title: Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Protein 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.
authors: Boyce Thompson Institute for Plant Research, Ithaca, NY 14853 USA.|Monsanto Company, St. Louis, MO 63141 USA.
authors: Patrick C
authors: PC
authors: Boyle
authors: Boyce Thompson Institute for Plant Research, Ithaca, NY 14853 USA.|Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY 14853 USA.
authors: Simon
authors: S
authors: Schwizer
authors: Boyce Thompson Institute for Plant Research, Ithaca, NY 14853 USA.
authors: Sarah R
authors: SR
authors: Hind
authors: Boyce Thompson Institute for Plant Research, Ithaca, NY 14853 USA.|Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY 14853 USA.
authors: Christine M
authors: CM
authors: Kraus
authors: Boyce Thompson Institute for Plant Research, Ithaca, NY 14853 USA.
authors: Susana
authors: S
authors: De la Torre Diaz
authors: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853 USA.|College of Pharmacy, Guiyang Medical University, Guiyang, 550004 Guizhou China.
authors: Bin
authors: B
authors: He
authors: Boyce Thompson Institute for Plant Research, Ithaca, NY 14853 USA.|Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY 14853 USA.
authors: Gregory B
authors: 0000-0003-0044-6830
authors: GB
authors: Martin
keywords: Arabidopsis thaliana; Click chemistry; Fatty acylation; Myristoylation; Nicotiana benthamiana; Palmitoylation; Pathogen effectors; Pattern recognition receptors; Plasma membrane; Resistance proteins; S-acylation; Stearylation