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

植原体是一种无细胞壁的微生物，由寄主植物韧皮部中的昆虫媒介传播，在一千多种植物中引起各种重要的经济疾病。植原体的体外培养方法还有待进一步的可行性验证，因此这些病原菌在生理生化基础上的特征较差。此外，由于缺乏复杂的技术，寄主-植原体相互作用的本质仍然知之甚少，主要涉及植物-植原体相互作用和植物寄主的定殖。对局部接种后植原体的移位或树木重新定殖的研究提供了证据，证明植原体的移位不能仅用通过同化液流的拉动来解释。然而，考虑到缺乏编码细胞骨架元素或鞭毛的基因，植原体的活跃运动似乎不太可能。最近的证据表明，植原体进行重组，将植物寄主筛选元件肌动蛋白单边锚定到植原体表面。宿主肌动蛋白的单极获取和聚合已经进化，特别是在革兰氏阳性细胞间致病菌中，以促进细胞间的传播。类似的宿主肌动蛋白的不对称聚合可能发生在植原体感染的筛子元件中，以使植原体在宿主中移动和易位。此外，其中一名申请者已经证明瞬时表达了“马里假丝酵母植原体”(‘Ca)的免疫优势膜蛋白(Imp)。P·马里)是一种植原体，由Cacophalla属昆虫媒介传播，在体内与植物肌动蛋白结合，最终支持植原体在韧皮部的移动。扫描力显微镜和光谱学(SFM/SFS)的最新发展使(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。因此，我们打算首次用这种方法测量植原体与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，并定位和确定结合域。此外，还将使用高速扫描力显微镜(HS-SFM)。这种方法在植原体研究中具有突出和创新的地位，直到今天才被用于植物病原菌与寄主的调查。特别是，HS-SFM将被用来解释和说明重组柑橘螺旋体暴露a‘Ca的运动。P.马里的小腿沿着植物和昆虫肌动蛋白的表面。这项研究的发现将有助于首次解决植质学中长期存在的问题，即植原体如何在宿主内定植和转移。

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.