RESUMO
The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into plant cells by a type III secretion system and interfere with cellular processes to the benefit of the pathogen. In this study, we analyzed eight T3Es from Xcv strain 85-10, six of which were newly identified effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress pathogen-associated molecular pattern (PAMP)-triggered plant defense gene expression. In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing defense responses related to both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in the suppression of ETI-related responses, but still interfered with vesicle trafficking and was only slightly affected with regard to the suppression of defense gene induction. This suggests that XopB-mediated suppression of PTI and ETI is dependent on different mechanisms that can be functionally separated.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Imunidade Vegetal , Xanthomonas campestris/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Bactérias/genética , Morte Celular , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes Bacterianos/genética , Estudos de Associação Genética , Complexo de Golgi/metabolismo , Células Vegetais/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/metabolismo , Transporte Proteico/genética , Solanaceae/citologia , Solanaceae/microbiologia , Virulência/genética , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidadeRESUMO
The pathogenicity of many bacteria depends on the injection of effector proteins via type III secretion into eukaryotic cells in order to manipulate cellular processes. TAL (transcription activator-like) effectors from plant pathogenic Xanthomonas are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind to DNA via a central domain of tandem repeats. Here, we show how target DNA specificity of TAL effectors is encoded. Two hypervariable amino acid residues in each repeat recognize one base pair in the target DNA. Recognition sequences of TAL effectors were predicted and experimentally confirmed. The modular protein architecture enabled the construction of artificial effectors with new specificities. Our study describes the functionality of a distinct type of DNA binding domain and allows the design of DNA binding domains for biotechnology.
Assuntos
Motivos de Aminoácidos , DNA de Plantas/química , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Ativação Transcricional , Xanthomonas/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Biotecnologia , Capsicum/genética , Genes de Plantas , Modelos Biológicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Sequências Repetitivas de Aminoácidos , Nicotiana/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Xanthomonas/patogenicidadeRESUMO
The Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) employs a type III secretion system to translocate effector proteins into plant cells where they modulate host signaling pathways to the pathogen's benefit. The effector protein AvrBs3 acts as a eukaryotic transcription factor and induces the expression of plant genes termed UPA (up-regulated by AvrBs3). Here, we describe 11 new UPA genes from bell pepper that are induced by AvrBs3 early after infection with Xcv. Sequence comparisons revealed the presence of a conserved AvrBs3-responsive element, the UPA box, in all UPA gene promoters analyzed. Analyses of UPA box mutant derivatives confirmed its importance for gene induction by AvrBs3. We show that DNA binding and gene activation were strictly correlated. DNase I footprint studies demonstrated that the UPA box corresponds to the center of the AvrBs3-protected DNA region. Type III delivery of AvrBs3 and mutant derivatives showed that some UPA genes are induced by the AvrBs3 deletion derivative AvrBs3Deltarep16, which lacks four repeats. We show that AvrBs3Deltarep16 recognizes a mutated UPA box with two nucleotide exchanges in positions that are not essential for binding and activation by AvrBs3.
Assuntos
Proteínas de Bactérias/metabolismo , Capsicum/genética , Proteínas de Ligação a DNA/metabolismo , Xanthomonas campestris/patogenicidade , Sequência de Bases , Capsicum/microbiologia , Análise Mutacional de DNA , DNA Complementar/genética , DNA de Plantas/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Família Multigênica , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas , Ligação Proteica , Elementos de Resposta , Efetores Semelhantes a Ativadores de Transcrição , Xanthomonas campestris/metabolismoRESUMO
Pathogenicity of Xanthomonas and most other Gram-negative phytopathogenic bacteria depends on a conserved type III secretion (T3S) system which injects more than 25 different effector proteins into the plant cell. Extensive studies in the last years on the molecular mechanisms of type III effector function revealed that effector proteins with enzymatic functions seem to play important roles in the interaction of Xanthomonas with its host plants, for example, the SUMO protease XopD. In addition, xanthomonads express a unique class of type III effectors to pursue another strategy. Effectors of the AvrBs3 family, so far only identified in Xanthomonas spp. and Ralstonia solanacearum, mimic plant transcriptional activators and manipulate the plant transcriptome.
Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Fatores de Virulência/metabolismo , Xanthomonas/patogenicidade , Ralstonia solanacearum/patogenicidadeRESUMO
Pathogenicity of many Gram-negative bacteria relies on the injection of effector proteins by type III secretion into eukaryotic cells, where they modulate host signaling pathways to the pathogen's benefit. One such effector protein injected by Xanthomonas into plants is AvrBs3, which localizes to the plant cell nucleus and causes hypertrophy of plant mesophyll cells. We show that AvrBs3 induces the expression of a master regulator of cell size, upa20, which encodes a transcription factor containing a basic helix-loop-helix domain. AvrBs3 binds to a conserved element in the upa20 promoter via its central repeat region and induces gene expression through its activation domain. Thus, AvrBs3 and likely other members of this family provoke developmental reprogramming of host cells by mimicking eukaryotic transcription factors.
Assuntos
Proteínas de Bactérias/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Capsicum/genética , Capsicum/microbiologia , Proteínas de Plantas/fisiologia , Xanthomonas campestris/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Capsicum/citologia , Crescimento Celular , Tamanho Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Dados de Sequência Molecular , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Nicotiana/genética , Transcrição Gênica , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidadeRESUMO
The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Análise de Sequência de DNA , Xanthomonas campestris/genética , Adesinas Bacterianas/genética , Composição de Bases , Cromossomos Bacterianos/genética , Coxiella burnetii/genética , Ordem dos Genes , Legionella pneumophila/genética , Dados de Sequência Molecular , Plasmídeos/genética , Polissacarídeos Bacterianos/genética , Transporte Proteico/genética , Sintenia , Virulência/genética , Fatores de Virulência/genética , Xanthomonas campestris/fisiologiaRESUMO
Xanthomonas campestris pv. armoraciae strain 5 is a Brassicaceae pathogen that expresses three members of the highly related avrBs3 gene family of type III effectors. Here, we report on the isolation and characterization of these genes, designated hax2, hax3, and hax4 (homolog of avrBs3 in Xanthomonas). All three Hax proteins are translocated from Xanthomonas spp. into the plant cell via the type III secretion system. Hax3 and Hax4 show the typical structure of AvrBs3-like effectors and contain a repetitive region in their central part consisting of 34-amino-acid (aa) repeats. By contrast, the Hax2 repeat region is composed of 35-aa repeats that are characterized by an additional proline residue. Hax2, Hax3, and Hax4 contain 21.5, 11.5, and 14.5 repeats, respectively. Genetic studies revealed an additive effect of hax2, hax3, and hax4 on disease symptom formation of X. campestris pv. armoraciae strain 5 on radish. The contribution of individual genes to the aggressiveness of strain 5 is quantitatively different, with hax2 showing the strongest effect on the development of chlorosis and necrosis. In addition, hax3 and hax4, but not hax2, have a Bs4-dependent avirulence activity in tomato and in transgenic Nicotiana benthamiana expressing the Bs4 resistance gene.