title: The tomato R gene products I-2 and MI-1 are functional ATP binding proteins with ATPase activity.
abstract: Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP.
authors.affiliation: Plant Pathology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1090 GB Amsterdam, The Netherlands.
authors.forename: Wladimir I L
authors.initials: WI
authors.lastname: Tameling
authors.forename: Sandra D J
authors.initials: SD
authors.lastname: Elzinga
authors.forename: Patricia S
authors.initials: PS
authors.lastname: Darmin
authors.forename: Jack H
authors.initials: JH
authors.lastname: Vossen
authors.forename: Frank L W
authors.initials: FL
authors.lastname: Takken
authors.forename: Michel A
authors.initials: MA
authors.lastname: Haring
authors.forename: Ben J C
authors.initials: BJ
authors.lastname: Cornelissen
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D000090263:Leucine-Rich Repeat Proteins; D018551:Solanum lycopersicum / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; C106157:I2C protein, Lycopersicon esculentum; D000090263:Leucine-Rich Repeat Proteins; C117349:MI-1 protein, Lycopersicon esculentum; D010940:Plant Proteins; D011506:Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases