The major site of the Pti1 kinase phosphorylated by the Pto kinase is located in the activation domain and is required for Pto–Pti1 physical interaction

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Abstract
The Pto and Pti1 serine/threonine protein kinases are key components of the signaling pathway leading to speck disease resistance in tomato. The two kinases physically interact in the yeast two‐hybrid system, and Pto specifically phosphorylates Pti1 in vitro. In this study, we identified and characterized the major Pti1 site phosphorylated by Pto. Pto was expressed in Escherichia coli as a maltose‐binding fusion protein (MBP‐Pto), and used to phosphorylate in vitro a kinase deficient Pti1 protein fused to glutathione S‐transferase (GST‐Pti1[K96N]). The major phosphopeptide derived from trypsin digestion of phosphorylated GST‐Pti1(K96N) was partially purified by reverse‐phase HPLC and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Its mass corresponded to phosphopeptide LHSTR, which lies in the Pti1 kinase activation domain at amino acid position 230–234. By phosphoamino acid analysis, Thr233 was determined to be the phosphorylation site of peptide LHSTR. Mutations of Thr233 reduced dramatically Pti1 phosphorylation by MBP‐Pto and Pti1 autophosphorylation, providing evidence that the same Pti1 site is involved in the two reactions. Moreover, phosphorylation of Thr233 appeared to be required for Pto–Pti1 physical interaction, as a mutation of this site to alanine, but not to aspartate, abolished the interaction between Pto and Pti1 in the yeast two‐hybrid system.