EFS12- Book of abstracts - Contact

SESSION 1: FUSARIUM – GENETICS, GENOMICS AND SYSTEMS BIOLOGY
Using redox-proteomics to identify targets of NADPH
oxidase-generated reactive oxygen species in
Fusarium graminearum.
C. Rampitsch, 1 G. Subramaniam 2 , M. Joshi 1,2 , T. Fan 1
1 Agriculture and Agri-Food Canada, Winnipeg MB; 2 Agriculture and Agri-Food Canada, Ottawa ON.
E-mail: crampitsch@agr.gc.ca
The regulated production of reactive oxygen species by NADPH oxidases NoxA
and NoxB in Fusarium graminearum (Fgr) is essential for the establishment of
Fusarium head blight in wheat. Knock-out mutants, FgrΔNoxAB, are nonpathogenic
and produce no perithecia in vitro, although normal levels of DON are
secreted. Nox A and B oxidize NADPH to generate O2
34
– and thence H2O2 during
cellular differentiation, creating an oxidizing environment intracellularly, in which
susceptible cysteine residues on target proteins are oxidized. This can profoundly
affect the activity of these proteins, and they are candidate participants in redoxmediated
control of cellular processes, including cellular differentiation and
pathology. Two strategies were used to identify targeted proteins in the redox
proteome: 1) 2-D electrophoresis, using monobromo-bimane to label reduced Cys
residues, followed by MS-based protein identification, and 2) an affinityenrichment
strategy based upon biotinylation of targeted Cys residues, with
relative quantification by spectral counting. Using these strategies, we have
identified several proteins which are potentially targeted, i.e. those which were
oxidized in WT but not in FgrΔNoxAB, under mycotoxin-inducing conditions in
vitro. Confirmation of biological activity through mutagenesis is underway, with
the aim of further understanding the role of redox regulation in Fgr pathogenesis.
Keywords: Nox, proteomics, redox