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The comprehension of the molecular mechanisms which give a pathogenic bacterium the ability to invade, colonize and reorient the physiopathology of its host is a goal of primary importance, from both academic and medical or agronomic points of view: such studies may direct the conception of new strategies to fight these pathogenic agents. The bacterium Ralstonia solanacearum is a widely accepted model organism for the study of pathogenicity in plants. This soil-borne bacterium, which belongs to the group of beta-proteobacteria, is responsible for bacterial wilts on more than 200 plant species from 50 botanical families. Several of the susceptible plants, such as potato (in which the disease caused by the infection is called brown rot), tomato, eggplant, pepper, tobacco and banana tree, are of prime agro-industrial importance. R. solanacearum in present in all the tropical and subtropical regions of the globe; recently there has been dissemination of strains with an optimal growth temperature corresponding to the temperate zones of Europe and the United States.
The study of R. solanacearum is complementary to that of the other phytopathogenic model bacteria Pseudomonas syringae, Xanthomonas campestris and Xanthomonas citri. These three species attack the aerial parts of the plant, whereas R. solanacearum, which is capable of living for prolonged periods in the soil, infects its hosts beginning with the root system and presents a very strong tropism for the xylem vessels. Its extensive multiplication in the water-conducting system leads to a systemic infection of the plant.
A comparison of the sequences of a group of genes -including 16S RNA- has revealed that the R. solanacearum species can be classified into four large evolutionary clades. The strains within a same clade can exhibit major differences in parasitic specificity. This makes R. solanacearum an excellent model for studying the determinants of parasitic specificity as well as the evolutionary mechanisms which have led to the emergence of pathogenicity. This latter research theme will also benefit from the knowledge of the genome of Ralstonia metallidurans, a bacterium which is taxonomically close to R. solanacearum, but is non-pathogenic and for which a draft genome sequence was assembled in 2003 by the Joint Genome Institute (Walnut Creek, California).
For a first exploration of the genome of R. solanacearum, Christian Boucher’s group at the Laboratoire Interactions Plantes-Microorganisms (LIPM, mixed CNRS/INRA unit) selected the CMI1000 strain. This strain, which was isolated in Guyana from a withered tomato plant, belongs to the Asiatic clade and has a wide host range. Furthermore, it can infect the model plant, Arabidopsis thaliana, whose genome has been entirely sequenced. This ability offers the possibility of studying host responses at the genomic scale. The genomic sequence of CMI1000 which was assembled at Genoscope has been annotated by C. Boucher’s group. It is 5.8 Mb long and is organized in two replicons, a chromosome of 3.7 Mb and a megaplasmid of 2.1 Mb.
The annotation work has led to the identification of numerous candidate genes for pathogenesis, including structural genes for more than 50 so-called “effector” proteins. Some of these proteins act by perturbing the metabolism of the plant cell, whereas others suppress the defense reactions of the plant. The effectors are injected by the bacterium into the plant cell using a type III secretion system, whose components had been characterized before sequencing. Among other factors of pathogenesis identified during the annotation are several lytic enzymes, enzymes which control the production of plant hormones and numerous factors implicated in cell adherence. This work, which was published in January 2002, also presented evidence for the type of role horizontal genetic transfer could play in the acquisition of pathogenicity.
The genomic sequence of CMI1000 only presents a small part of the genetic diversity of R. solanacearum, as shown by a sampling of the genome of another strain. Almost 100 kb from the genome of the strain Molk2, which belongs to the American clade and is strictly limited to the banana tree, were sequenced at random. Comparison of the sequences indicates that up to 30% of the genome of Molk2 is absent in the genome of CMI1000. This observation has justified the launching of a new sequencing project at Genoscope in 2004, in collaboration with C. Boucher’s team. The objective is to obtain a “draft” of the genomic sequence of two new strains, Molk2 and 1609. Like Molk2, this latter strain belongs to the American clade, but differs in that it does not attack banana trees, and in that it is especially well-adapted to the potato. The 1609 strain is considered to be the type strain for strains introduced in Europe.
The research in progress today is mainly oriented toward functional analysis of the effectors and the characterization of their molecular targets in the plant cell. The sequencing of two additional strains will create a new research axis: the analysis of determinants of parasitic specificity. This work will utilize a pangenomic chip from CMI1000 which is available today. The capacity of this chip will soon be extended with new genes which will be identified in the genomic sequences of the Molk2 and 1609 strains. Thus increased, the chip will enable correlations between genes present and expressed in about 50 strains of R. solanacearum ( collection maintained at LIPM) and the nature of plant hosts which can be infected by these strains. Using the data obtained in this way, C. Boucher’s team will try to develop a molecular tool which will make it possible to rapidly evaluate the infectious potential of any new R. solanacearum strain.