Figure 4.

Expression of NopM and variants in tobacco. A, photographs of tobacco leaves expressing NopM, NEL, C338A, and C338S, respectively. A. tumefaciens carrying the empty vector (ev) was used as a negative control. Photographs were taken 3 days after infiltration of the bacterial suspensions. Harvested leaves were decolorized by boiling in ethanol (ROH) to visualize necrotic tissue. Bars, 0.5 cm. B, Western blot (WB) analysis of expressed proteins. Crude proteins from transformed tobacco leaves were isolated 2 days after infiltration with agrobacteria. Western blots were performed with 5 μl of the prepared extract and anti-NopM or anti-GFP antibodies. The upper bands (above NopM and C338S; marked by an arrow) likely represent mono-ubiquitinated forms. C, treatment of extracted C338S with 100 mm NaOH (final concentration) resulted in disappearance of the upper band, suggesting ubiquitination on the serine residue by formation of an oxyester bond.