The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange

Stepan Fenyk,Christopher H Dixon,William H Gittens,Philip D Townsend,Gary J Sharples,Lars-Olof Pålsson,Frank L.W Takken,Martin J Cann

doi:10.1074/jbc.m115.698589

Abstract

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize 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. Because DNA binding by I-2 was decreased in the presence of ADP when compared with 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.

Highlights

Gen effectors or the effect these virulence factors exert on host proteins [3, 5, 6]
Plant nucleotide-binding leucine-rich repeat (NLR) are named after their central nucleotide-binding (NB) and C-terminal leucine-rich repeat (LRR) domains
Redistribution of MLA10, N, RPS4, and SNC1 from the nucleus to the cytoplasm compromises their immune signaling [24, 25, 27, 31], suggesting that their signaling target resides in the nucleus

Summary

Introduction

Gen effectors or the effect these virulence factors exert on host proteins [3, 5, 6]. The N terminus of a plant NLR is extremely divergent and typically encodes either a coiled-coil (CC) or Toll interleukin receptor domain [3] In addition to these core domains, other domains can be present such as a WRKY, Squamosa promoter binding protein-like heavy metal-associated or Ratx (related to ATX1) domains that, in conjunction with the LRR, are proposed to aid in effector perception (9 –11). Together with the recent notion that many NLRs work in pairs, such as RRS-1/RPS4 and RGA4/RGA5 or require “downstream” or “helper” NLRs such as ADR1, NRG1, or NRC members, this suggests the interesting hypothesis that, even for NLRs not located themselves in the nucleus, the conserved signaling target for NLR signaling might be a nuclear component [32,33,34,35,36] In line with this hypothesis, we recently identified DNA as a molecular target for an activated NLR. These experiments will allow an appraisal of the mode of interaction and could reveal NLR-specific differences in nucleic acid interactions

Methods

Results

Conclusion