Figure 7.

Silencing of bZIP60 results in enhanced cell death induced by P25/helper component‐proteinase (HC‐Pro). Nicotiana benthamiana leaves were infiltrated with Agrobacterium cultures expressing pTRV2:bZIP60 or pTRV2:00 (vector control). (A) Silencing of bZIP60 transcripts was monitored by reverse transcription‐polymerase chain reaction (RT‐PCR) in the uppermost leaves at 20 days after inoculation (dai). The same RT reactions were used to amplify 18S RNA gene transcripts as a control. The number of PCR cycles is indicated below the treatments. RT‐, control without RT. (B) At 20 dai, leaves of pTRV2:bZIP60 and control plants were agroinfiltrated with green fluorescent protein (GFP) alone or the combination of T7‐tagged P25 plus HC‐Pro in opposite leaf patches. Leaf discs from bZIP60‐silenced and control leaves were excised and assayed for electrolyte leakage at 8 days post‐infiltration (dpi). Data represent the means ± standard errors of six replicates, each consisting of four plants that received the same treatment. Statistically significant differences between means were determined by employing Scheffé’s multiple range test. Different letters indicate significant differences at P < 0.05. (C) Leaves from bZIP60‐silenced and control leaves were stained with 3,3′‐diaminobenzidine (DAB) solution at 8 dpi. (D) Western blot analysis of extracts derived from leaf patches at 8 dpi, using antibodies against the T7 epitope. The lower panel below the western blot shows the Ponceau S‐stained membrane after blotting, as a loading control. The intensity of the P25 bands, normalized for the loading controls, was quantified by densitometric analysis. [Colour figure can be viewed at wileyonlinelibrary.com]