Stage:120

From Master 2 Recherche Biosciences Végétales

There is currently no text in this page. You can search for this page title in other pages, search the related logs, or edit this page.

Molecular mechanisms involved in the regulation of tolerance and resistance towards Verticillium wilt in the model legume plant Medicago truncatula

Laboratoire d'accueil : UMR 5245 Ecolab, Pôle de Biotechnologie Végétale Auzeville
Equipe d'accueil : Dynabio
Encadrant(e)(s) : Martina Rickauer (tel. 0532343887, mail: martina.rickauer@ensat.fr)

Introduction
Vascular wilt diseases cause losses in crop yields worldwide. They are characterised by the colonisation of the plant’s xylem vessels by soil-borne microorganisms (bacteria or fungi) such as fungal species of the genus Fusarium, and Verticillium. Verticillium species have a large range of host plants and infect more than 200 plant species, some of them of high economic impact (e.g. potato, hop, tomato, olive, rapeseed).
V. albo-atrum (Va) is the causal agent of vascular wilt in alfalfa (Medicago sativa), a major forage crop in temperate climates. Alfalfa yields in Europe are strongly reduced by the impact of Verticillium wilt and although tolerant cultivars are available, the bases of resistance or tolerance are not well understood. The closely related wild species Medicago truncatula has been established during the last 15 years as a model for legume plants. It is diploid, self-fertile, has a short generation cycle and a small genome size, and shows high synteny with cultivated legume crops. International efforts providing genomic and genetic resources such as genome sequence, EST databases, collections of pure lines representing the biodiversity of the species, as well as of mutants, make M. truncatula one of the most interesting model plant for the study of plant-microbe interactions.

Project
Our group was the first to describe the interaction between M. truncatula and Va. We identified resistant and susceptible lines and localised major QTLs involved in resistance to the fungus (Ben et al., 2013). The sequenced reference line A17 is one of the most resistant to Va whereas many other lines are susceptible. A highly diverse response was observed among M. truncatula lines regarding disease parameters and colonisation of aerial parts (Ben et al., 2013; Negahi et al., 2013; and unpublished results). In line A17 the fungus is eliminated after initial infection whereas in other symptomless or susceptible lines it can be detected in aerial parts.
The internship project will make use of these different interactions (resistance, tolerance, susceptibility) with one host plant in order to study the different mechanisms underlying resistance (no colonisation by pathogen) and tolerance (absence of symptoms despite colonisation). The responses of some resistant and some tolerant lines will be compared by cytological and molecular approaches. We want to obtain answers to the following questions: 1) are colonisation patterns in tolerant lines similar, and which is the infection stage where tolerant and resistant lines start to differ? 2) What is the difference between defence mechanisms in resistance and tolerance, i.e. is it possible to identify those which are killing the fungus?

The tools available for this studies are:
- a GFP-expressing Va strain to study the colonisation of plant roots and shoots
- RNAseq data recently obtained from roots of the resistant line A17 and the susceptible line F83005.5, at the moment when the fungus is eliminated from roots of the resistant line

The colonisation patterns of tolerant and resistant lines will be finely characterised with a GFP-expressing Va strain by confocal laser scanning microscopy. Defence reactions in these lines will be assessed first by a cytological approach (reactive oxygen species, NO, accumulation of phenolic compunds). Expression of an array of selected defence genes (based on analysis of the RNAseq data) will be assessed by qRT-PCR or by Fluidigm technology in roots of the tolerant and resistant lines at various time points after inoculation, determined by the microscopic study in part 1 of the project.
This work will be performed in collaboration with the PhD student who produced RNAseq data and described the colonisation patterns in A17 and F83005.5. (Maoulida Toueni, 3rd year of PhD)

Techniques to be used during the project:
Microbiology: Cultures of fungi, preparation of fungal spore suspensions
Plant pathology: inoculation assay, in-vitro plant culture, symptom scoring, statistical analysis
Molecular biology: RNA extraction, reverse transcription, qRT- PCR
Microscopy: confocal laser scanning microscopy, cytology

References:
BEN C. , TOUENI M. , MONTANARI S. , TARDIN M.C., FERVEL M., NEGAHI A. , SAINT-PIERRE L., MATHIEU G., GRAS M.C., NOËL D., PROSPÉRI J.M., PILET-NAYEL M.L., BARANGER A., HUGUET T., JULIER B., RICKAUER M., GENTZBITTEL L., 2013. Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt. J. Exp. Bot, 64, 317-332.
NEGAHI A, SARRAFI A., EBRAHIMI A., MAURY P., PROSPÉRI J.M., BEN C., RICKAUER M., 2013. Genetic variability of tolerance to Verticillium albo-atrum and Verticillium dahliae in Medicago truncatula. Eur. J. Plant Pathol. 136, 135-143