Qualitative and quantitative investigation of the interaction of the immunodominant transmembrane protein from ´Candidatus Phytoplasma mali´ with host actin by scanning force microscopy

植原体是无细胞壁的微生物，通过昆虫媒介传播，其驻留在宿主植物的韧皮部中，在一千多种植物物种中引起各种经济上重要的疾病。植原体的体外培养方法有待进一步确认其可行性，因此这些病原体在生理和生化基础上的特征很差。此外，宿主-植原体相互作用的本质仍然是知之甚少，尤其是由于缺乏先进的技术，主要是当它涉及植物-植原体相互作用和植物宿主的定殖。对植原体在局部接种或树木再定殖后的易位的研究提供了证据，植原体的易位不能仅通过同化物流的拉动来解释。然而，植原体的主动运动似乎不太可能，考虑到缺乏编码细胞骨架元件或鞭毛的基因。最近的证据表明植原体施加重组，锚定植物宿主筛元件肌动蛋白单方面植原体表面。宿主肌动蛋白的单极获得和聚合已经进化，特别是在革兰氏阳性细胞间致病菌中，以促进细胞到细胞的传播。在植原体感染的筛元件中，宿主肌动蛋白也可能发生类似的不对称聚合，从而使植原体能够在宿主中运动和移位。此外，其中一个申请人已经证明了瞬时表达的‘苹果植原体’（‘Ca.苹果疫霉（Phytoplasma mali '）是一种由卡蚤属昆虫传播的植原体，在体内与植物肌动蛋白结合，最终支持植原体在韧皮部中的运动。扫描力显微镜和光谱学（SFM/SFS）的最新发展使得联合收割机（亚）分子成像与物理、化学和生物相互作用的定量图谱相结合成为可能。因此，我们打算用这种方法首次测量植原体与其宿主之间的分子间力，以测量肌动蛋白与Imp和Imp突变体之间的结合力，并定位和确定结合结构域。此外，还将使用高速扫描力显微镜（HS-SFM）。该方法在植原体研究中具有突出的创新地位，直到今天才用于植物病原体及其宿主的调查。特别是HS-SFM将被用来解释和说明重组螺旋体柑橘暴露的'Ca的运动。苹果疫霉在其表面沿着有植物和昆虫的肌动蛋白。这项研究的结果将有助于首次解决植原体学中长期存在的问题，植原体如何在其宿主中定殖和移位。

Phytoplasmas are cell wall less microorganisms, transmitted by insect vectors, which reside in the phloem of host plants, causing a variety of economically important diseases in more than a thousand plant species. Methods for in vitro cultivation of phytoplasmas await further confirmation of feasibility, therefore these pathogens are poorly characterized on a physiological and biochemical basis. Moreover, the essentials of host-phytoplasma interaction are still poorly understood amongst others due to lack of sophisticated techniques, mainly when it concerns the plant-phytoplasma interaction and the colonization of the plant host. Studies on the translocation of phytoplasmas after localized inoculation or the re-colonization of trees provided evidence that the translocation of phytoplasmas cannot be explained only by a pulling through the assimilate flow. Active movement of phytoplasmas seems, however, unlikely, considering the lack of genes coding for cytoskeleton elements or flagella. Recently evidence was shown that phytoplasmas impose a reorganization that anchors the plant host sieve element actin unilaterally to the phytoplasma surface. Unipolar acquisition and polymerization of host actin has evolved, in particular, in gram-positive intercellular pathogenic bacteria, to facilitate cell to cell spread. A similar asymmetric polymerization of host actin may occur in phytoplasma infected sieve elements to enable phytoplasma motility and translocation in the host. Moreover, one of the applicants could already demonstrate that transiently expressed immunodominant membrane protein (Imp) of ´Candidatus Phytoplasma mali´ (´Ca. P. mali´), a phytoplasma transmitted by insect vectors belonging to the genus Cacopsylla, binds with plant actin in vivo thus eventually supporting the movement of phytoplasmas in the phloem.Recent developments in scanning force microscopy and spectroscopy (SFM/SFS) allow to combine (sub)molecular imaging with quantitative mapping of physical, chemical and biological interactions. Therefore, we intend to measure for the first time the intermolecular forces between phytoplasmas and their hosts by this method to measure the binding force between actin and Imp and Imp mutants and to localize and determine the binding domain. In addition, high-speed scanning force microscopy (HS-SFM) will be used. This method has an outstanding and innovative position in the phytoplasma research and was not used for investigations of plant pathogens with their host until today. Especially the HS-SFM will be utilized to explain and illustrate the movement of a recombinant Spiroplasma citri exposing a ´Ca. P. mali´ Imp on its surface along the plant and insect actin. The findings out of this research will contribute to solve for the first time long standing questions in phytoplasmology, how phytoplasmas colonize and translocate within their hosts.