Función y origen de los plásmidos en especies de Erwinia patógenas y epífitas de frutales de pepita
Fire blight is considered the most serious disease affecting pome fruit and ornamental and wild rosaceae. The causal agent is the bacterium Erwinia amylovora, considered a quarantine organism in the EU. This species has been extensively studied, but at the genomic level there is still much to know,...
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| Formato: | tesis doctoral |
| Fecha de publicación: | 2017 |
| País: | España |
| Recursos: | Universitat Politècnica de València (UPV) |
| Repositorio: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
| Idioma: | español |
| OAI Identifier: | oai:riunet.upv.es:10251/86146 |
| Acesso em linha: | https://riunet.upv.es/handle/10251/86146 |
| Access Level: | acceso abierto |
| Palavra-chave: | plásmidos especies de Erwinia Erwinia amylovora Erwinia piriflorinigrans microarray detección expresión diferencial de genes PCR |
| Resumo: | Fire blight is considered the most serious disease affecting pome fruit and ornamental and wild rosaceae. The causal agent is the bacterium Erwinia amylovora, considered a quarantine organism in the EU. This species has been extensively studied, but at the genomic level there is still much to know, as there are currently only two genomes published and another thirteen were assembled in scaffolds. Its pangenoma is considered open, although with a core with a high sequence identity. There is very little intraspecific variability, which is manifested in a low genotypic diversity, being noticed that the plasmids are the major source of genetic variability. This could explain the differences in virulence in strains, as well as their better adaptation to the different environmental conditions. The plasmid pEA29 described in the majority of the strains of E. amylovora, and with a quantitative effect in virulence, was not found in some Spanish isolates of the bacterium. The study of these strains gave way to the discovery of another plasmid, pEI70, which is present in strains of several European countries. After pEA29, pEI70 is the one with the highest presence. The function, distribution and genetic content of this plasmid, as well as the effect of pEA29 and pEI70 on the expression of the chromosomal genes in strain bearing them, have been studied. The inoculation experiments on fruit with the strains to which the plasmid pEI70 or pEA29 had been introduced compared to that same strain without plasmids showed an increase in virulence, which was manifested in a reduction in the time of the emergence of symptoms and in which they appeared more aggressively. An experiment was carried out using a microarray, in order to study if the presence of each one of these plasmids could affect the expression on certain chromosomal genes that would explain that variation in virulence of the carrier strain, using a microarray. The results demonstrated the role of both plasmids to affect gene expression, between 120 and 180 chromosomal genes according to the plasmid carrying the strain, in each case enriching different functional categories, although 28 of them were coincident in the two cases. E. piriflorinigrans is a newly described pathogenic species that produces necrosis only in pear blossoms but does not appear to affect other organs. Both species share phenotypic and molecular characteristics, making their distinction difficult. Its detection and correct identification was a challenge because the symptoms it causes are practically indistinguishable from those caused by E. amylovora. In this work new plasmid pEPIR37 found in this new species was studied also. This is present in all analyzed strains. When this plasmid was introduced into strains of the species E. amylovora cured of plasmids, they showed an increase in virulence comparable to that observed with pEA29, suggesting that pEPIR37 produces a similar effect. Two specific and sensitive real-time and conventional PCR protocols have also been developed to identify, detect and differentiate E. piriflorinigrans from E. amylovora and other species of this genus using primers designed from specific sequences, annoted in this same work, from plasmid pEPIR37. This has allowed to identify this new species in other hosts as Pyracantha sp., besides pear tree and in other regions where previously it had not been detected. Likewise, these results have allowed to know biological and epidemiological aspects of E. piriflorinigrans that contribute to have new key scientific information to establish strategies for its control in pome fruit trees. The study of the plasmids and their functions in these two phylogenetically related species and their role in the adaptation to the environment in which these species live, as well as in the virulence of the strains that carry them, could give new clues about the origin of both pathogens, their evolution, their biological cycle and interaction with the host plant |
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