Redox signalling from NADPH oxidase targets metabolic enzymes and developmental proteins in Fusarium graminearum
Citation
Fernando, U., Chatur, S., Joshi, M., Thomas Bonner, C., Fan, T., Hubbard, K., Chabot, D., Rowland, O., Wang, L., Subramaniam, R., Rampitsch, C. (2019). Redox signalling from NADPH oxidase targets metabolic enzymes and developmental proteins in Fusarium graminearum. Molecular Plant Pathology, [online] 20(1), 92-106. http://dx.doi.org/10.1111/mpp.12742
Plain language summary
NADPH oxidase, or NOX, is an enzyme that plants, animals and fungi use to generate oxygen radicals. The enzyme is used both as part of their immune system, and to send signals to their cells to activate various biological processes. The oxygen radicals can act as small switches which can be used to turn enzymes on or off, and this can in turn effect biological changes inside the cell. We compared healthy Fusarium graminearum, a fungus that causes the wheat disease Fusarium Head Blight (FHB), with a mutant Fusarium that does not have the NOX enzyme and therefore can’t make the oxygen radicals and can’t send the signals. These mutants do not cause FHB on wheat. Using proteomics and mass spectrometry we were able to find many of the enzymes that are switched by the oxygen radicals. Furthermore, in some cases we were able to verify that these targeted enzymes also have a role in FHB, because if we removed them from the genome of healthy Fusarium we once again got mutants that did not cause FHB. We were also able to show – using mass spectrometry – exactly which amino acid on the target protein was activated by the oxygen radical signal. This work demonstrates that signalling through reactive oxygen radicals is important in permitting Fusarium graminearum to cause FHB.
Abstract
NADPH oxidase (NOX) is one of the sources of reactive oxygen species (ROS) that modulates the activity of proteins through modifications of their cysteine residues. In a previous study, we demonstrated the importance of NOX in both the development and pathogenicity of the phytopathogen Fusarium graminearum. In this article, comparative proteomics between the wild-type and a Nox mutant of F. graminearum was used to identify active cysteine residues on candidate redox-sensing proteins. A two-dimensional gel approach based on labelling with monobromobimane (mBBR) identified 19 candidate proteins, and was complemented with a gel-free shotgun approach based on a biotin switch method, which yielded 99 candidates. The results indicated that, in addition to temporal regulation, a large number of primary metabolic enzymes are potentially targeted by NoxAB-generated ROS. Targeted disruption of these metabolic genes showed that, although some are dispensable, others are essential. In addition to metabolic enzymes, developmental proteins, such as the Woronin body major protein (FGSG_08737) and a glycosylphosphatidylinositol (GPI)-anchored protein (FGSG_10089), were also identified. Deletion of either of these genes reduced the virulence of F. graminearum. Furthermore, changing the redox-modified cysteine (Cys325) residue in FGSG_10089 to either serine or phenylalanine resulted in a similar phenotype to the FGSG_10089 knockout strain, which displayed reduced virulence and altered cell wall morphology; this underscores the importance of Cys325 to the function of the protein. Our results indicate that NOX-generated ROS act as intracellular signals in F. graminearum and modulate the activity of proteins affecting development and virulence in planta.