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. 2013 Feb 14;9(2):e1003177. doi: 10.1371/journal.ppat.1003177

Figure 2. Pit2 interacts with different apoplastic maize cysteine proteases and inhibits their activity.

Figure 2

(A) Y2H assay showing interaction of Pit2 with the maize cysteine proteases CP1A, CP1B, CP2 and XCP2 but not with CatB. BD: Gal4 Binding Domain. AD: Gal4 Activation Domain. (B) Protease activity in fractionated apoplastic fluid of maize leaves after treatment with SA. Highest activity was observed in fractions at elution volume 19.5 ml and 20.5 ml. Protease activity was inhibited by treatment with 10 µM recombinant Pit2 or 5 µM E-64, respectively. (C) Protease activity in apoplastic fluid fractions 19.5 ml (dark grey) and 20.5 ml (light grey). Application of 1–10 µM recombinant Pit2 resulted in a concentration-dependent inhibition of protease activity. D) Activity based protein profiling of cysteine proteases in apoplastic fractions 19.5 ml and 20.5 ml using the specific probe DCG-04 shows inhibition of the apoplastic proteases by Pit2 and E-64. LC: loading control; error bars represent SEM.