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Team leader : Pascal Sirand-Pugnet (pascal.sirand-pugnet@inrae.fr - (33) 5 57 12 23 59)

Research objectives : 

The Mollicutes team works on bacteria that are pathogenic to plants (phytoplasmas, spiroplasmas) and animals (mycoplasmas). Phytoplasmas, Spiroplasmas and mycoplasmas belong to the class Mollicutes, a group of « minimal » bacteria characterized by a lack of cell wall and very small genomes.

The main goal of the team is the production of knowledge on the biology and evolution of these bacteria with a specific focus on the molecular mechanisms involved in the interaction with their eukaryotic hosts. This knowledge stands as a basis for the development of new strategies towards the control of the diseases involving these bacteria, especially on grapevine (Flavescence dorée phytoplasma) and in ruminants (mycoplasmas from the mycoides cluster of species).

Using integrated approaches including epidemiology, etiology, molecular biology, biochemistry, biophysics and cutting-edge technologies of synthetic biology (genome-scale engineering and genome transplantation), the team develops a wide range of research projects, from the most fundamental (minimal cell concept, production of bacterial chassis, construction of synthetic vesicles mimicking cells, structure of bacterial membranes) to the most applied (diagnostic methods and control strategies of phytoplasmas and corresponding insect vectors, construction of new vaccine strains).

Through the integrative studies carried out on various mollicutes and active collaborations the Mollicutes team is nationally and internationally recognized as a major expert on these organisms.

Lines of research :

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

Contacts :
- Phloem-limited bacteriosis : Xavier Foissac (xavier.foissac@inrae.fr)
- Flavescence dorée : Sylvie Malembic-Maher (sylvie.malembic-maher@inrae.fr)

Phloem-limited bacteriosis are incurable diseases and their control is based on prophylactic methods. We develop molecular diagnostics targeting the bacterial DNA to allow the detection and the elimination of infected plants. Sequencing the genome of these bacteria enabled the development of bacterial genotyping tools used to trace their propagation pathways. We describe new phytoplasma, liberibacter or phlomobacter species, demonstrate their etiological role and identify their main plant reservoirs and insect vectors. Finally, we develop and promote 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 carrot proliferation. They are conducted in collaboration with the laboratories of the Euro-Mediterranean region, the plant protection services, technical institutes and researchers in the human and social sciences specialized in the management of epidemics in agriculture. We participate together in the co-construction of multidisciplinary projects dedicated to optimizing the management of these diseases. 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.

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

Contacts :
- Interactions with plants :  Sandrine Eveillard (sandrine.eveillard@inrae.fr)
- Effectors : Christophe Garcion (christophe.garcion@inrae.fr)
- Interactions with insects :
Nathalie Arricau-Bouvery (nathalie.bouvery@inrae.fr) Xavier Foissac (xavier.foissac@inrae.fr)

To improve control of phytoplasma diseases (e.g. 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. We compare the disturbances of gene expression, in particular those implied in the defense and metabolism of the grapevine, in Cabernet-Sauvignon and Merlot, two varieties which present a contrasted susceptibility.

In parallel, we are investigating the proteins (termed as "effectors") produced and excreted by the flavescence dorée phytoplasma. The involvement of these proteins in the interaction with the host has been largely demonstrated for many pathogens, including phytoplasma. Through an analysis of the genomic sequence of the flavescence dorée phytoplasma, we established a list candidate effectors. Current work now aims at determining their role and understanding how they participate in the infection and colonization process of the hosts by the phytoplasma.

The transmission of the phytoplasma by the insect vector begins with the colonization of the midgut during the acquisition phase and ends with the invasion of the salivary glands which then makes the insect infectious. These processes involve mechanisms of cell adhesion, internalization and intracellular multiplication. Our research aims first to identify both the adhesins of the bacterium and their receptors in the insect, then to determine how this adhesin-receptor recognition facilitates the invasion of the cells of the leafhopper. Electron and confocal microscopy imaging allows us to visualize and quantify the effectiveness of the adhesion and internalization steps on leafhopper organs or their cells in culture. The inhibition of the expression of the leafhopper genes by RNAi allows us to validate the implication of insect proteins and to try to block the phytoplasma transmission.

3 - Synthetic biology for the engineering of mollicutes genomes

Contacts :
- Carole Lartigue (carole.lartigue-prat@inrae.fr)
- Yonathan Arfi (yonathan.arfi@u-bordeaux.fr)
- Pascal Sirand-Pugnet (pascal.sirand-pugnet@inrae.fr)
- Alain Blanchard (alain.blanchard@inrae.fr)

Mollicutes are arduous to study, owing in large part to the limited number of genetic tools available for these bacteria.

Recently, new methods in Synthetic Biology have been developed in order to transfer (“Cloning”) and maintain a mycoplasma chromosome in a yeast. Once in the yeast, this bacterial genome can be precisely modified (“Editing”) using the large array of tools available, including CRISPR/Cas9. In a third step, the bacterial genome can be transferred back in a bacteria (“Transplantation”) to bring to life a new, genetically-modified cell.

This “Cloning-Editing-Transplantation” cycle (“C-E-T”) has currently been achieved for a small number of mycoplasma, and has allowed tremendous improvement of our understanding of these organisms. The ability to generate mutants allows us to dissect the various mechanisms involved in mycoplasma virulence, and to produce genetically attenuated strains to use as basis for new and improved veterinary vaccines. 

Together with Synthetic Biology approaches, we also use comparative genomics to study the evolution of mollicutes and build hypotheses to understand the genetic basis of the metabolism and pathogenicity of these minimal bacteria. In collaboration with the Bordeaux Bioinformatics Center (CBiB), we are developing a database dedicated to the comparative genomics and genome engineering of mollicutes, MolliGen (molligen.org).

We are trying to perform the “C-E-T” cycle and other genome engineering technics to several mycoplasma species, in order to improve our understanding of these diversified pathogens, often exhibiting species-specific virulence mechanisms.

We also wish to apply synthetic biology tools to the study of phytoplasmas. The phyto-pathogenic bacteria are currently impossible to grow in a laboratory, greatly impeding our ability to study them. The “C-E-T” cycle could pave the way to the creation of genetically engineered phytoplasma able to grow in axenic conditions.

We are also attempting to apply our genome engineering platform outside of the Mollicutes Class, in particular in the model bacterium Bacillus subtilis. These tools would open the way to the rapid manufacturing of rationally designed strained for industrial applications in the fields of green chemistry and biosynthesis.

 4 – Biochemistry and biophysics of mollicutes

Contacts :
- Biochemistry and biophysics of biological membranes : Laure Béven (laure.beven@inrae.fr)
- Physical chemistry of synthetic cells : Jean-Paul Douliez (jean-paul.douliez@inrae.fr)

The formulation of compartments that can encapsulate biological materials (enzymes or structural proteins, DNA, etc.) is of considerable interest, not only from an industrial point of view, but also for fundamental research aimed at the development of artificial cells. We are interested in the formulation of artificial compartmentalized systems mimicking the mycoplasma cell. This choice of model is mainly based on the "minimal" aspect of these bacteria, which harbor a small-sized genome, a single cell membrane and which lack a cell wall as well as many biosynthetic pathways commonly found in other bacteria. Once these compartments have been formulated, our studies aim to characterize them, to understand the mechanisms of encapsulation and concentration of biomolecules, and to identify ways to control exchanges at the envelope level. We have developed different systems (colloidosomes, capsules, vesicles formed after coacervate-to-vesicle transition) allowing the encapsulation and the concentration of small molecules, enzymes and DNA. In order to feed them with molecules, we now focus on the control of their permeability and morphology. In order to meet this challenge, knowledge of the organization of biological membranes and the biological processes responsible for the exchanges taking place there is essential. We are therefore conducting parallel studies on the membrane organization of mycoplasmas and spiroplasmas as minimal cell models whose membrane permeability is perfectly controlled, and try to apply the lessons of these studies to the development of artificial minimal cells. This is a bottom-up strategy of synthetic biology aimed at reconstructing cellular mimics from elementary bricks of biological or chemical origin.

The data on membrane organization and composition of spiroplasmas and mycoplasmas that we obtain are also very useful for better understanding the physiology, maintenance of morphology and pathogenicity of mollicutes. Our favorite model bacterium is Spiroplasma citri, a phytopathogenic, helical and motile mollicute. They allow us to better understand how, despite an apparent structural simplicity and a small genome, these bacteria have adapted to many hosts. We were able to identify and characterize several membrane-associated, molecular factors involved in the transmission of this spiroplasma to plants by its insect vector, or in the maintenance of the bacterial shape. As they progress, our research uncovers various molecular mechanisms that allow this bacterium to gain maximum benefit from a minimal genome, indicating that behind this structural and metabolic simplicity hides great adaptability of these bacteria.

We apply a multidisciplinary approach using the biochemistry of biological membranes, the physicochemistry of polymers, microbiology and use various resolving technologies of microscopy. This research is part of a transverse axis in the team and is more specifically closely related to the axes 2 and 3 described above.