Renaudin Joël, Béven Laure, Batailler Brigitte, Duret Sybille, Desqué Delphine, Arricau-Bouvery Nathalie, Malembic-Maher Sylvie, Foissac Xavier
INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.
Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.
BMC Microbiol. 2015 Apr 2;15:82. doi: 10.1186/s12866-015-0417-5.
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.
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.
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.
葡萄黄化病（FD）是一种威胁欧洲葡萄园的韧皮部细菌病害。该病害与一种不可培养的柔膜菌纲微生物有关，即一种植原体，它由葡萄叶蝉葡萄叶蝉以持久、增殖的方式传播。昆虫传播的特异性大概是通过宿主组织与植原体表面蛋白（即所谓的可变膜蛋白（Vmps））之间的相互作用介导的。植物螺原体和植原体共享相同的生态位，即宿主植物的韧皮部筛管分子和昆虫载体的血腔。然而，与植原体不同的是，螺原体，特别是柑橘螺原体，可以在无细胞培养基中生长并进行基因工程改造。作为研究植原体与昆虫细胞相互作用的一种新方法，我们试图通过构建在细胞表面展示FD植原体Vmps的重组螺原体来模拟植原体。
在此，我们报道了FD植原体VmpA在柑橘螺原体中的表达。用质粒载体转化柑橘螺原体，其中vmpA编码序列受柑橘螺原体tuf基因启动子控制，导致VmpA的积累高于天然启动子。通过直接菌落免疫印迹和免疫金标记电子显微镜显示，通过将vmpA编码序列与柑橘螺原体粘附素ScARP3d的信号肽序列融合，实现了VmpA在螺原体表面的表达。通过Triton X-114蛋白分配和Western免疫印迹进一步证明了VmpA锚定在螺原体膜上。使用相同的策略，重组螺原体实现了将游离的、具有功能活性的β-内酰胺酶（用作模型蛋白）分泌到培养基中。
成功构建了在其表面携带FD植原体可变膜蛋白VmpA的重组螺原体，这为研究植原体表面蛋白与宿主细胞的相互作用提供了一种新的生物学方法。同样，重组螺原体分泌功能性β-内酰胺酶，确立了柑橘螺原体表达系统在将植原体效应蛋白递送至宿主细胞方面的巨大前景。
