Heterologous expression and processing of the flavescence dorée phytoplasma variable membrane protein VmpA in Spiroplasma citri

BMC Microbiology

Xavier Foissac²  Sylvie Malembic-Maher²  Nathalie Arricau-Bouvery²  Delphine Desqué²  Sybille Duret²  Brigitte Batailler¹  Laure Béven²  Joël Renaudin² 

[1] INSERM, Bordeaux Imaging Center, Bordeaux, US 004, France;Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d’Ornon, France

关键词: Phytoplasma effector;    Protein secretion;    Surface expression;    Spiroplasma citri;    Variable membrane protein;    Flavescence dorée;    Phytoplasma;   

Others  :  1206336
DOI  :  10.1186/s12866-015-0417-5

 received in 2014-12-10, accepted in 2015-03-18,  发布年份 2015

【 摘 要 】

Background

Flavescence dorée (FD) of grapevine is a phloem bacterial disease that threatens European vineyards. The disease is associated with a non-cultivable mollicute, a phytoplasma that is transmitted by the grapevine leafhopper Scaphoideus titanus in a persistent, propagative manner. The specificity of insect transmission is presumably mediated through interactions between the host tissues and phytoplasma surface proteins comprising the so-called variable membrane proteins (Vmps). Plant spiroplasmas and phytoplasmas share the same ecological niches, the phloem sieve elements of host plants and the hemocoel of insect vectors. Unlike phytoplasmas, however, spiroplasmas, and Spiroplasma citri in particular, can be grown in cell-free media and genetically engineered. As a new approach for studying phytoplasmas-insect cell interactions, we sought to mimic phytoplasmas through the construction of recombinant spiroplasmas exhibiting FD phytoplasma Vmps at the cell surface.

Results

Here, we report the expression of the FD phytoplasma VmpA in S. citri. Transformation of S. citri with plasmid vectors in which the vmpA coding sequence was under the control of the S. citri tuf gene promoter resulted in higher accumulation of VmpA than with the native promoter. Expression of VmpA at the spiroplasma surface was achieved by fusing the vmpA coding sequence to the signal peptide sequence of the S. citri adhesin ScARP3d, as revealed by direct colony immunoblotting and immunogold labelling electron microscopy. Anchoring of VmpA to the spiroplasma membrane was further demonstrated by Triton X-114 protein partitioning and Western immunoblotting. Using the same strategy, the secretion of free, functionally active β-lactamase (used as a model protein) into the culture medium by recombinant spiroplasmas was achieved.

Conclusions

Construction of recombinant spiroplasmas harbouring the FD phytoplasma variable membrane protein VmpA at their surface was achieved, which provides a new biological approach for studying interactions of phytoplasma surface proteins with host cells. Likewise, the secretion of functional β-lactamase by recombinant spiroplasmas established the considerable promise of the S. citri expression system for delivering phytoplasma effector proteins into host cells.

【 授权许可】

   
2015 Renaudin et al.; licensee BioMed Central.

【 图 表 】

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