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The tomato R gene products I-2 and Mi-1 are functional ATP binding proteins with ATPase activity

Tameling, W.I.L., Elzinga, S.D.J., Darmin, P.S., Vossen, J.H., Takken, F.L.W., Haring, M.A., Cornelissen, B.J.C.
plant cell 2002 v.14 no.11 pp. 2929-2939
Solanum lycopersicum var. lycopersicum, binding proteins, adenosine triphosphate, guanosine triphosphate, adenosinetriphosphatase, enzyme activity, disease resistance, pH
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.