The tomato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor I-2 couples DNA-Binding to Nucleotide-Binding Domain Nucleotide Exchange

Fenyk, S.; Dixon, C. H.; Kittens, W. H.; Townsend, P. D.; Sharples, G .J.; Pålsson, L. -O.; Takken, F. L. W.; Cann, M. J.

Abstract

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognise and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA≈dsDNA>ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Since DNA binding by I-2 was decreased in the presence of ADP when compared to ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA-binding is a general property of at least a subset of NLR proteins and NLR activation is directly linked to its activity at DNA.

Citation

Fenyk, S., Dixon, C. H., Kittens, W. H., Townsend, P. D., Sharples, G. .., Pålsson, L. .-O., Takken, F. L. W., & Cann, M. J. (2016). The tomato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor I-2 couples DNA-Binding to Nucleotide-Binding Domain Nucleotide Exchange. Journal of Biological Chemistry, 291(3), 1137-1147. https://doi.org/10.1074/jbc.m115.698589

• Journal Article Type: Article
• Acceptance Date: Nov 24, 2015
• Online Publication Date: Nov 24, 2015
• Publication Date: Jan 15, 2016
• Deposit Date: Nov 17, 2015
• Publicly Available Date: Feb 1, 2016
• Journal: Journal of Biological Chemistry
• Print ISSN: 0021-9258
• Electronic ISSN: 1083-351X
• Publisher: American Society for Biochemistry and Molecular Biology
• Peer Reviewed: Peer Reviewed
• Volume: 291
• Issue: 3
• Pages: 1137-1147
• DOI: https://doi.org/10.1074/jbc.m115.698589
• Keywords: ATPases associated with diverse cellular activities (AAA), Cellular immune response, DNA binding protein, Nod-like receptor (NLR), Nucleotide Plant biochemistry
• Public URL: https://durham-repository.worktribe.com/output/1398038