Figure 5. Fen has higher kinase activity than Pto and Pto(G50S) has little or no kinase activity.

(A) In vitro kinase assay for tomato Pto, Fen, and Pto(G50S) at pH6.8. At this pH, no brown discoloration was visible upon addition of 10 mM MnCl₂, indicating a better availability of Mn²⁺. Kinase buffers were supplemented with 10 mM MnCl₂, 10 mM MgCl₂ or 10 mM of each. Under these conditions, Fen showed a higher kinase activity than Pto and Pto(G50S) had little or no kinase activity. Coomassie Brilliant Blue (CBB) staining showed similar amounts of the kinases were present. (B) Phosphoprotein-specific ProQ staining of Pto, Fen and Pto(G50S) to assess their phosphorylation status in bacteria. Pto and Fen were expressed in E. coli, pulled down using MBP-agarose, resolved by SDS-PAGE and subjected to ProQ staining. Stronger staining of Fen indicates a higher autophosphorylation activity in situ. Pto(G50S) had little or no kinase activity in this in vivo assay. Coomassie Brilliant Blue (CBB) staining showed similar amounts of the kinases were present.