Bacterial pathogens of plants and animals utilize conserved type III delivery systems to traffic effector proteins into host cells. Plant innate immune systems evolved disease resistance (R) genes to recognize some type III effectors, termed avirulence (Avr) proteins. On disease-susceptible (r) plants, Avr proteins can contribute to pathogen virulence. We demonstrate that several type III effectors from Pseudomonas syringae are targeted to the host plasma membrane and that efficient membrane association enhances function. Efficient localization of three Avr proteins requires consensus myristoylation sites, and Avr proteins can be myristoylated inside the host cell. These prokaryotic type III effectors thus utilize a eukaryote-specific posttranslational modification to access the subcellular compartment where they function.

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Coimmunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Coimmunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.

The Arabidopsis thaliana disease resistance genes RPS2 and RPM1 belong to a class of plant disease resistance genes that encode proteins that contain an Nterminal tripartite nucleotide binding site (NBS) and a Cterminal tandem array of leucine-rich repeats. RPS2 and RPM1 confer resistance to strains of the bacterial phytopathogen Pseudomonas syringae carrying the avirulence genes avrRpt2 and avrB, respectively. In these gene-for-gene relationships, it has been proposed that pathogen avirulence genes generate specific ligands that are recognized by cognate receptors encoded by the corresponding plant resistance genes. To test this hypothesis, it is crucial to know the site of the potential molecular recognition. Mutational analysis of RPS2 protein and in vitro translation͞translocation studies indicated that RPS2 protein is localized in the plant cytoplasm. To determine whether avirulence gene products themselves are the ligands for resistance proteins, we expressed the avrRpt2 and avrB genes directly in plant cells using a novel quantitative transient expression assay, and found that expression of avrRpt2 and avrB elicited a resistance response in plants carrying the corresponding resistance genes. This observation indicates that no bacterial factors other than the avirulence gene products are required for the specific resistance response as long as the avirulence gene products are correctly localized. We propose that molecular recognition of P. syringae in RPS2-and RPM1-specified resistance occurs inside of plant cells.In plants, robust defense responses to invading phytopathogens often conform to a gene-for-gene relationship: resistance to a pathogen is only observed when the pathogen carries a specific avirulence (avr) gene and the plant carries a corresponding resistance (R) gene (1-3). Because avr-R gene-forgene relationships are observed in many plant-pathogen systems and are accompanied by a characteristic set of defense responses, a common molecular mechanism underlying avr-R gene mediated resistance has been postulated (4). One simple model which explains gene-for-gene relationships is that pathogen avr genes directly or indirectly generate a specific molecular signal (ligand) that is recognized by cognate receptors encoded by plant R genes. Recent cloning of plant resistance genes and corresponding pathogen avirulence genes provided the tools for a direct test of this ligand-receptor model (5).In the phytopathogenic interaction between the small flowering plant Arabidopsis thaliana and the bacterial phytopathogen Pseudomonas syringae, two R genes, RPS2 (6, 7) and RPM1 (8), and three corresponding avr genes, avrRpt2 (9), avrRpm1 (10), and avrB (11), have been isolated. RPS2 confers resistance to P. syringae strains expressing avrRpt2 (12, 13) and RPM1 confers resistance to P. syringae expressing avrRpm1 (14) or avrB (15). RPS2 and RPM1 belong to a major class of plant resistance genes which encode proteins containing nucleotide binding sites (NBS) and leucine-rich repeats (LRR) and which confer ...