RESUMEN

Many plant-pathogenic bacteria of considerable economic importance rely on type III secretion systems (T3SSs) of the Hrc-Hrp 1 family to subvert their plant hosts. T3SS gene expression is regulated through the HrpG and HrpV proteins, while secretion is controlled by the gatekeeper HrpJ. A link between the two mechanisms was so far unknown. Here, we show that a mechanistic coupling exists between the expression and secretion cascades through the direct binding of the HrpG/HrpV heterodimer, acting as a T3SS chaperone, to HrpJ. The ternary complex is docked to the cytoplasmic side of the inner bacterial membrane and orchestrates intermediate substrate secretion, without affecting early substrate secretion. The anchoring of the ternary complex to the membranes potentially keeps HrpG/HrpV away from DNA. In their multiple roles as transcriptional regulators and gatekeeper chaperones, HrpV/HrpG provide along with HrpJ potentially attractive targets for antibacterial strategies.IMPORTANCE On the basis of scientific/economic importance, Pseudomonas syringae and Erwinia amylovora are considered among the top 10 plant-pathogenic bacteria in molecular plant pathology. Both employ type III secretion systems (T3SSs) of the Hrc-Hrp 1 family to subvert their plant hosts. For Hrc-Hrp 1, no functional link was known between the key processes of T3SS gene expression and secretion. Here, we show that a mechanistic coupling exists between expression and secretion cascades, through formation of a ternary complex involving the T3SS proteins HrpG, HrpV, and HrpJ. Our results highlight the functional and structural properties of a hitherto-unknown complex which orchestrates intermediate T3SS substrate secretion and may lead to better pathogen control through novel targets for antibacterial strategies.

Asunto(s)

Erwinia amylovora/metabolismo , Expresión Génica , Transporte de Proteínas , Pseudomonas syringae/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Sistemas de Secreción Tipo III/metabolismo , Erwinia amylovora/genética , Pseudomonas syringae/genética , Sistemas de Secreción Tipo III/genética

RESUMEN

This article recounts the experiences that shaped my career as a molecular plant pathologist. It focuses primarily on technical and conceptual developments in molecular phytobacteriology, shares some personal highlights and untold stories that impacted my professional development, and describes the early years of agricultural biotechnology. Writing this article required reflection on events occurring over several decades that were punctuated by a mid-career relocation across the Atlantic. I hope it will still be useful, informative, and enjoyable to read. An extended version of the abstract is provided in the Supplemental Materials , available online.

Asunto(s)

Botánica/historia , Selección de Profesión , Enfermedades de las Plantas , Plantas/genética , Agricultura , Bacterias/patogenicidad , Biotecnología , Genes de Plantas , Historia del Siglo XX , Historia del Siglo XXI , Familia de Multigenes , Sistemas de Secreción Tipo III

RESUMEN

Prophage sequences became an integral part of bacterial genomes as a consequence of coevolution, encoding fitness or virulence factors. Such roles have been attributed to phage-derived elements identified in several Gram-negative species: The type VI secretion system (T6SS), the R- and F-type pyocins, and the newly discovered Serratia entomophila antifeeding prophage (Afp), and the Photorhabdus luminescens virulence cassette (PVC). In this study, we provide evidence that remarkably conserved gene clusters, homologous to Afp/PVC, are not restricted to Gram-negative bacteria but are widespread throughout all prokaryotes including the Archaea. Even though they are phylogenetically closer to pyocins, they share key characteristics in common with the T6SS, such as the use of a chaperon-type AAA+ ATPase and the lack of a host cell lysis mechanism. We thus suggest that Afp/PVC-like elements could be classified as phage-like-protein-translocation structures (PLTSs) rather than as pyocins. The reconstruction of phylogeny and the conserved gene content suggest that the diversification of prophage sequences to PLTS occurred in bacteria early in evolution and only once, but PLTS clusters have been horizontally transferred to some of the bacterial lineages and to the Archaea. The adaptation of this element in such a wide host range is suggestive of its versatile use in prokaryotes.

Asunto(s)

Bacteriófagos/genética , Genoma Arqueal/genética , Genoma Bacteriano/genética , Genómica , Filogenia , Secuencia Conservada , Evolución Molecular

RESUMEN

Countries of Southern Europe are currently suffering from severe socio-economic pain resulting from high debt levels and austerity measures which constrain investment in innovation-based recovery strategies that are essential for entry into a long-term sustainable period of increasing employment and wealth creation. Young university-educated people are particularly innovative, and hence vital to the development of such strategies, but employment opportunities are poor and many are forced to seek employment that neither profits from their training nor satisfies their justified career expectations, or to emigrate. They are the 'lost generation'. A strategy is proposed here for the creation of Pipelines for New Chemicals, national centre-network partnerships for the discovery-synthesis of new chemicals obtained though harvesting new biological diversity, and their exploitation to develop new medicines, agrochemicals, materials, and other products and applications. The goal is to create new regional motors of economic growth and development, by harnessing the knowledge, motivation and innovation potential of the excellently educated young people of Europe to catalyse the development of new small, medium and large enterprises centred around novel chemicals, and the value chains that will evolve with them, and thereby develop a powerful sector of sustainable growth in employment and social and economic prosperity in Southern Europe.

Asunto(s)

Productos Biológicos/economía , Biotecnología/economía , Emigración e Inmigración , Empleo/economía , Europa (Continente) , Recursos Humanos

RESUMEN

Comparative genomics of closely related pathogens that differ in host range can provide insights into mechanisms of host-pathogen interactions and host adaptation. Furthermore, sequencing of multiple strains with the same host range reveals information concerning pathogen diversity and the molecular basis of virulence. Here we present a comparative analysis of draft genome sequences for four strains of Pseudomonas cannabina pathovar alisalensis (Pcal), which is pathogenic on a range of monocotyledonous and dicotyledonous plants. These draft genome sequences provide a foundation for understanding host range evolution across the monocot-dicot divide. Like other phytopathogenic pseudomonads, Pcal strains harboured a hrp/hrc gene cluster that codes for a type III secretion system. Phylogenetic analysis based on the hrp/hrc cluster genes/proteins, suggests localized recombination and functional divergence within the hrp/hrc cluster. Despite significant conservation of overall genetic content across Pcal genomes, comparison of type III effector repertoires reinforced previous molecular data suggesting the existence of two distinct lineages within this pathovar. Furthermore, all Pcal strains analyzed harbored two distinct genomic islands predicted to code for type VI secretion systems (T6SSs). While one of these systems was orthologous to known P. syringae T6SSs, the other more closely resembled a T6SS found within P. aeruginosa. In summary, our study provides a foundation to unravel Pcal adaptation to both monocot and dicot hosts and provides genetic insights into the mechanisms underlying pathogenicity.

Asunto(s)

Proteínas Bacterianas/genética , Genoma Bacteriano , Genómica , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/microbiología , Pseudomonas/genética , Avena/microbiología , Proteínas Bacterianas/clasificación , Bromus/microbiología , Mapeo Cromosómico , Islas Genómicas , Especificidad del Huésped , Solanum lycopersicum/microbiología , Familia de Multigenes , Filogenia , Pseudomonas/clasificación , Pseudomonas/patogenicidad , Pseudomonas aeruginosa/genética , Pseudomonas syringae/genética , Análisis de Secuencia de ADN , Especificidad de la Especie

RESUMEN

Plant- and animal-pathogenic bacteria deploy a variable arsenal of type III effector proteins (T3EP) to manipulate host defense. Specific biochemical functions and molecular or subcellular targets have been demonstrated or proposed for a growing number of T3EP but remain unknown for the majority of them. Here, we show that transient expression of genes coding certain bacterial T3EP (HopAB1, HopX1, and HopF2), which did not elicit hypersensitive response (HR) in transgenic green fluorescent protein (GFP) Nicotiana benthamiana 16C line, enhanced the sense post-transcriptional gene silencing (S-PTGS) triggered by agrodelivery of a GFP-expressing cassette and the silencing enhancement could be blocked by two well-known viral silencing suppressors. Further analysis using genetic truncations and site-directed mutations showed that the receptor recognition domains of HopAB1 and HopX1 are not involved in enhancing silencing. Our studies provide new evidence that phytobacterial pathogen T3EP manipulate the plant small interfering RNA pathways by enhancing silencing efficiency in the absence of effector-triggered immunity signaling and suggest that phytopathogenic bacterial effectors affect host RNA silencing in yet other ways than previously described.

Asunto(s)

Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Nicotiana/metabolismo , Interferencia de ARN/fisiología , Agrobacterium tumefaciens , Arabidopsis/genética , Proteínas Bacterianas/genética , Dexametasona , Proteínas Fluorescentes Verdes , Interacciones Huésped-Patógeno , Mutación , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Hojas de la Planta/virología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nicotiana/genética

RESUMEN

The type VI secretion system (t6ss) is a recently characterized secretion system which appears to be involved in bacterial pathogenesis as a potential nano-syringe for the translocation of effector proteins into the eukaryotic host cell cytoplasm. Until now no evidence was provided for the presence of t6ss in the genomes of the sequenced representatives of Klebsiella spp., including the human opportunistic pathogen Klebsiella pneumoniae. However, in a previous study by Lawlor et al. (2005), were revealed two insertion mutants in hypothetical proteins of K. pneumoniae with decreased ability to infect mouse spleen. Interestingly, these two putative proteins appear to be homologues with two characterized t6ss core proteins of Yersinia pestis. In order to investigate the presence of genes encoding for putative t6ss core components and putative effectors in Klebsiella spp., we have undertaken an in silico genome mining in three fully and one partially sequenced strains of K. pneumoniae, as well as a strain of the Klebsiella variicola. Moreover, we have investigated the phylogenetic relatedness of three core proteins of the Klebsiella t6ss with their orthologues of various bacteria species. Our analysis evidenced three distinguishable, conserved syntenies in Klebsiella spp. genomes that contain the recognised as putative t6ss genes. The results of our work taken together with the results on the functional analysis of insertion mutants, strongly suggest the existence of an organised t6ss mechanism that likely accounts of the host-pathogen interaction.

Asunto(s)

Genes Bacterianos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/clasificación , Filogenia

RESUMEN

Type VI secretion systems (T6SS) of Gram-negative bacteria form injectisomes that have the potential to translocate effector proteins into eukaryotic host cells. In silico analysis of the genomes in six Pseudomonas syringae pathovars revealed that P. syringae pv. tomato DC3000, pv. tabaci ATCC 11528, pv. tomato T1 and pv. oryzae 1-6 each carry two putative T6SS gene clusters (HSI-I and HSI-II; HSI: Hcp secretion island), whereas pv. phaseolicola 1448A and pv. syringae B728 each carry one. The pv. tomato DC3000 HSI-I and pv. tomato T1 HSI-II possess a highly similar organization and nucleotide sequence, whereas the pv. tomato DC3000, pv. oryzae 1-6 and pv. tabaci 11528 HSI-II are more divergent. Putative effector orthologues vary in number among the strains examined. The Clp-ATPases and IcmF orthologues form distinct phylogenetic groups: the proteins from pv. tomato DC3000, pv. tomato T1, pv. oryzae and pv. tabaci 11528 from HSI-II group together with most orthologues from other fluorescent pseudomonads, whereas those from pv. phaseolicola, pv. syringae, pv. tabaci, pv. tomato T1 and pv. oryzae from HSI-I group closer to the Ralstonia solanacearum and Xanthomonas orthologues. Our analysis suggests multiple independent acquisitions and possible gene attrition/loss of putative T6SS genes by members of P. syringae.

Asunto(s)

Proteínas Bacterianas/metabolismo , Biología Computacional , Pseudomonas syringae/metabolismo , Proteínas Bacterianas/química , Filogenia , Pseudomonas syringae/clasificación , Pseudomonas syringae/genética

RESUMEN

With the advent of recombinant DNA techniques, the field of molecular plant pathology witnessed dramatic shifts in the 1970s and 1980s. The new and conventional methodologies of bacterial molecular genetics put bacteria center stage. The discovery in the mid-1980s of the hrp/hrc gene cluster and the subsequent demonstration that it encodes a type III secretion system (T3SS) common to Gram negative bacterial phytopathogens, animal pathogens, and plant symbionts was a landmark in molecular plant pathology. Today, T3SS has earned a central role in our understanding of many fundamental aspects of bacterium-plant interactions and has contributed the important concept of interkingdom transfer of effector proteins determining race-cultivar specificity in plant-bacterium pathosystems. Recent developments in genomics, proteomics, and structural biology enable detailed and comprehensive insights into the functional architecture, evolutionary origin, and distribution of T3SS among bacterial pathogens and support current research efforts to discover novel antivirulence drugs.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Interacciones Huésped-Patógeno/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética

RESUMEN

Chemical or biological synthesis of plant secondary metabolites has attracted increasing interest due to their proven or assumed beneficial properties and health promoting effects. Resveratrol, a stilbenoid, naringenin, a flavanone, genistein, an isoflavone, and the flavonols kaempferol and quercetin have been shown to possess high nutritional and agricultural value. Four metabolically engineered yeast strains harboring plasmids with heterologous genes for enzymes involved in the biosynthesis of these compounds from phenylalanine have been constructed. Time course analyses of precursor utilization and end-product accumulation were carried out establishing the production of 0.29-0.31 mg/L of trans-resveratrol, 8.9-15.6 mg/L of naringenin, 0.1-7.7 mg/L of genistein, 0.9-4.6 mg/L of kaempferol and 0.26-0.38 mg/L of quercetin in defined media under optimal growth conditions. The recombinant yeast strains can be used further for the construction of improved flavonoid- and stilbenoid-overproducers.

Asunto(s)

Flavonoides/biosíntesis , Mejoramiento Genético/métodos , Modelos Biológicos , Ingeniería de Proteínas/métodos , Saccharomyces cerevisiae/fisiología , Transducción de Señal/fisiología , Estilbenos/metabolismo , Simulación por Computador , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología

RESUMEN

Polyamine oxidase (PAO) catalyzes the oxidative catabolism of spermidine and spermine, generating hydrogen peroxide. In wild-type tobacco (Nicotiana tabacum 'Xanthi') plants, infection by the compatible pathogen Pseudomonas syringae pv tabaci resulted in increased PAO gene and corresponding PAO enzyme activities; polyamine homeostasis was maintained by induction of the arginine decarboxylase pathway and spermine was excreted into the apoplast, where it was oxidized by the enhanced apoplastic PAO, resulting in higher hydrogen peroxide accumulation. Moreover, plants overexpressing PAO showed preinduced disease tolerance against the biotrophic bacterium P. syringae pv tabaci and the hemibiotrophic oomycete Phytophthora parasitica var nicotianae but not against the Cucumber mosaic virus. Furthermore, in transgenic PAO-overexpressing plants, systemic acquired resistance marker genes as well as a pronounced increase in the cell wall-based defense were found before inoculation. These results reveal that PAO is a nodal point in a specific apoplast-localized plant-pathogen interaction, which also signals parallel defense responses, thus preventing pathogen colonization. This strategy presents a novel approach for producing transgenic plants resistant to a broad spectrum of plant pathogens.

Asunto(s)

Adaptación Fisiológica , Ingeniería Genética , Nicotiana/microbiología , Oomicetos/fisiología , Poliaminas/metabolismo , Pseudomonas syringae/fisiología , Cucumovirus/patogenicidad , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno , Peróxido de Hidrógeno/metabolismo , Datos de Secuencia Molecular , Oomicetos/patogenicidad , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/microbiología , Hojas de la Planta/virología , Plantas Modificadas Genéticamente , Pseudomonas syringae/patogenicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ácido Salicílico/metabolismo , Estrés Fisiológico , Nicotiana/enzimología , Nicotiana/genética , Nicotiana/virología , Virulencia , Poliamino Oxidasa

RESUMEN

Plant natural products derived from phenylalanine and the phenylpropanoid pathway are impressive in their chemical diversity and are the result of plant evolution, which has selected for the acquisition of large repertoires of pigments, structural and defensive compounds, all derived from a phenylpropanoid backbone via the plant-specific phenylpropanoid pathway. These compounds are important in plant growth, development and responses to environmental stresses and thus can have large impacts on agricultural productivity. While plant-based medicines containing phenylpropanoid-derived active components have long been used by humans, the benefits of specific flavonoids and other phenylpropanoid-derived compounds to human health and their potential for long-term health benefits have been only recognized more recently. In this part of the review, we discuss the diversity and biosynthetic origins of phenylpropanoids and particularly of the flavonoid and stilbenoid natural products. We then review data pertaining to the modes of action and biological properties of these compounds, referring on their effects on human health and physiology and their roles as plant defense and antimicrobial compounds. This review continues in Part II discussing the use of biotechnological tools targeting the rational reconstruction of multienzyme pathways in order to modify the production of such compounds in plants and model microbial systems for the benefit of agriculture and forestry.

Asunto(s)

Biología/tendencias , Biotecnología/tendencias , Flavonoides/metabolismo , Flavonoides/uso terapéutico , Extractos Vegetales/uso terapéutico , Plantas/metabolismo , Propanoles/metabolismo , Productos Biológicos/metabolismo , Productos Biológicos/uso terapéutico

RESUMEN

Plant natural products derived from phenylalanine and the phenylpropanoid pathways are impressive in their chemical diversity and are the result of plant evolution, which has selected for the acquisition of large repertoires of pigments, structural and defensive compounds, all derived from a phenylpropanoid backbone via the plant-specific phenylpropanoid pathway. These compounds are important in plant growth, development and responses to environmental stresses and thus can have large impacts on agricultural productivity. While plant-based medicines containing phenylpropanoid-derived active components have long been used by humans, the benefits of specific flavonoids and other phenylpropanoid-derived compounds to human health and their potential for long-term health benefits have only been recognized more recently. In this part of the review, we discuss in detail the recent strategies and achievements used in the reconstruction of multienzyme pathways in plants and microbes in an effort to be able to attain higher amounts of the desired flavonoids and stilbenoids exploiting their beneficial properties as analyzed extensively in Part I of this review.

Asunto(s)

Biotecnología/tendencias , Flavonoides/metabolismo , Complejos Multienzimáticos/metabolismo , Plantas/genética , Plantas/metabolismo , Propanoles/metabolismo , Ingeniería de Proteínas/métodos , Productos Biológicos/metabolismo , Productos Biológicos/uso terapéutico , Flavonoides/uso terapéutico , Complejos Multienzimáticos/genética , Extractos Vegetales/uso terapéutico

RESUMEN

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.

Asunto(s)

Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/fisiología , Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/fisiología , Pseudomonas/enzimología , Secuencia de Aminoácidos , Membrana Celular/enzimología , Membrana Celular/metabolismo , Cromatografía , Dicroismo Circular , Reactivos de Enlaces Cruzados/farmacología , Detergentes/farmacología , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Iones , Cinética , Microscopía Electrónica , Datos de Secuencia Molecular , Plásmidos/metabolismo , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Fracciones Subcelulares , Temperatura , Agua/metabolismo

RESUMEN

High throughput assays have been developed to measure the ice nucleation activity of transgenic tobacco, Nicotiana tabacum L. cv. Petit Havana SR1 plants expressing the ice nucleation gene, inaZ, from Pseudomonas syringae at a young seedling stage, as well as in leaf tissue. Both assays are carried out in 96-well microtitre plates. The first assay involves direct seeding in vitro, one seed per microtitre plate well containing Murashige-Skoog agar. When seedlings reach the two-leaf stage, they are exposed to freezing temperatures by floating the plates on a circulating alcohol bath set at temperatures colder than -9 degrees C. The second assay involves placing small leaf discs individually in microtitre plate wells containing sterile distilled water. The assays complement each other, give highly reproducible results, are technically simple and enable the detection of freezing events in large numbers of plants. The utility and limitations of these assays are discussed.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/genética , Congelación , Nicotiana/fisiología , Pseudomonas/genética , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Plantas Modificadas Genéticamente , Temperatura , Volumetría/métodos , Nicotiana/genética

RESUMEN

The avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola determines incompatibility, manifested as a hypersensitive reaction (HR), on bean cultivars carrying the R3 resistance gene and also confers avirulence on other plants. The AvrPphB protein carries an embedded consensus myristoylation motif and is cleaved in bacteria and certain plants to yield fragments of about 6 and 28 kDa. We investigated plant recognition and type III translocation determinants in AvrPphB by constructing three N-terminally truncated and two site-directed mutants carrying substitutions in the conserved G63 residue of the myristoylation motif, which lies adjacent to the proteolytic cleavage site. The peptides were either delivered to plant cells by pseudomonads or were expressed transiently in planta via the Agrobacterium tumefaciens or Potato virus X. The 63 amino terminal residues were required for type III-mediated translocation from Pseudomonas strains to the plant, but were partially dispensable for effector recognition following in planta expression. Substitution of the G63 residue resulted in differential HR phenotypes in two different R3 cultivars of bean and abolished effector processing in Pseudomonas strains. Agrobacterium-mediated expression of the mutant proteins elicited HR in resistant bean hosts and in tomato but elicited no reaction in Nicotiana species.

Asunto(s)

Proteínas Bacterianas/genética , Fabaceae/genética , Plantas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Secuencia de Consenso , Cisteína Endopeptidasas , Cartilla de ADN , Datos de Secuencia Molecular , Enfermedades de las Plantas/microbiología , Reacción en Cadena de la Polimerasa , Virulencia/genética