Figure 2.

The predicted 14-3-3 binding motif of HopQ1 is phosphorylated by plant kinases. A and B, HopQ1-Strep tag II fusion protein expressed in N. benthamiana leaves was affinity purified and subjected to LC-MS/MS analyses. C and D, HopQ1-6xHis expressed in bacteria was incubated with total protein extracts from N. benthamiana and then affinity purified and analyzed by LC-MS/MS. A and C show the fragmentation spectra with peak assignment to b, y, b-H₃PO₄, and y-H₃PO₄, with “–P” denoting loss of an H₃PO₄ group. Major signals of the MS/MS spectra are identified by their corresponding fragment tags of the b, y series, but also of the y-H₃PO₄ and b-H₃PO₄ series, which were expected in the case of a phosphorylated peptide. B and D show the peptide sequences with daughter ions of the b, y, b-H₃PO₄, and y-H₃PO₄ series found in the spectra. In the peptide derived from HopQ1 phosphorylated in vitro (C and D), the presence of the full series of b₂ to b₁₀ fragments and the expected b-H₃PO₄ series allows for unequivocal localization of the phosphate at Ser-51. In the peptide derived from HopQ1 expressed in planta (A and B), the majority of b and b-H₃PO₄ pairs are also present. In addition, the presence of strong y, y-PO₃, and y-H₃PO₄ signals indicates that the site of phosphorylation is Ser-51, not Ser-49.