Characterisation of protein-protein interactions of membrane proteins from Hordeum vulgare and Arabidopsis thaliana involved in plant immunity

• Charakterisierung von Protein-Protein-Interaktionen von Membranproteinen aus Hordeum vulgare und Arabidopsis thaliana beteiligt an der Pflanzenabwehr

Sabelleck, Björn; Panstruga, Ralph (Thesis advisor); Schaffrath, Ulrich (Thesis advisor)

Aachen (2019, 2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019

Abstract

Plants possess a sophisticated immune system, which is capable of defending a plant against the vast majority of microbes. For the recognition of different microbes, plants use receptor-like kinases (RLKs) and receptor-like proteins (RLPs), which are localised at the plasma membrane. These receptors can bind microbe-specific molecules on the extracellular side and can transmit the signal by phosphorylation reactions into a cellular signal. A defence signalling cascade is started to prevent the growth of the microbe. Not only RLKs are present in the plasma membrane, but also other proteins that play a crucial role in plant immunity. How these proteins interact with other proteins and the meaning of this interaction is only partially solved. The overall aim of this thesis was the identification of new physical interaction partners of plasma membrane-localised proteins involved in plant immunity. The main approach that was used in this thesis was the split-ubiquitin yeast two-hybrid (Y2H) system. This yeast-based system was designed to identify protein-protein interactions at the plasma membrane, wherefore the classical Y2H system is not applicable. Two different split-ubiquitin Y2H systems were tested, and various cDNA library screens were performed. A library composed of effector proteins of the powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was used to screen it against barley and wheat RLKs, which should have a proposed role in non-host resistance. Potential interaction partners with two of these RLKs could be identified and validated in yeast. The split-ubiquitin Y2H system was further applied for the identification of new interaction partners of the barley Mlo protein. The biochemical function of this seven transmembrane-containing protein is to date unknown, but a knock-out of specific genes of MLO family leads to complete powdery mildew resistance. The interaction screen should help to find new indications of the biochemical function. Two new potential interacting proteins of barley could be identified, which have to be further validated in the future. The overexpression of one of the candidates, an O-methyltransferase, leads to enhanced resistance against Bgh. Two potential interacting CSEPs could be determined, although one candidate (CSEP0515) could not be validated in an in planta bimolecular fluorescence complementation (BiFC) assays. The other effector needs to be tested. Finally, the yeast system was used for the identification of new interaction partners of the Arabidopsis thaliana chitin receptor CERK1. Yeast and in planta split-luciferase experiments validated that CERK1 interacts with calmodulin (CaM), a plasma membrane H+ ATPase (AHA2) and the protein PCC1 (PATHOGEN AND CIRCADIAN CONTROL 1). The function of the interaction of CaM and AHA2 needs further investigation in the future. PCC1 belongs to a family of biotic and abiotic stress response proteins in eukaryotes. It possesses a specific domain, which is called cysteine-rich transmembrane domain (CYSTM). In silico analysis and interaction data indicate that different CYSTM family members form homo- and heterodimers and that specific members show a similar expression pattern. For the CERK1-PCC1 interaction could be shown: 1. The potential interaction site of PCC1 was narrowed down; 2. It could be shown that also other family CYSTM members can interact with CERK1; 3. Indication of likely phosphorylation of the N-terminus of PCC1 could be revealed and 4. It could be displayed that also other RLKs involved in plant immunity, like LYK4, LYK5, BAK1, EFR and FLS2, can interact with PCC1. Two Arabidopsis mutant lines of PCC1 revealed an unfortunately inconclusive ROS burst phenotype. A knock-down mutant showed a reduced ROS response after chitin and elf18 trigger, whereas the knock-out mutant exhibited a wild type-like reaction.