Analysis of Thionin Proprotein Processing in Arabidopsis
Abstract
Thionins are a group of plant-specific antimicrobial peptides which are often stabilized by 3 or 4 disulfide bridges. They are produced as preproproteins and the mature peptides are found in cell wall, vacuoles, and protein bodies. In a previous FWF project we have isolated a TPPE (Thionin Proprotein Processing Enzyme) from barley. We produced the TPPE in E. coli and showed that it was able to process a barley leaf thionin proprotein (which was also produced in E. coli) in vitro. The acidic domain was cleaved at several sites, which explains why a peptide corresponding to an acidic domain has never been isolated from plants. The thionin was protected from processing by the TPPE by its threedimensional structure which is stabilised by disulfide bridges. In this project it is proposed to further study thionin proprotein processing in planta in order to confirm the in vitro results. This would be done in Arabidopsis which is a much better experimental system than barley for this purpose. Arabidopsis contains 4 thionin genes which are expressed in different tissues. Our unpublished results indicate that Arabidopsis contains a single TPPE (AtSbt1.4) which is homologous to the barley TPPE. The AtSbt1.4 and 2 related subtilases (AtSbt1.2 and AtSbt1.7) have already been cloned as have the proproteins for all 4 Arabidopsis thionins. In the proprosed project processing of the Arabidopsis proproteins by the TPPE would be first studied in vitro using especially mass spectrometry as has been done before for the barley TPPE. These studies would then be extended to study processing in planta using available T-DNA mutants and the AGROBEST system for transient expression in Arabidopsis seedlings. On top of this, transgenic lines which express FLAG-tagged proproteins will be produced which will be useful to study processing in tissues other than seedlings. These experiments should confirm AtSbt1.4 as Arabidopsis TPPE. Furthermore, sequence requirements for the cleavage site of the proprotein will be analyzed using a range of different peptides which will be incubated with the TPPE and cleavage analysed by mass spectroscopy. Thionins have been found in vacuoles, cell walls and protein bodies but it is not clear where in the cell processing occurs. Therefore, the localization of the Arabidopsis thionins will be analyzed using antibodies and the localization of the TPPE will be studied using also antibodies as well as tagging with GFP and FLAG. Finally, it is proposed to study the function of the PA domain and the C-terminal domain of AtSbt1.4 using a mutant approach and analysis of the function of these mutants in vitro. Those mutants with an effect in vitro would then be expressed in the AtSbt1.4 mutant background to test them in planta.
Arabidopsis Thionin Antimicrobial Peptide Subtilase
Publikationen
Project staff
Holger Bohlmann
Assoc. Prof. Dr. Holger Bohlmann
holger.bohlmann@boku.ac.at
Project Leader
01.03.2016 - 30.06.2021