Analysis of the catalytic activity and function of Pto and Fen proteins and subcellular localization of Pto interactors, Pti4, Pti5, Pti6, and AvrPto
Abstract
Resistance of tomato to Pseudomonas syringae pv. tomato strains that express the avirulence gene avrPto is conferred by the Pto gene (Martin et al., 1993). Pto is a member of a small gene family that is clustered on chromosome 5. Another member of this family, Fen, confers sensitivity to an organophosphorous insecticide, fenthion (Martin et al., 1994). The deduced proteins encoded by Pto and Fen contain the hallmarks of protein kinases, namely 15 conserved amino acid residues located in eleven subdomains. I investigated the catalytic activity and amino acid specificity of the tomato Pto and Fen proteins. In an in vitro assay, Pto and Fen proteins phosphorylated serine and threonine (but not tyrosine) residues. A mutation at a conserved lysine residue of subdomain II of Pto and Fen abolished in vitro kinase activity. Pto contains a putative myristylation site characterized by a penultimate glycine (Gly2) residue at its amino terminus. Myristylation is the addition of myristic acid to a Gly2 residue and plays a role in the localization of proteins to the plasma membrane. I investigated the role of the myristylation moiety and its effect on conferring resistance to bacterial speck disease. I altered the invariant glycine residue in the myristylation motif to an alanine and tested the function of mutant Pto in transgenic tomato plants. The mutant Pto protein conferred resistance to Pseudomonas syringae pv. tomato expressing avrPto as effectively as the control wild-type Pto plants. This result indicates that the myristylation motif of Pto is not required for bacterial speck disease resistance. In the yeast two-hybrid system, the Pto kinase interacts with AvrPto and three transcription factors Pti4, Pti5, and Pti6. The AvrPto protein appears to be secreted into the plant cell where it functions by physically interacting the Pto. Pti5/6 bind to a cis-element in the promoters of many pathogenesis-related (PR) genes. Pti415/6 have putative nuclear localization signals (NLSs) in the primary amino acid sequence characterized by clusters of the basic residues lysine and arginine whereas AvrPto does not have any apparent NLSs. I investigated the subcellular localization of Pti4/5/6 and AvrPto. In a transient expression system in tobacco suspension cells, Pti4/5/6 were localized to the nucleus, while AvrPto was retained in the cytoplasm. These data suggest that the AvrPto interacts with Pto in the cytoplasm, while Pti4/5/6 are actively targeted to the nucleus to regulate the expression of PR genes.
Degree
Ph.D.
Advisors
Martin, Purdue University.
Subject Area
Plant pathology|Molecular biology|Genetics|Microbiology
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