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

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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.