A Family of Bacterial Cysteine Protease Type III Effectors Utilizes Acylation-dependent and -independent Strategies to Localize to Plasma Membranes

doi:10.1074/jbc.M900519200

A Family of Bacterial Cysteine Protease Type III Effectors Utilizes Acylation-dependent and -independent Strategies to Localize to Plasma Membranes*

From the ^‡Departments of Pharmacology, Cellular and Molecular Medicine, and Chemistry and Biochemistry, the
^§Biomedical Sciences Graduate Program, and
^‖The Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92093-0721 and the
^¶Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037

² To whom correspondence may be addressed. Tel.: 858-453-4100 (ext. 1795); Fax: 858-558-6379; E-mail: ecker{at}salk.edu.
³ To whom correspondence may be addressed:
9500 Gilman Dr., La Jolla, CA 92093-0721.
Tel.: 858-822-0491; Fax: 858-822-5888; E-mail: jedixon{at}ucsd.edu.

↵1 Current address: National Institute of Biological Sciences, Beijing 102206, China.

Abstract

Bacterial phytopathogens employ a type III secretion system to deliver effector proteins into the plant cell to suppress defense pathways; however, the molecular mechanisms and subcellular localization strategies that drive effector function largely remain a mystery. Here, we demonstrate that the plant plasma membrane is the primary site for subcellular localization of the Pseudomonas syringae effector AvrPphB and five additional cysteine protease family members. AvrPphB and two AvrPphB-like effectors, ORF4 and NopT, autoproteolytically process following delivery into the plant cell to expose embedded sites for fatty acylation. Host-dependent lipidation of these three effectors directs plasma membrane localization and is required for the avirulence activity of AvrPphB. Surprisingly, the AvrPphB-like effectors RipT, HopC1, and HopN1 utilize an acylation-independent mechanism to localize to the cellular plasma membrane. Although some AvrPphB-like effectors employ acylation-independent localization strategies, others hijack the eukaryotic lipidation machinery to ensure plasma membrane localization, illustrating the diverse tactics employed by type III effectors to target specific subcellular compartments.

Footnotes

↵* This work was supported, in whole or in part, by National Institutes of Health Grant AI060662 (to J. E. D.) and Pharmacology Training Grant 2 T32 GM07752-25 (to R. H. D.).
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7 and additional “Experimental Procedures.”
↵4 The abbreviations used are:

TTSS

type III secretion system

AvrPphB

avirulence gene Pseudomonas syringae pv. phaseolicola B

ORF4

open reading frame 4

NopT

nodulation outer protein T

RipT

Ralstonia effector injected into plant cells T

HopC1

Hrp outer protein C1

HopN1

Hrp outer protein N1

YopT

Yersinia outer protein T

PBS1

AvrPphB susceptible 1

RPS5

resistant to Pseudomonas syringae 5

PIP2A

plasma membrane intrinsic protein 2A

HR

hypersensitive response

R gene

resistance gene

R protein

resistance protein

Avr

avirulence

YFP

yellow fluorescent protein

CFP

cyan fluorescent protein

PM

plasma membrane

Pst

Pseudomonas syringae pv. tomato DC3000

Pf

Pseudomonas fluorescens

PR1

pathogenesis-related gene 1

cfu

colony forming unit

HA

hemagglutinin epitope

NPTII

neomycin phosphotransferase II

h.p.i.

hours post-infection

NMT

N-myristoyl transferase

PAT

palmitoyl acyltransferase.
- Received January 26, 2009.
- Revision received March 16, 2009.