Figure 5.

Silencing of the group III E2 genes diminishes the suppression of Fen-mediated PCD by AvrPtoB and AvrPtoB-promoted degradation of Fen in N. benthamiana. A, Knocking down the expression of group III E2 genes diminished the suppression of Fen-mediated PCD by AvrPtoB in N. benthamiana. Transient coexpression of AvrPtoB and Fen-HA in fully expanded N. benthamiana leaves was performed by syringe infiltrating Agrobacterium tumefaciens carrying T-DNA with the AvrPtoB and Fen genes. Transient coexpression of the empty pBTEX vector (EV) and Fen was used as a control. The infiltrated area of each leaf is outlined by the black circle. TRV-group III was used to knock down the expression group III E2 genes. TRV vector only was used as a control. Photographs were taken on day 4 after infiltration. The experiment was repeated two times with similar results. Bar = 1 cm. B, The group III E2s were required for AvrPtoB-promoted degradation of Fen. Transient coexpression of AvrPtoB and Fen-HA in N. benthamiana leaves was performed as described in A. The leaf samples were collected at 36 h after infiltration. Western blotting was performed using anti-HA and anti-AvrPtoB antibody, respectively, to detect Fen-HA and AvrPtoB. Staining of Rubisco subunits by Coomassie Blue demonstrated equal loading. The experiment was repeated two times with similar results. C, Fen was ubiquitinated in the presence of AvrPtoB and a member of group III E2s. The in vitro ubiquitination assays were performed with recombinant E1, E2, AvrPtoB, ubiquitin (Ub), and Fen. Reactions without E2 or AvrPtoB (as denoted with ‒) were used as controls. Polyubiquitinated forms of AvrPtoB [AvrPtoB-(Ub)n] confirmed the presence of E3 ligase activity.