HcPro effects on 20S proteasome catalytic activities.

A. Quantification by western blotting of the steady-state levels of accumulation of total ubiquitinated protein in the infiltrated patch using a rabbit polyclonal anti-ubiquitin antiserum. The total plant protein (10 µg) was fractionated in a 12% SDS-PAGE in each case. A range of proteins from 17 to 130 kDa was detected by immunoblotting. Immunoblot detection of free ubiquitin was done with 5 µg of protein in a 14% SDS-PAGE. The lower panel shows a Ponceau S stained membrane. The right panel shows the graph depicting relative band intensities of HcPro, HcPro (M1) and HcPro(M2). B. (i) Proteasome activity assay of HcPro along with MG132 after different time of incubations. The assay was started by addition of fluorogenic substrate after incubation of 100 µg of proteosomal pellet with 30 µg HcPro without ATP. (ii) Comparison of proteasome inhibitory action of wt HcPro with its mutants after 120 minutes of incubation. The MBP was taken as control. The wt HcPro and HcPro (M1) showed a similar trend whereas HcPro could not inhibit the proteasomal protease function after 120 minutes of incubation. All the data are means ±SD of three repeat assays. Asterisks indicate statistically significant difference (* P<0.001 and ** P<0.005 by one way ANOVA). C. Relative expression levels of PRSV CP and GFP mRNA estimated by qPCR using the 2−ΔΔCtmethod. The accumulation of RNAs in N.benthamiana leaves was compared from leaf samples co-infiltrated with Agrobacterium harboring a binary plasmid (empty vector) along with binary vectors expressing either the PRSV CP or GFP genes, with leaf samples co-infiltrated with Agrobacterium harboring a plasmid expressing wt HcPro, HcPro (M1) or HcPro (M2) along with plasmids expressing either the PRSV CP or GFP genes. The actin mRNA level was used as an internal standard. The data represented are means of three independent experiments in each case. The error bars represents deviation observed in three repeat assays. Asterisks indicate statistically significant differences (* P<0.001 and ** P<0.005 by one way anova) D. Detection of HcPro RNA transcript by RNA gel blot. The total RNAs extracted from the various CP and GFP expressed samples were analyzed by agarose gel electrophoresis and blotted onto nylon membrane. The blot was hybridized by radioactive HcPro probes.