Our research aims at understanding the molecular mechanisms enabling the root pathogen Ralstonia solanacearum to promote disease on a wide array of host plants. R. solanacearum is probably one of the most destructive plant pathogenic bacterium worldwide, infecting more than 200 plant species in over 50 families, including major crops such as tomato, potato but also peanut and banana. Owing to the fact that several model plants are also hosts (Arabidopsis, Tomato, Medicago) the mechanisms involved in this plant-pathogen interaction are getting better understood.
We developed genetic and genomic approaches on the model R. solanacearum strain GMI1000 to identify essential pathogenicity determinants such as the Type 3 protein secretion system. This secretion system, present in many pathogenic bacteria, is a sophisticated tool allowing the pathogen to manipulate eukaryotic host cells with the direct transport of bacterial virulence proteins (effectors) into the host cells. We have identified more than seventy effectors in R. solanacearum. The molecular activities of most of these effectors are still unknown and represent an exciting prospect for our team in order to better understand the underlying mechanisms of this disease. For this, we are looking for plant targets of these effectors, direct or indirect targets related to resistance or susceptibility in Arabidopsis and Tomato plants.
We are also interested in the mechanisms of adaptation of the bacterium to its environment, and in particular its exceptional ability to colonize a wide range of hosts. We have developed an experimental evolution approach in which, by serial inoculations on a given plant species, variants with fitness gains have been identified. The characterization of genetic or epigenetic alterations of these variants has already shown the key role of a virulence regulatory network in these adaptation processes. This work has recently been supplemented by a system’s biology approach aimed at reconstructing both the metabolic and virulence regulatory networks in order to study more finely the close interconnection between metabolism and virulence and to initiate a modeling of the infectious dynamics of the pathogen.
Functional analysis of Type 3 effectors
R. solanacearum possesses an abundant repertoire of Type 3 effectors (T3Es). A newly created database contains all the predicted T3Es for a large collection of strains. Since these effectors are injected by the bacterium into plant cells, the elucidation of Type 3 effector functions require the identification and characterization of their plant targets in order to understand their mode of action in the host cell.
Through comparative genomic approaches on the many available genomes, we defined a group of 'core' T3Es whose presence is conserved in the strains representative of the biodiversity of the species. Systematic searches of protein interactors of these effectors in tomato are currently being carried out using yeast-two-hybrid. Using this knowledge we are performing reverse genetics (mainly on Solanaceae) to identify key players involved in the control of this bacterial disease. The objectives are to identify either (i) susceptibility genes and/or (ii) alleles able to escape the recognition by T3Es, in order to propose new means to improve plant tolerance/resistance to this bacterial pest.
The search for resistance or susceptibility genes by screening natural plant diversity (GWA approach) is also underway (Arabidopsis, tomato).
Several of these effectors have been functionally characterized such as the RipG effector family, which have LRR and F-box domains and which probably mimic the action of some plant components with E3 ubiquitin ligase activity. Another way of investigation concerns the regulation of the translocation process of Type 3 effectors into plant cells. We identified Type 3 chaperones controlling secretion of effectors and some observations suggest that alteration of the secretion process can be detrimental on specific hosts.
Our group also characterized several effector proteins that specify host range of R. solanacearum GMI1000 towards several plants, such as, for example, the AvrA and PopP1 avirulence proteins which are recognized by the tobacco immune system and trigger a defensive hypersensitive response.
Adaptation of R. solanacearum to his environment
This approach has unraveled genetic alterations targeting essential regulatory genes impacting bacterial fitness in planta. The functional characterization of these genes is currently being carried out. More recently, we developed experiments aimed at determining the importance of epigenetic alterations in adaptation to the host.
In order to explore the metabolic adaptation of the pathogen to physiological host conditions in a global context, we developed a system’s biology approach aiming at reconstructing the bacterial metabolic and virulence regulatory networks. All the metabolic reactions identified through the genome annotation were manually curated to generate a high-quality genome-scale metabolic reconstruction. Predictive model-based approaches with these reconstructed networks will be used in the future to study how environmental variables constraint pathogenic fitness.
Fernandez de Grado Quentin
Master INSA (2021-2022)