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Stage:169
From Master 2 Recherche Biosciences Végétales
Laboratoire d'accueil : LIPM (Laboratoire des Interactions Plantes - Microorganismes) UMR CNRS-INRA)
Equipe d'accueil : Plant response to environment and immunity networks
Encadrant(e)(s) : Dominique ROBY (Dominique.Roby@toulouse.inra.fr) et Ullrich DUBIELLA (Ullrich.Dubiella@toulouse.inra.fr)
As humans, plants are constantly exposed to micro-organisms. During a coevolutionary arms race, plants developed an elaborated, multilayered system to detect the presence of pathogenic micro-organisms and to defend themselves. A lot of progress has been made in the understanding of R-gene related defense (ETI, Effector Triggered Immunity) (Qi & Innes 2013; Gill et al. 2015). In this case the plant reacts with a very strong defense reaction (hypersensitive response, HR), which results in the death of the infected cells and therefore prevents the spread of pathogens. This consequently creates a strong evolutionary pressure for the selection of virulent pathogen strains that can bypass recognition.
Because ETI fails to provide durable and broad-spectrum resistance in an agricultural context in most cases, increased attention has been recently devoted to quantitative disease resistance (QDR; Roux et al., 2014). QDR is a form of resistance which leads to a reduction in disease, rather than an absence of disease (Poland et al., 2009); and is typically polygenic (Roux et al., 2014). While QDR is the most prevalent strategy of defense in crops and natural plant populations, only very little is known about the molecular mechanisms underlying this form of resistance.
Recently our lab identified the atypical kinase RKS1 as a major component of QDR against the bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) (Huard-Chauveau et al. 2013). Xcc infects a broad range of plants within the Brassicaceae family, which includes crop plants like broccoli, cauliflower, mustard and radish but also the model organism Arabidopsis thaliana. By using Arabidopsis thaliana as a model, we are now interested in deciphering the molecular mechanisms by which RKS1 confers resistance against Xcc.
The project will consist in the identification of new components of the RKS1-dependent resistance pathways, using two complementary strategies.
I) The identification and characterization of direct interacting partners of RKS1
A yeast two hybrid (Y2H) screen is currently underway in the lab,and will allow to isolate candidate genes encoding proteins potentially interacting with RKS1. Direct interaction of some of these candidates will be validated in the lab, and the project during the Master will focus on one candidate:
- further confirmation of Y2H results in planta
- intracellular localization studies
- characterization of KO lines (single and double mutants)
- biochemical characterization of the interaction partner either as substrate for RKS1 kinase activity or RKS1 dependent modulation of its biochemical activity (dependent on the nature of the interacting protein)
- evaluation of a potential co-evolution of RKS1 and its interaction partner.
(II) The identification and functional analysis of putative components of the RKS1 signalling pathway
Candidate genes have been identified by transcriptome analysis of Arabidopsis lines mis-expressing RKS1 using the RNA-sequencing (RNA-seq) methodology. . Validation by Q RT-PCR of the expression profiles of the candidate genes, using independent inoculation experiments, will be performed in the lab and the project during the Master will include:
- Search for, characterization and phenotyping of KO mutants for the candidate genes.
- In the case of only one KO mutant line is available, complementation will be performed.
Methods:
Molecular biology: cloning, PCR, Q-RT PCR…..
Plant pathology: Pathogen assays with Xcc in Arabidopsis thaliana
Biochemistry: Co-immunoprecipitation, SDS-PAGE, Western blot, protein affinity-chromatography,
in-vitro kinase assays, protein expression in E. coli and in planta
Plant genetics: Stable transformation of Arabidopsis thaliana
Literature:
Gill, U.S., Lee, S. & Mysore, K.S., 2015. Host Versus Nonhost Resistance?: Distinct Wars with Similar Arsenals. Phytopathology, 10(5): 580–587.
Poland, JA, Balint-Kurti, PJ, Wisser, RJ, Pratt, RC, Nelson, RJ. (2008) Shades of gray; the world of quantitative disease resistance. Trends in Plant Science, 14 (1): 21-29
Roux, F. et al., 2014. Resistance to phytopathogens e tutti quanti: Placing plant quantitative disease resistance on the map. Molecular Plant Pathology, 15(5): 427–432.
Huard-Chauveau, C. et al., 2013. An Atypical Kinase under Balancing Selection Confers Broad-Spectrum Disease Resistance in Arabidopsis. PLoS Genetics, 9(9).
Qi, D. & Innes, R.W., 2013. Recent Advances in Plant NLR Structure, Function, Localization, and Signaling. Frontiers in immunology, 4(348): 1–10.