Subproject D04: Plant receptor kinases as targets for microbial effectors
Principal investigators:
Prof. Dr. Thorsten Nürnberger
Universität Tübingen
ZMBP, Pflanzenbiochemie
Auf der Morgenstelle 5, 72076 Tübingen
Tel 07071 - 29 76658
Fax 07071 - 29 5226
nuernberger@zmbp.uni-tuebingen.de
Prof. Dr. Thilo Stehle
Universität Tübingen
Interfakultäres Institut für Biochemie (IFIB)
Hoppe-Seyler-Str. 4, 72076 Tübingen
Tel 07071 - 29 73043
Fax 07071 - 29 5565
Summary:
Suppression of host immunity is a characteristic feature of microbial infection strategies. During infection, the Pseudomomas syringae effector protein AvrPto is translocated into the cytoplasm of its plant host, Arabidopsis thaliana. There, AvrPto inhibits complex formation of the pattern recognition receptor kinase FLS2 and its co-receptor kinase BAK1 thereby suppressing activation of host immune responses by bacterial flagellin. Ectopic (over)-expression of the tagged effector in planta combined with co-immunoprecipitation has provided controversial scientific evidence for interaction of AvrPto with either FLS2 or BAK1 kinase domains. We have assumed that effector protein concentrations in infected host plants are rather small and that primary, biologically most significant effector targets are most likely those exhibiting the highest affinity to AvrPto. A combination of surface plasmon resonance (SPR) and microscale thermophoresis (MST) approaches was employed to determine kinetic constants of effector-effector target interactions in vitro. Not surprisingly, AvrPto bound to structurally closely related kinase domains of both (and likely many more) receptor kinases, albeit with significantly different affinities and KON and KOFF rates, suggesting that BAK1 is the physiologically more relevant target for AvrPto. In the second funding period, we attempt to verify these data by measurements of protein affinities in planta by employing dual colour confocal fluorescence cross correlation spectroscopy (FCCS). Successful implementation of this technology for plant biology purposes will not only help answer the question why structurally conserved plant proteins become (or not) specific targets for microbial effectors, but will be more widely applicable to better understand, for example, stimulus-induced changes in protein complex assemblies that are under study in this CRC. Further, X-ray structural analyses of AvrPto in complex with the structurally closely related BAK1 and FLS2 protein kinase domains will provide atomic-resolution insight into the determinants governing high-affinity binding. The structural analyses will also help to identify other potential protein kinase targets of the AvrPto effector in other host plants, such as tomato. This work is part of a longstanding interest of our laboratories to apply structural biochemistry tools to elucidate the functionality of host immune receptors.