Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry.

Patrick C Boyle,Gregory B Martin,Sarah R Hind,Susana De La Torre Diaz,Bin He,Simon Schwizer,Christine M Kraus

doi:10.1186/s13007-016-0138-2

Abstract

BackgroundThe plant plasma membrane is a key battleground in the war between plants and their pathogens. Plants detect the presence of pathogens at the plasma membrane using sensor proteins, many of which are targeted to this lipophilic locale by way of fatty acid modifications. Pathogens secrete effector proteins into the plant cell to suppress the plant’s defense mechanisms. These effectors are able to access and interfere with the surveillance machinery at the plant plasma membrane by hijacking the host’s fatty acylation apparatus. Despite the important involvement of protein fatty acylation in both plant immunity and pathogen virulence mechanisms, relatively little is known about the role of this modification during plant-pathogen interactions. This dearth in our understanding is due largely to the lack of methods to monitor protein fatty acid modifications in the plant cell.ResultsWe describe a rapid method to detect two major forms of fatty acylation, N-myristoylation and S-acylation, of candidate proteins using alkyne fatty acid analogs coupled with click chemistry. We applied our approach to confirm and decisively demonstrate that the archetypal pattern recognition receptor FLS2, the well-characterized pathogen effector AvrPto, and one of the best-studied intracellular resistance proteins, Pto, all undergo plant-mediated fatty acylation. In addition to providing a means to readily determine fatty acylation, particularly myristoylation, of candidate proteins, this method is amenable to a variety of expression systems. We demonstrate this using both Arabidopsis protoplasts and stable transgenic Arabidopsis plants and we leverage Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves as a means for high-throughput evaluation of candidate proteins.ConclusionsProtein fatty acylation is a targeting tactic employed by both plants and their pathogens. The metabolic labeling approach leveraging alkyne fatty acid analogs and click chemistry described here has the potential to provide mechanistic details of the molecular tactics used at the host plasma membrane in the battle between plants and pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0138-2) contains supplementary material, which is available to authorized users.

Highlights

The plant plasma membrane is a key battleground in the war between plants and their pathogens
The experimental steps outlined here can be completed within a few days and we describe below the successful application of this approach to detect fatty acid modifications in a variety of candidate proteins using different expression methods and plant systems
We describe the development of a click chemistry-based method using metabolic labeling with ω-alkynyl fatty acid analogs to study the fatty acylation, especially myristoylation, of both host and pathogen proteins in the plant cell

Summary

Introduction

The plant plasma membrane is a key battleground in the war between plants and their pathogens. N-myristoylation describes the irreversible amide bond formation between myristate, a saturated 14-carbon fatty acid, and the N-terminal amine of a glycine residue exposed as a result of co-translational N-terminal methionine excision, or more rarely, post-translational proteolytic processing [1, 3, 4] This modification is mediated by N-myristoyltransferases, cytosolic entities often associated with ribosomes since protein myristoylation is typically a co-translational modification [5,6,7,8]. Protein S-acylation with palmitate or stearate is sufficient for stable interaction with the membrane [2, 15] This modification is suggested to serve roles in retaining proteins at various membranes and trafficking previously myristoylated proteins to the PM, in addition to dynamically regulating protein activity, stability, and complex assembly [1, 9, 14]. Proteins bearing both myristoylation and proximal S-acylation are said to be N-terminally dual fatty acylated and this combination of lipid modifications appears to drive stable association with the PM [1]

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Conclusion