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Grapevine flavescence dorée symptoms © Sandrine Eveillard

Grapevine flavescence dorée

Using spiroplasmas to study flavescence dorée by proxy

Flavescence dorée, a type of grapevine yellows, is a disease that is threatening European vineyards. It is caused by a phytoplasma, a bacterium that cannot be cultured in the lab. This challenge can be circumvented by studying a more amenable bacterial relative, notably a spiroplasma that was characterized by INRA scientists in the 1970s.

By Pascale Mollier, translated by Jessica Pearce
Updated on 09/06/2016
Published on 08/29/2016

The flavescence dorée phytoplasma – an uncooperative study subject

Phytoplasmas are bacteria belonging to the taxonomic class Mollicutes; they have the particularity of lacking a cell wall. They are also obligate intracellular parasites that live in plant phloem and are transmitted by phloem-feeding insects. All efforts to date to grow phytoplasmas in the lab have failed (1), rendering molecular studies extremely difficult. Yet such data are necessary if we wish to understand how the pathogen’s surface proteins, called VMPs (2), interact with the cells of the insect vector. Such interactions are key in host specificity.

The spiroplasma – a more accommodating relative

Spiroplasma citri is a spiroplasma with a similar ecological niche to the flavescence dorée phytoplasma. It also lives in plant phloem and is vectored by phloem-feeding insects. However, in contrast to the phytoplasma, S. citri can be cultured in the lab and thus be genetically modified. As a result, it is an ideal candidate for conducting recombinant experiments involving genetic material from the flavescence dorée phytoplasma.

Making the switch

Transmission electron microscope image of Spiroplasma citri expressing VmpA. The presence of the VmpA proteins is revealed by gold nanoparticles coupled with the secondary antibodies targeting the anti-VmpA antibodies produced by the phytoplasma.. © INRA, Joël Renaudin et Brigitte Batailler
Transmission electron microscope image of Spiroplasma citri expressing VmpA. The presence of the VmpA proteins is revealed by gold nanoparticles coupled with the secondary antibodies targeting the anti-VmpA antibodies produced by the phytoplasma. © INRA, Joël Renaudin et Brigitte Batailler

The first step in the process is to construct recombinant spiroplasmas that can express genes for different proteins that are secreted or found on the surface of the phytoplasma.

The results obtained for one type of membrane protein, VmpA, are very promising. The recombinant spiroplasmas successfully produced VmpA and, furthermore, secreted functional proteins into the extracellular environment.

This preliminary study sought to evaluate how feasible it was to use recombinant spiroplasmas to see how VMPs affect host specificity in the flavescence dorée phytoplasma.

This research should ultimately enhance our understanding of this pathogen, facilitate the identification of other vectors, and help evaluate the risk of future outbreaks.
 

(1) The rudimentary metabolism of this taxon likely explains their inability to reproduce in vitro, as well as their status as obligate parasites.
(2) VMPs: variable membrane proteins

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Plant Health and Environment
Associated Centre(s):
Nouvelle-Aquitaine-Bordeaux

Reference

Renaudin, J., L. Béven, B. Batailler, S. Duret, D. Desqué, N. Arricau-Bouvery, S. Malembic-Maher and X. Foissac. 2015. Heterologous expression and processing of the flavescence dorée phytoplasma variable membrane protein VmpA in Spiroplasma citri. BMC Microbiol 15:82-93.

Joseph-Marie Bové sur le terrain en verger. © INRA, Inra

INRA RESEARCHERS DISCOVER A NEW SPIROPLASMA

In the 1970s, Joseph-Marie Bové and colleagues characterized Spiroplasma citri, the pathogen responsible for stubborn disease, which is vectored by leafhoppers and severely affects citrus species, notably orange and grapefruit. The advance took place at the INRA center in Bordeaux. The researchers managed to culture these bacteria in an environment similar to the one used for growing mycoplasmas, bacteria hosted by animal species, including humans. Once S. citri had been cultured, long-time international collaborators of the INRA team were able to confirm the bacterium’s helical form, which is unique to the Spiroplasma genus and responsible for its corkscrew-like motion. S. citri was officially recognized as a new species by the international scientific community in 1973. The genus Spiroplasma would continue to swell with new members, becoming extremely large. Spiroplasmas are rarely found in plants; they more often occur in a wide variety of arthropods, from the mosquito to the crawfish. Read this article (in French) for more information.