RESUMEN
Molecular mechanisms underlying quantitative variations of pathogenicity remain elusive. Here, we identified the Xanthomonas campestris XopJ6 effector that triggers disease resistance in cauliflower and Arabidopsis thaliana. XopJ6 is a close homolog of the Ralstoniapseudosolanacearum PopP2 YopJ family acetyltransferase. XopJ6 is recognized by the RRS1-R/RPS4 NLR pair that integrates a WRKY decoy domain mimicking effector targets. We identified a XopJ6 natural variant carrying a single residue substitution in XopJ6 WRKY-binding site that disrupts interaction with WRKY proteins. This mutation allows XopJ6 to evade immune perception while retaining some XopJ6 virulence functions. Interestingly, xopJ6 resides in a Tn3-family transposon likely contributing to xopJ6 copy number variation (CNV). Using synthetic biology, we demonstrate that xopJ6 CNV tunes pathogen virulence on Arabidopsis through gene dosage-mediated modulation of xopJ6 expression. Together, our findings highlight how sequence and structural genetic variations restricted at a particular effector gene contribute to bacterial host adaptation.
RESUMEN
The Yersinia outer protein J (YopJ) family effectors are widely deployed through the type III secretion system by both plant and animal pathogens. As non-canonical acetyltransferases, the enzymatic activities of YopJ family effectors are allosterically activated by the eukaryote-specific ligand inositol hexaphosphate (InsP6). However, the underpinning molecular mechanism remains undefined. Here we present the crystal structure of apo-PopP2, a YopJ family member secreted by the plant pathogen Ralstonia solanacearum. Structural comparison of apo-PopP2 with the InsP6-bound PopP2 reveals a substantial conformational readjustment centered in the substrate-binding site. Combining biochemical and computational analyses, we further identify a mechanism by which the association of InsP6 with PopP2 induces an α-helix-to-ß-strand transition in the catalytic core, resulting in stabilization of the substrate recognition helix in the target protein binding site. Together, our study uncovers the molecular basis governing InsP6-mediated allosteric regulation of YopJ family acetyltransferases and further expands the paradigm of fold-switching proteins.
Asunto(s)
Acetiltransferasas/química , Apoproteínas/química , Arabidopsis/microbiología , Proteínas Bacterianas/química , Ácido Fítico/química , Ralstonia solanacearum/química , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Regulación Alostérica , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Ácido Fítico/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Ralstonia solanacearum/enzimología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Nicotiana/microbiologíaRESUMEN
Protein lysine acetylation is a highly conserved posttranslational modification that plays key roles in many biological processes such as the regulation of gene expression, chromatin dynamics, and metabolic pathways. Recent studies revealed that various pathogens use lysine acetylation to interfere with host immune responses. Identification of lysine-acetylated host proteins resulting from virulence activities of pathogen effectors is therefore essential for understanding their biological functions. Here we provide a method for immunoprecipitating lysine-acetylated proteins transiently expressed in planta under non-denaturing or denaturing conditions and detecting them by immunoblotting. To illustrate this rapid and simple procedure, immunoprecipitation of the lysine-acetylated WRKY domain of the RRS1-R immune receptor, a substrate of the Ralstonia solanacearum PopP2 effector, is presented as a typical example.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Inmunoprecipitación/métodos , Lisina/química , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/patogenicidad , Acetilación , Arabidopsis/microbiología , Proteínas de Arabidopsis/química , Desnaturalización Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
DNA-binding proteins are involved in the dynamic regulation of various cellular processes such as recombination, replication, and transcription. For investigating dynamic assembly and disassembly of molecular complexes in living cells, fluorescence microscopy represents a tremendous tool in biology. A fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM) has been used recently to monitor protein-DNA associations in plant cells. With this approach, the donor fluorophore is a GFP-tagged binding partner expressed in plant cells. A Sytox® Orange treatment converts nuclear nucleic acids to FRET acceptors. A decrease of GFP lifetime is due to FRET between donor and acceptor, indicating close association of the GFP binding partner and Sytox® Orange-stained DNA. In this chapter, we present a step-by-step protocol for the transient expression in N. benthamiana of GFP-tagged proteins and the fixation and permeabilization procedures used for the preparation of plant material aimed at detecting protein-nucleic acid interactions by FRET-FLIM measurements.
