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Darino, M.

Publications and source records attributed to Darino, M..

2 recordsLinked to original sources

The Fusarium graminearum effector protease FgTPP1 suppresses immune responses and facilitates Fusarium Head Blight Disease

Most plant pathogens secrete effector proteins to circumvent host immune responses, thereby promoting pathogen virulence. One such pathogen is the fungus Fusarium graminearum, which causes Fusarium Head Blight (FHB) disease on wheat and barley. Transcriptomic analyses revealed that F. graminearum expresses many candidate effector proteins during early phases of the infection process, some of which are annotated as proteases. However, the contributions of these proteases to virulence remains poorly defined. Here, we characterize a F. graminearum endopeptidase, FgTPP1 (FGSG_11164), that is highly upregulated during wheat spikelet infection and is secreted from fungal cells. To elucidate the potential role of FgTPP1 in F. graminearum virulence, we generated FgTPP1 deletion mutants ({Delta}Fgtpp1) and performed FHB infection assays. While the number of completely bleached spikes infected by F. graminearum wild-type reached 50% of total infected spikes, the number of fully bleached spikes infected by{Delta} Fgtpp1 mutants was 25%, suggesting FgTPP1 contributes to fungal virulence. Transient expression of green fluorescent protein (GFP)-tagged FgTPP1 revealed that FgTPP1 localizes, in part, to chloroplasts and attenuates chitin-mediated activation of mitogen-activated protein kinase (MAPK) signaling, reactive oxygen species production, and cell death induced by an autoactive disease resistance protein when expressed in planta. Notably, the FgTPP1 protein is conserved across the Ascomycota phylum, making it a core effector among ascomycete plant pathogens. These properties make FgTPP1 an ideal candidate for decoy substrate engineering, with the goal of engineering resistance to FHB, and likely other crop diseases caused by ascomycete fungi.

plant biology↗

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↗