Functional characterization of Arabidopsis Extra Large G proteins (XLGs) and their role as signal transducers downstream of receptor kinases
Final Report Abstract
We identified XLG2 as an essential mediator of cell death elicited by cerk1-4, a mutated allele of the chitin receptor kinase CERK1. XLG2 is an unusual heterotrimeric G-protein α subunit and we could show that its role in cerk1-4 signalling partially depends on the canonical β and γ subunits AGB1 and AGG1/AGG2. In contrast, the well-studied pattern recognition co-receptor BAK1 and the adaptor kinase SOBIR1 are dispensable for the process. XLG2 promotes PAMP-induced ROS generation and analyses of a xlg triple mutant revealed that it fulfils this function redundantly with its close homologs XLG1 and XLG3. Mutations in XLG2 alone supress the cerk1-4 cell death phenotype and multiple lines of evidence suggest that the underlying mechanism is NADPH oxidase-independent. We extensively investigated the subcellular localisation of XLG2 in stably transformed Arabidopsis plants expressing Venus-XLG2 from its native promoter. These analyses revealed that XLG2 localizes to the cell periphery in unchallenged cells but can accumulate in nuclei upon biotic stress. XLG2 interacts with NADPH oxidases at the plasma membrane to regulate ROS production. We performed detailed analyses with a set of Venus-XLG2 variants showing altered subcellular localisation. The results indicated that the function of XLG2 required for cerk1-4 cell death signalling is also coupled to cell periphery localization. Furthermore, our analyses revealed that the GTPase activity of XLG2 is not required for the cerk1-4 phenotype, but a conserved cysteine-rich motif in the N-terminal domain is. Mass spectrometry studies on the cysteine rich region are currently under way. Biochemical analyses showed that XLG2 variants that fail to mediate cerk1-4 cell death and have lost normal cell periphery localisation, show reduced phosphorylation in the N-terminus at sites distinct from those required for ROS-regulation. The project faced a number of challenges during its course. We had to adapt the original plan to a single PhD position and had to make some changes due to research activities of other groups. Our first attempt to create fully functional XLG2-fluorescence protein fusions was not successful, which considerably increased the workload. Finally, the Corona crisis caused significant delays at the end of the project. Despite our work and the work of others, the role of XLG2 in the nucleus is still not understood. It seems likely that it is a process masked by redundancy with XLG1 and XLG3. We have now established tools such as the xlg triple mutant and the collection of Venus-XLG2 localisation variants to address this issue in the future.
Publications
- “Extra-large G-protein 2 (XLG2) plays an important role in cerk1-4 cell death formation”, International Plant Immunity Symposium 2018 IRTG 2172 PRoTECT, 14.-15.6. 2018, Göttingen, Germany
Ronja Hacke
- “The Role of Arabidopsis Extra-Large G-Protein 2 in cerk1-4-dependent cell death signaling”, ASPB Plant Biology 2020 Worldwide Summit, 27.-31.7. 2020
Julia Anders
