Doctorate thesis of Montpellier University

Wednesday november 8 2023  at 2 pm, Amphi 208

Role and function of membrane signalling platforms containing the Rho GTPase ROP6 during cell signalling

Doctoral school : GAIA – Biodiversité, Agriculture, Alimentation, Environnement, Terre, Eau
Spéciality : BIDAP – Biologie, Interactions, Diversité Adaptative des Plantes
University : Université Montpellier
Reasearch unit : IPSiM –  Institut for Plant Sciences of Montpellier

Team: Aqua

Jury:

Yohann BOUTTE, Directeur de recherche CNRS, Bordeaux – Rapporteur
Marie-Cécile CAILLAUD, Directrice de recherche CNRS, Lyon – Rapporteur
Marie BOUDSOCQ, Chargée de recherche CNRS, Gif-sur-Yvette – Examinatrice
Laurent LAPLAZE, Directeur de recherche IRD, Montpellier – Examinateur
Alexandre MARTINIERE, Directeur de recherche CNRS, Montpellier – Directeur de thèse

Abstract:

The cell membrane is composed of a myriad of different lipids and proteins. It is known to be both heterogeneous (juxtaposition of domains) and dynamic (diffusion of these constituents). In addition, the plasma membrane is at the interface of the cell and control the perception and integration of biological signals. The molecular players can be compartmentalised to respond to different signals (Jaillais and Ott, 2020). This is the case for Rho of Plant (ROP) GTPases, which have been described as a family of key players in the response to different signals: Auxin, ABA, response to pathogen response, osmotic signalling and symbiosis (Basu et al., 2008; Lin et al, 2012 ; Poraty-Gavra et al., 2013 ; Choi et al., 2014 ; Lin et al., 2015 ; Engelsdorf et al., 2018; Feiguelman et al., 2018; Platre et al., 2019; Smokvarska et al., 2020). Interestingly, this pleiotropic role also exists at the level of a single ROP isoform. ROP6 has been described as a regulator of auxin and osmotic signalling (Platre et al., 2019; Smokvarska et al., 2020). In roots, we found that ROP6 forms nanodomains during these two signalling processes. During osmotic
signalling, ROP6 forms nanodomains in association with RBOHDs and F allowing the accumulation of reactive oxygen species (ROS). However, in response to auxin, ROP6 nanodomains do not associate with RBOHs (Platre et al., 2019; Smokvarska et al., 2020). Thus, a question is emerging: How can the organisation of ROP6 nanodomains induce a specific response downstream of the different signals? We therefore set out to determine how ROP6 activators can help encode a specific response at the cellular level. Using a reverse genetic screen, we found that guanine exchange factor 14 (GEF14) is necessary and specific for inducing osmotic signalling in plant cells. Based on genetic and GTPase activation sensor experiments, we show that GEF14 acts as an activator of ROP6 in planta. Using super-resolution microscopy, we showed that ROP6 nanodomains induced by the osmotic signal, but not those induced by auxin, require GEF14 to form. Finally, we show that GEF14 restructures into clusters after an osmotic signal. We extended our model to immune signalling where we similarly identified a specific GEF isoform. In a second part, I sought to determine how GEF14 was activated by the osmotic signal. Using a comparative biochemical approach, I identified 5 phosphorylation sites in the PRONE domain of GEF14. The GEF14 interactome led us to identify candidate kinases and phosphatases that need to be confirmed by reverse genetic approaches. Our results suggest that the activation of a GEF isoform can determine the early stages of signalling and act on the specific recruitment of effector proteins. This mechanism makes it possible to maintain and control early cell signalling in plants.