheat scab. We report here the annotated genome of a deoxynivalenol/15-acetyl-deoxynivalenol-producing Brazilian strain called CML3066, isolated from FHB-symptomatic wheat spikes collected in 2009.