FIGURE 4. Serine 258 is the major in vivo phosphorylation site of AvrPtoB_1–307.

A, phosphorylation assay of wild type (WT) AvrPtoB_1–307 and the AvrPtoB_1–307(S258A) mutant in RG-prf3 tomato protoplasts. In vivo ³²P labeling was performed as described in the legend to Fig. 2. Incorporation of ³² P into AvrPtoB_1–307 and AvrPtoB_1–307(S258A) mutant is shown by autoradiography after SDS-PAGE separation (top panel). Equal expression of WT AvrPtoB_1–307 and AvrPtoB_1–307(S258A) mutant in plant cells was confirmed by Western blotting with an anti-HA antibody using a small amount of lysate before immunoprecipitation (bottom panel). B, relative ³²P incorporation level of WT AvrPtoB_1–307 and AvrPtoB_1–307(S258A) mutant. The data are shown as percentages of ³²P incorporation with WT set to 100%. The bar represents standard error from three biological replicates.