Plasmids are a major source of horizontal gene transfer among bacteria contributing to their evolution and ecology. The known collection of plasmids carried by the plant pathogen Xylella fastidiosa increases as new strains from different origins and hosts are sampled and sequenced. Here we performed an extensive comparison of 61 publicly available sequences annotated as plasmids belonging to 38 X. fastidiosa strains isolated from different plant species and distinct geographical locations. Twenty-six strains exhibited at least one plasmid and up to four plasmids were found in a single strain. Plasmids sizes varied widely from 1.3 kbp to 64.3 kbp, ranging from 1 to 70 protein-coding sequences (CDS) encompassing 324 orthologs. Based on the presence of specific mobility proteins such as relaxases and type 4 secretion system-related genes, respectively 40 and 8 of the X. fastidiosa plasmids were classified as conjugative and mobilizable, while 13 were classified as non-mobilizable. X. fastidiosa plasmids did not carry known antibiotic resistance or virulence genes, and their stability seems to take advantage of toxin/antitoxin systems. The comparative analyses described here revealed similarity among plasmids of X. fastidiosa from different subspecies, geographical regions, and hosts, as well as with sequences found in plasmids from other bacterial species. Altogether, our results provide an in silico analysis of X. fastidiosa plasmid content and their main features, with applications in future studies of epidemiology, ecology, and evolution of this phytopathogen.

Xylella fastidiosa causes diseases in many plant species. Originally confined to the Americas, infecting mainly grapevine, citrus, and coffee, X. fastidiosa has spread to several plant species in Europe causing devastating diseases. Many pathogenicity and virulence factors have been identified, which enable the various X. fastidiosa strains to successfully colonize the xylem tissue and cause disease in specific plant hosts, but the mechanisms by which this happens have not been fully elucidated. Here we present thorough comparative analyses of 94 whole-genome sequences of X. fastidiosa strains from diverse plant hosts and geographic regions. Core-genome phylogeny revealed clades with members sharing mostly a geographic region rather than a host plant of origin. Phylogenetic trees for 1605 orthologous CDSs were explored for potential candidates related to host specificity using a score of mapping metrics. However, no candidate host-specificity determinants were strongly supported using this approach. We also show that X. fastidiosa accessory genome is represented by an abundant and heterogeneous mobilome, including a diversity of prophage regions. Our findings provide a better understanding of the diversity of phylogenetically close genomes and expand the knowledge of X. fastidiosa mobile genetic elements and immunity systems.

Xylella fastidiosa subsp. pauca, once confined to South America and infecting mainly citrus and coffee plants, has been found to be associated with other hosts and in other geographic regions. We present high-quality draft genome sequences of X. fastidiosa subsp. pauca strains J1a12, B111, U24D, and XRB isolated from citrus plants in Brazil, strain Fb7 isolated from a citrus plant in Argentina and strains 3124, Pr8x, and Hib4 isolated, respectively, from coffee, plum, and hibiscus plants in Brazil. Sequencing was performed using Roche 454-GS FLX, MiSeq-Illumina or Pacific Biosciences platforms. These high-quality genome assemblies will be useful for further studies about the genomic diversity, evolution, and biology of X. fastidiosa.

por me aceitar em seu grupo de pesquisa e pelo suporte e incentivo que me deu durante o Doutorado. Ao Dr. Paulo Zaini, por me apoiar no desenvolvimento das minhas ideias e pelas excelentes imagens de microscopia eletrônica de varredura. Ao colega Paulo Pierry pela parceria neste trabalho, pela companhia nas viagens para Cordeirópolis e pela ajuda em vários experimentos. Aos colegas Fernando Tria e Dr. Joaquim Martins Junior, pelo apoio nas análises in silico. Aos demais colegas de laboratório Oséias Feitosa, Patrícia Pessoa, Rodrigo Duarte, Gustavo Chaves, Luciana Principal, Ana Paula Souza e Deibs Barbosa pela amizade e ajuda em alguns experimentos. À Dra. Layla Farage, técnica do Centro Avançado de Tecnologias em Genômica (CATG) e aos amigos Felipe Beckedorff e Murilo Sena, pelo valioso apoio na realização do sequenciamento dos genomas da X. fastidiosa descrito neste trabalho. Ao Dr. João Paulo Kitajima pela imensa atenção e dedicação, além do excelente e heróico trabalho de montagem dos genomas das cepas descritas neste trabalho. Aos técnicos de laboratório Alexandre Sanchez, grande amigo, Marivon Pereira e Dóris Araújo, pelo imprescindível serviço prestado.

The Gram-negative bacterium Xylella fastidiosa colonizes plant xylem vessels and is obligately vectored by xylem sap-feeding hemipteran insects. X. fastidiosa causes diseases in many plant species but in a variety of its plant hosts this bacterium behaves as a commensal endophyte. Originally confined to the Americas, infecting mainly grapevine, citrus and coffee plants, X. fastidiosa has spread to several plant species in Europe, causing devastating crop diseases. Although many pathogenicity and virulence factors have been identified in X. fastidiosa which enable the bacterium to successfully establish in the xylem tissue, the mechanisms by which distinct X. fastidiosa strains colonize and cause disease in specific plant hosts have not been fully elucidated. Here we present comparative analyses of 94 publicly available whole-genome sequences of X. fastidiosa strains with the goal of providing insights into plant host specificity determinants for this phytopathogen as well as of expanding the knowledge of its mobile genetic elements (MGE) content, mainly prophages. Our results revealed a pangenome of 4,549 protein coding sequences (CDSs) which is still open. The core- and accessory genomes comprise 954 and 2,219 CDSs, respectively. Phylogenetic tree construction using all core genome CDSs grouped the strains in three major clades of subspecies fastidiosa, multiplex and pauca, with subclades related to the strains sequence type (ST) obtained from multi-locus sequence typing (MLST). The geographic region where the strains were collected showed stronger association with the clades of X. fastidiosa strains rather than the plant species from which they were isolated. Among the CDSs related to virulence and pathogenicity found in the core genome, those related to lipopolysaccharide (LPS) synthesis and trimeric autotransporter adhesins (TAA) are somewhat related with the plant host of a given strain according to phylogenetic inference. The X. fastidiosa accessory genome is represented by an abundant and heterogeneous mobilome, which includes a diversity of prophage regions. In summary, the genome comparisons reported here will enable a better understanding of the diversity of phylogenetically close genomes and warrant further investigation of LPS and TAAs as potential X. fastidiosa host-specificity determinants.