The Ustilago maydis Effector Pep1 Suppresses Plant Immunity by Inhibition of Host Peroxidase Activity
Figure 5
Pep1 directly inhibits peroxidase activity.
(A) Measurement of HRP activity using an in vitro DAB assay. Dark coloration indicates peroxidase activity, visualized by the precipitation of DAB. The addition of purified GFP as well as heat inactivated Pep1 (Pep1IA) does not interfere with HRP activity. Addition of 5 µM native Pep1 results in reduced DAB precipitation, indicating suppressed HRP activity. (B) Quantification of DAB based HRP activity assay. Different concentrations of Pep1 were added to the assay solution at two different relevant pH values. Pep1 exhibits the ability of concentration dependent suppression of HRP activity at pH 6.5 and 7.5. (C) Far Western blot shows physical interaction of HRP with Pep1 (18.5 kDa) but not GFP (29.8 kDa) (upper panel). As a loading control, a separate gel was equally loaded and stained with coomassie blue (lower panel). (D) Peroxidase activity of maize apoplastic fluid was determined in a quantitative DAB assay. Recombinant native Pep1 and heat inactivated Pep1 (Pep1IA) were added to the reaction in respective concentrations. Data represent three biological replicates. P values have been calculated by an unpaired t test. Error bars show SEM. * P≤0.05.