Research topics
1. Genetic determinants of Disease.
For this part of our research, we use the wide-host range pathogen Ralstonia solanacearum as a model. R solanacearum is a soil-borne pathogen whose major virulence determinant is the type III secretion system (T3SS). This secretion machinery delivers bacterial effector proteins into host cells, in an engaging example of information transfer between evolutionary distant organisms. In addition, the main regulatory cascades controlling expression of the T3SS and other virulence genes is well characterised. We are currently addressing the following questions:
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1.1 Molecular characterisation of R. solanacearum type III pathogenicity determinants
We are characterising novel R. solanacearum effectors so as to unravel their molecular function and their contribution to disease. The process includes a) study of the expression profile of candidate genes, b) disruption of each structural gene and analysis of the resulting phenotype regarding pathogenicity, c) demonstration of effector translocation into plant cells, d) determination of the sub-cellular localisation of bacterial effectors inside the plant and e) identification of molecular targets in the eukaryotic host. We have thus far studied theawr gene family, which encodes type III effectors involved in virulence and avirulence activities on solanaceous plants (Solé et al. MPMI 2012)
Bacterial wilt on different host plants of Ralstonia solanacearum
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1.2 Environmental cues controlling the pathogenicity of R. solanacearum
In order to study the molecular mechanisms controlling R. solanacearum pathogenicity, we developed set of genetic tools: the pRC series, which allows the determination of promoter activities both in vitro andin planta, complementation of mutants and protein overexpression (Monteiro et al. MPMI 2012). The pRC system has already been applied successfully to strains GMI1000 (phylotype I) and UW551 (phylotype II) and it is freely available to the community. Recently, we reported expression of the T3SS regulator HrpB and the effector protein PopA at advanced stages of wilting (Monteiro et al. Microbiology 2012). We are now using the generated pRC constructs to identify the environmental signals triggering the expression/repression of type III secretion genes both in planta and synthetic media. In the long term, the data we are gathering from transcriptional profiling experiments will help us establish the genetic program deployed by the pathogen during infection, including the timings and intensities of expression of the main virulence activities. This knowledge can lead to the rational design of crop protection strategies, or at least, provide a means to evaluate resistance and susceptibility.
Expression of R. solanacearum genes during plant infection (GFP reporter)
Time-course evaluation of bacterial wilt on single plants of S. commersonii accessions inoculated with R. solanacearum UY031 (lux reporter) dai – days after inoculation
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1.3 Characterization of Ralstonia solanacearum type III effectors toxicity in Saccharomyces cerevisiae
Understanding the function of R. solanacearum pathogenicity determinants in plants may be difficult because of the redundancy in the effector repertoire of the bacterial strain and the presence of plant resistance genes. Expression Ralstonia type III effectors of in the effector repertoire of the bacterial strain and the presence of plant resistance genes. Expression Ralstonia type III effectors of in the budding yeast S. cerevisiae overstep these limitations. This research project aims to use yeast as a genetic system to identify the processes that type III effectors target to cause cell death. To this end, gain-of-function and loss-of-function screenings are under way.
2. Metacaspases as executioners of programmed cell death.
Programmed cell death (PCD) is an essential process for normal development and defense against pathogens in all multicellular organisms. Some of its key regulators evolved from primordial molecules likely serving homeostatic functions in a unicellular common ancestor. In animals and plants, millions of years of separate evolution have resulted in unique and exquisitely fine-tuned PCD systems which still serve the most important function: keeping the organism alive. In animals, the execution systems that control cell death involve a limited number of pathways that converge on the activation of the caspase family of proteases. In contrast, in plants very little is known about the mechanisms that commit a cell to die after perception of a cell death-trigger. We have recently shown that metacaspases, distant relatives of caspases, are main regulators of PCD in plants: Type I metacaspases AtMC1 and AtMC2 antagonistically control pathogen-triggered cell death upon innate immune receptor activation (Coll et al. Science 2010), also known as the hypersensitive response (HR) cell death. Despite the fact that the metacaspases are divergent from animal caspases, they control similar processes, suggesting an ancient evolutionary link between cell death control and innate immune system, analogous in plants and animals. We are currently studying:
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2.1 Protein complex dynamics during HR cell death:
What is the composition of supramolecular protein complexes (deathosomes) that regulate execution of HR PCD? How do these protein hubs dynamically respond to pathogen recognition? How do plant deathosomes transduce the signal downstream once they have been formed? What are their mechanisms of action? What is the role of AtMC3, an atypical type I metacaspase, during HR cell death?
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2.2 The role of autophagy during metacaspase-regulated cell death.
Autophagy participates in immune cell death control in both animals and plants. In plants, it is still not well understood how these two pathways are interconnected. We are investigating what are the functional links between autophagy and pathogen-triggered cell death.
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2.3 Melon metacaspases and their role in HR cell death triggered by economically relevant pathogens in this crop species.
The mechanisms leading to disease resistance in horticultural species are still poorly understood. Taking advantage of the molecular and genetic tools developed for melon, an agriculturally relevant crop in Spain, we are currently studying the role of metacaspases during HR cell death in this species, triggered upon pathogen recognition.
Metacaspase networks in Arabidopsis thaliana