RESUMEN
The genus Clavibacter comprises one species and five subspecies of plant-pathogenic bacteria, four of which are classified as quarantine organisms due to the high economic threat they pose. Clavibacter michiganensis subsp. michiganensis is one of the most important pathogens of tomato, but the recommended diagnostic tools are not satisfactory due to false-negative and/or -positive results. To provide a robust analysis of the genetic relatedness among a worldwide collection of C. michiganensis subsp. michiganensis strains, relatives (strains from the four other C. michiganensis subspecies), and nonpathogenic Clavibacter-like strains isolated from tomato, we performed multilocus sequence-based analysis and typing (MLSA and MLST) based on six housekeeping genes (atpD, dnaK, gyrB, ppK, recA, and rpoB). We compared this "framework" with phenotypic and genotypic characteristics such as pathogenicity on tomato, reaction to two antisera by immunofluorescence and to five PCR identification tests, and the presence of four genes encoding the main C. michiganensis subsp. michiganensis pathogenicity determinants. We showed that C. michiganensis subsp. michiganensis is monophyletic and is distinct from its closest taxonomic neighbors. The nonpathogenic Clavibacter-like strains were identified as C. michiganensis using 16S rRNA gene sequencing. These strains, while cross-reacting with C. michiganensis subsp. michiganensis identification tools, are phylogenetically distinct from the pathogenic strains but belong to the C. michiganensis clade. C. michiganensis subsp. michiganensis clonal complexes linked strains from highly diverse geographical origins and also strains isolated over long periods of time in the same location. This illustrates the importance of seed transmission in the worldwide dispersion of this pathogen and its survival and adaptation abilities in a new environment once introduced.
Asunto(s)
Actinomycetales/clasificación , Actinomycetales/genética , Semillas/microbiología , Solanum lycopersicum/microbiología , Actinomycetales/aislamiento & purificación , Actinomycetales/patogenicidad , Proteínas Bacterianas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Datos de Secuencia Molecular , Tipificación de Secuencias Multilocus , Filogenia , Enfermedades de las Plantas/microbiología , ARN Ribosómico 16S/genética , Factores de Virulencia/genéticaRESUMEN
Molecular markers linked to phenotypically important traits are of great interest especially when traits are difficult and/or costly to be observed. In tomato where a strong focus on resistance breeding has led to the introgression of several resistance genes, resistance traits have become important characteristics in distinctness, uniformity and stability (DUS) testing for Plant Breeders Rights (PBR) applications. Evaluation of disease traits in biological assays is not always straightforward because assays are often influenced by environmental factors, and difficulties in scoring exist. In this study, we describe the development and/or evaluation of molecular marker assays for the Verticillium genes Ve1 and Ve2, the tomato mosaic virus Tm1 (linked marker), the tomato mosaic virus Tm2 and Tm2 ( 2 ) genes, the Meloidogyne incognita Mi1-2 gene, the Fusarium I (linked marker) and I2 loci, which are obligatory traits in PBR testing. The marker assays were evaluated for their robustness in a ring test and then evaluated in a set of varieties. Although in general, results between biological assays and marker assays gave highly correlated results, marker assays showed an advantage over biological tests in that the results were clearer, i.e., homozygote/heterozygote presence of the resistance gene can be detected and heterogeneity in seed lots can be identified readily. Within the UPOV framework for granting of PBR, the markers have the potential to fulfil the requirements needed for implementation in DUS testing of candidate varieties and could complement or may be an alternative to the pathogenesis tests that are carried out at present.
Asunto(s)
Ligamiento Genético , Inmunidad Innata/genética , Mapeo Físico de Cromosoma/métodos , Enfermedades de las Plantas/inmunología , Solanum lycopersicum/genética , Solanum lycopersicum/inmunología , Genes de Plantas/genética , Sitios Genéticos/genética , Marcadores Genéticos , Solanum lycopersicum/parasitología , Solanum lycopersicum/virología , Enfermedades de las Plantas/parasitología , Enfermedades de las Plantas/virología , Reproducibilidad de los Resultados , Selección GenéticaRESUMEN
We show here that MtMMPL1, a Medicago truncatula nodulin gene previously identified by transcriptomics, represents a novel and specific marker for root and nodule infection by Sinorhizobium meliloti. This was established by determining the spatial pattern of MtMMPL1 expression and evaluating gene activation in the context of various plant and bacterial symbiotic mutant interactions. The MtMMPL1 protein is the first nodulin shown to belong to the large matrix metalloendoproteinase (MMP) family. While plant MMPs are poorly documented, they are well characterized in animals as playing a key role in a number of normal and pathological processes involving the remodeling of the extracellular matrix. MtMMPL1 represents a novel MMP variant, with a substitution of a key amino acid residue within the predicted active site, found exclusively in expressed sequence tags corresponding to legume MMP homologs. An RNA interference approach revealed that decreasing MtMMPL1 expression leads to an accumulation of rhizobia within infection threads, whose diameter is often significantly enlarged. Conversely, MtMMPL1 ectopic overexpression under the control of a constitutive (35S) promoter led to numerous abortive infections and an overall decrease in the number of nodules. We discuss possible roles of MtMMPL1 during Rhizobium infection.
Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Medicago truncatula/enzimología , Nódulos de las Raíces de las Plantas/enzimología , Sinorhizobium meliloti/fisiología , Simbiosis/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Medicago truncatula/microbiología , Datos de Secuencia Molecular , Interferencia de ARN , Nódulos de las Raíces de las Plantas/microbiología , Transcripción GenéticaRESUMEN
Upon submergence, Azorhizobium caulinodans infects the semiaquatic legume Sesbania rostrata via the intercellular crack entry process, resulting in lateral root-based nodules. A gene encoding a gibberellin (GA) 20-oxidase, SrGA20ox1, involved in GA biosynthesis, was transiently up-regulated during lateral root base nodulation. Two SrGA20ox1 expression patterns were identified, one related to intercellular infection and a second observed in nodule meristem descendants. The infection-related expression pattern depended on bacterially produced nodulation (Nod) factors. Pharmacological studies demonstrated that GAs were involved in infection pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation, and that they were also needed for nodule primordium development. Moreover, GAs inhibited the root hair curling process. These results show that GAs are Nod factor downstream signals for nodulation in hydroponic growth.
Asunto(s)
Giberelinas/metabolismo , Sesbania/fisiología , Azorhizobium caulinodans/fisiología , Transporte Biológico , Botrytis , Clormequat/farmacología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Giberelinas/antagonistas & inhibidores , Giberelinas/biosíntesis , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Raíces de Plantas/citología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sesbania/crecimiento & desarrollo , Sesbania/microbiología , Triazoles/farmacología , Regulación hacia Arriba/genéticaRESUMEN
Establishment of a successful symbiosis between rhizobia and legumes results from an elaborate molecular dialogue between both partners. Bacterial nodulation (Nod) factors are indispensable for initiating plant responses, whereas bacterial surface polysaccharides are important for infection progression and nodule development. The mutant ORS571-oac2 of Azorhizobium caulinodans, affected in its surface polysaccharides, provokes a defective interaction with its host Sesbania rostrata. ORS571-oac2 induced structures with retarded development and continued generation of infection centers and organ primordia, leading to multilobed ineffective nodules. Bacterial development throughout the interaction occurred without major defects. A functional bidirectional complementation was obtained upon coinfection of ORS571-oac2 and a Nod factor-deficient mutant, indicating that the Fix- phenotype of ORS571-oac2-induced nodules resulted from the absence of a positive signal from ORS571-oac2. Indeed, the Fix- phenotype could be complemented by coinoculation of ORS571-oac2 with lipopolysaccharides (LPSs) purified from A. caulinodans. Our data show that Nod factors and LPSs are consecutive signals in symbiosis. Nod factors act first to trigger the onset of the nodulation and invasion program; LPSs inform the plant to proceed with the symbiotic interaction and to develop a functional fixation zone.
Asunto(s)
Azorhizobium caulinodans/metabolismo , Fabaceae/metabolismo , Fabaceae/microbiología , Lipopolisacáridos/metabolismo , Simbiosis/fisiología , Azorhizobium caulinodans/genética , Genes Bacterianos , Prueba de Complementación Genética , Mutación , Fenotipo , Transducción de Señal , Simbiosis/genéticaRESUMEN
Lateral root base nodulation on the tropical, semiaquatic legume Sesbania rostrata results from two coordinated, Nod factor-dependent processes: formation of intercellular infection pockets and induction of cell division. Infection pocket formation is associated with cell death and production of hydrogen peroxide. Pharmacological experiments showed that ethylene and reactive oxygen species mediate Nod factor responses and are required for nodule initiation, whereby induction of division and infection could not be uncoupled. Application of purified Nod factors triggered cell division, and both Nod factors and ethylene induced cavities and cell death features in the root cortex. Thus, in S. rostrata, ethylene and reactive oxygen species act downstream from the Nod factors in pathways that lead to formation of infection pockets and initiation of nodule primordia.