Abstract
BackgroundFlavescence 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.ResultsHere, 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.ConclusionsConstruction 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0417-5) contains supplementary material, which is available to authorized users.
Highlights
Flavescence dorée (FD) of grapevine is a phloem bacterial disease that threatens European vineyards
In ‘Ca. P. asteris’ strain CYP, Amp interacts with actin and ATP synthase of leafhopper vectors, suggesting that this protein plays a critical role in insect transmission specificity [15], whereas in ‘Ca. Phytoplasma mali’, the immunodominant membrane protein immunodominant membrane proteins (Imp) binds to plant actin, which supports the hypothesis that Imp-actin binding plays a role in phytoplasma motility in its host plant [16]
One contained the entire vmpA gene, including its own promoter, ribosome binding site (RBS), and signal peptide sequences, whereas, in the other, the signal peptide depleted-vmpA coding sequence fused to the signal peptide sequence of the adhesin S. citri adhesionrelated protein 3d (ScARP3d) was under the control of the S. citri tuf gene promoter and RBS (Figure 1 and Additional file 1: Figure S1)
Summary
Flavescence dorée (FD) of grapevine is a phloem bacterial disease that threatens European vineyards. Studying the molecular components that govern interactions of phytoplasmas with their host plants and insects is severely limited because phytoplasmas cannot be cultured in cell-free media and, cannot be genetically engineered. The immunodominant protein Amp of ‘Candidatus phytoplasma asteris’ strain OY has been shown to interact with actin microfilaments of leafhopper vectors, but not with those of non-vector insects [14]. In ‘Ca. P. asteris’ strain CYP, Amp interacts with actin and ATP synthase of leafhopper vectors, suggesting that this protein plays a critical role in insect transmission specificity [15], whereas in ‘Ca. Phytoplasma mali’, the immunodominant membrane protein Imp binds to plant actin, which supports the hypothesis that Imp-actin binding plays a role in phytoplasma motility in its host plant [16]
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