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 (Cys ) 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 Cys 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.

Fusarium graminearum is a fungal plant pathogen that causes Fusarium Head Blight (FHB) on important food and feed cereal crops including wheat, maize and barley.Earlier studies identified distinct roles for NADPH oxidase (NOX) genes in F. graminearum. NOX enzymes generate reactive oxygen species (ROS) including hydrogen peroxide (H 2 O 2 ), which are important in signal transduction. To elucidate the mechanism of NOX dependent signaling in pathogenicity, a proteomics approach was used to examine redox changes in the ΔnoxA/B mutant and compared to wildtype F. graminearum strain. Candidate substrates of NOX enzymes were characterized by genetic analysis. Deletion and overexpression of one of the candidate genes, FGSG_10089 with modified cysteine residues confirmed that it is likely a genuine substrate of the NOX enzyme complex. Bioinformatics and expression analysis indicate that this protein may function as a virulence factor. Deletion of FGSG_10089 as well as modification of the cysteine residue C 325 resulted in reduced virulence on wheat. In addition, there was a decrease in production of 15-acetyl deoxynivalenol in culture.iii Acknowledgements I would like to thank my supervisor Dr. Gopal Subramaniam for his guidance and patience in helping me develop my research and writing skills. His enthusiasm for scientific progress and innovation is infectious and inspiring. Furthermore, I am grateful to my co-supervisor Owen Rowland and committee members Dr. Willmore and Dr.Vierula for their insights and assistance.