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UMR 1332 Biologie du Fruit et Pathologie

Team Mollicutes

Leader: Pascal Sirand-Pugnet - Phone: (33) 5 57 12 23 59 - E-Mail: pascal.sirand-pugnet@inra.fr
Molli-visuel-Equipe-2018

Research objectives : 

The researches involve both plant pathogenic bacteria (phytoplasmas, spiroplasmas and proteobacteria), most of which are uncultivated , and animal pathogens (mycoplasmas). Phytoplasmas, spiroplasmas and mycoplasmas belong to the class Mollicutes, a group of cell wall-less bacteria with small genomes that includes pathogens of man, animals and plants. Through the integrative studies carried out on various mollicutes and active collaborations the Mollicutes team is nationally and internationally recognized as a major pole of expertise for these organisms.

The overall objective of the team is to generate fundamental knowledge on the evolution of genomes of mollicutes and the molecular mechanisms that govern the interactions of these "minimal" bacteria with their eukaryotic hosts. In addition, part of our researches, more field-oriented, is devoted to the etiology and epidemiology of emerging phloem bacterial diseases.

Functional studies focus on a limited number of organisms: Spiroplasma citri (a model organism which can be cultured in cell-free medium and genetically engineered), and the stolbur and flavescence dorée grapevine phytoplasmas. For animal mycoplasmas, the current studies focus on ruminant mycoplasmas and especially Mycoplasma mycoides subsp. mycoides, responsible for contagious bovine pleuropneumoniae.

Lines of research :

1 - Etiology, diagnosis and epidemiology of phloem-limited bacteriosis

Contacts :

- Xavier Foissac (xavier.foissac@inra.fr)

- Sylvie Malembic (sylvie.malembic@inra.fr)

Phloem-limited bacteriosis are incurable diseases and their control is based on prophylactic methods. We develop molecular diagnostics targetting the bacterial DNA to allow the detection and the elimination of infected plants. Sequencing the genome of these bacteria has enabled the development of bacterial genotyping tools used to trace the pathways of bacterial propagation. We describe new bacterial species, demonstrate their etiological role and identify their main plant reservoirs and insect vectors. Finally, we develop the use of genetic markers predictive of phytoplasma epidemic properties to sustain a lower use of insecticide. Our research projects focus on grapevine flavescence dorée and bois noir, but also on lavender decline, marginal chlorosis of strawberry and citrus Huanglongbing. They are conducted in collaboration with the laboratories of the Euro-Mediterranean region, the plant protection services and technical institutes. The team hosts a unique collection of phytoplasma maintained on Madagascar periwinkle in safety containment greenhouse providing resources for international research and supplying diagnostic laboratories with DNA controls.

Studying genetic diversity of the group V phytoplasmas highlights exchanges between the vineyard and the wild compartment

Studying genetic diversity of the group V phytoplasmas highlights exchanges between the vineyard and the wild compartment.

2 - Interactions of phytopathogenic mollicutes with their host plant and insect vector

Contacts :

- Interactions with plants :  Sandrine Eveillard (sandrine.eveillard@inra.fr)

- Interactions with insects : Nathalie Arricau-Bouvery (nathalie.bouvery@inra.fr)

To improve control of phytoplasma diseases (eg grapevine Flavescence Dorée (FD) and Bois Noir (BN), lavender decline), we study the interactions between phytoplasmas and their host plants and insect vectors.

Our research aims at characterizing the susceptibility range to FD disease within the genus Vitis in terms of phytoplasma multiplication and diffusion in the plant. We inoculate FD phytoplasma to different grapevine cultivars and species through transmission by its natural insect vector Scaphoideus titanus. We study the deregulation of gene expression and metabolism which can explain the differences of susceptibility. The final output aims at identifying resistance sources that can be used for grapevine breeding. From the FD phytoplasma genome sequence, we seek and characterize phytoplasma effectors, i.e. secreted proteins having a role in pathogenesis. For maximum environmental safety, all operations are carried out in a high confinement greenhouse (link). 

Insect transmission of phytoplasmas to healthy plant comprises different steps: crossing the intestinal barrier, proliferation into different insect tissues and invasion of the salivary glands. Understanding these mechanisms requires studying both partners, the phytoplasma and its vector. In the case of the FD phytoplasma, we aim at characterizing both the phytoplasma components and the insect receptors implicated into insect cell invasion. The populations of the stolbur/BN phytoplasma insect vector, Hyalesthes obsoletus, are characterized in relation with their symbiotic bacterial community and phytoplasma transmission.

Axe 2 - Mollicutes

First picture on left, grapevines in High Containment greenhouse.
2nd picture, fluorescence microscopy of a salivary gland of FD phytoplasma infected insect Euscelidius variegatus.
3rd picture, Scaphoideus titanus (fifth instar nymph), the insect vector of FD phytoplasma.
4th picture, observation by confocal microscopy of an epidermal cell of Nicotiana benthamiana expressing an FD phytoplasma effector fused to YFP

3 - Synthetic biology of mollicutes

Contacts :

- Carole Lartigue (carole.lartigue-prat@inra.fr)

- Pascal Sirand-Pugnet (pascal.sirand-pugnet@inra.fr)

Using new approaches of synthetic biology, it is now possible to clone a bacterial genome into yeast, to modify it and to transplant it back to a recipient cell. These approaches are particularly promising for mollicutes for which very few genetic tools are available. Currently, genome transplantation is only restricted to very few species of mycoplasmas. Combining comparative genomics and synthetic biology, we aim at extending genome engineering and transplantation technics to other bacterial species and use them in applied projects such as construction of new vaccinale strains for the control of animal mycoplasma disease and study of phytoplasmas that are phytopathogenic, uncultivated bacteria.

Axe 3 - Mollicutes

“Genomic platform” for producing Mycoplasma mutants using synthetic biology tools
First, Mycoplasmacells are transformed with a yeast/mycoplasma integrative vector (red rectangle). This vector generally contains (i) a Centromere, an Autonomously replicating sequence and an auxotrophic marker for Histidine for selection and propagation of centromeric plasmids in yeast and (ii) a selectable-resistant marker for screening in mycoplasma cells. Second, newly marked genomes are gently isolated from mycoplasma cells and transferred into yeast spheroplasts using conventional yeast transformation procedure. After cloning, the large repertoire of yeast genetic tools is used to modify the incoming genome at will. Third, the engineered genome is then isolated and transplanted back into suitable recipient cell to generate mycoplasma mutant strains that will be further genotypically and phenotypically characterized. In some cases, methylation of the donor DNA before the transplantation step might be necessary to protect it from the recipient cell’s restriction system(s). This cycle can be repeated starting from the newly engineered genome (dashed arrow) to create a multiple knock-out and/or knock-in strains. Figure adapted  from Lartigue et al, 2009