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.

Acid-resistant genes of oral plaque microbiome from the functional metagenomics

“…Subsequent analysis revealed that it is a novel nucleotide binding protein related to zinc carboxypeptidases. In addition, the proteins encoded by disease-resistant genes in most plants contain ATP/GTP binding motifs, such as kinase-1a, kinase-2, and kinase-3a motifs, which confer resistance on the host tomato against Fusarium oxysporum , root-knot nematodes or potato aphids [39]. However, this study is the first to confirm the acid-resistance of the ATP/GTP binding protein.…”

Guarding the Goods. New Insights into the Central Alarm System of Plants

“…Given its central role in NB-LRR function, surprisingly little is known about the role of nucleotide binding in NB-LRR activation. In fact, nucleotide binding has been demonstrated unequivocally for only two plant NB-LRR proteins, I-2 and Mi-1, both from tomato (Tameling et al, 2002). The NB domains of these two proteins were expressed in Escherichia coli and shown in a filter-binding assay to specifically bind ATP rather than other nucleoside triphosphates.…”

Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen

“…The TIR subclass is found in dicot species whereas the non-TIR subclass with a coiled-coil (CC) structure occurs among both the monocot and dicot plants [11,10]. The CC domain with a coiled-coil structure is implicated in specific interactions with other proteins and also facilitates dimerization [12]. The CC-NB-LRR protein Rx of potato is found to interact with the Potato virus X (PVX) coat protein conferring resistance to PVX [13] and the Arabidopsis RPS4 gene is involved in resistance to Avr Rps4 of bacterial pathogens [14].…”