Transient expression of XopJ in N. benthamiana leaves inhibits proteasome activity.

(A) Upper panel: Proteasome activity in N. benthamiana leaves transiently expressing XopJ-myc proteins. XopJ protein variants along with an empty vector (EV) control were transiently expressed in leaves of N. benthamiana using Agro-infiltration. After 48 h, relative proteasome activity in total protein extracts was determined by monitoring the breakdown of the fluorogenic peptide Suc-LLVY-AMC at 30°C in a fluorescence spectrophotometer. The empty vector (EV) control was set to 100%. Data represent the mean SD (n = 3). The experiment has been repeated more than three times with similar results. Lower panel: immunodetection of transiently expressed XopJ variants in the same leaves that were used for proteasome activity measurements. After immunodetection of proteins the membrane was stained with amido black to control for equal protein loading. (B) Upper panel: Proteasome activity in the presence of cysteine protease inhibitor E64 in N. benthamiana leaves transiently expressing XopJ-myc. XopJ protein along with an empty vector (EV) control was transiently expressed in leaves of N. benthamiana using Agro-infiltration. After 48 h, relative proteasome activity was determined by monitoring the breakdown of the fluorogenic peptide Suc-LLVY-NH-AMC at 30°C in a fluorescence spectrophotometer. Plant extracts were incubated with water or 100 µM E64 for 15 min at 30°C before measurements. The empty vector (EV) control was set to 100%. Data represent the mean SD (n = 4). Significant differences were calculated using Student's t-test and are indicated by: **, PN. benthamiana using Agro-infiltration. After 48 h, relative proteasome activity was determined by monitoring the breakdown of the fluorogenic peptide Suc-LLVY-NH-AMC at 30°C in a fluorescence spectrophotometer. Plant extracts were incubated with 1% EtOH or 50 µM MG132 for 15 min at 30°C before measurements. The empty vector (EV) control was set to 100%. Data represent the mean SD (n = 3). (C) Distribution of ubiquitin conjugates in N. benthamiana leaves transiently expressing XopJ-myc proteins. Total proteins were extracted 48 h after infiltration with Agrobacteria harbouring the respective XopJ expression constructs. Ubiquinated proteins were detected using an anti-ubiquitin antibody. Expression of the XopJ variants was verified using an anti myc-antibody. After immunodetection of proteins the membrane was stained with amido black to control for equal protein loading.