GTP binding by Arabidopsis extra-large G protein 2 is not essential for its functions

Abstract

The extra-large GTP-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gβγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action.

Heterotrimeric G Proteins in Plants: Canonical and Atypical Gα Subunits

“…Adding to the challenge, the aforementioned XLGs have low or no affinity to GTP and can work without it. Analyses of a wide range of XLGs from various plants revealed very weak conservation within the nucleotide-binding motifs and apparent losses of crucial amino acids and entire motifs [14,16]. Confirming this notion, non-quantitative [11,14,16,66] and quantitative in vitro assays showed that XLGs have very low steady-state GTPγS binding, up to two orders of magnitude lower than that of AtGPA1 [12].…”

“…Functional diversification driven by sequence divergence within the superfamily resulted in saltational evolution of these atypical plant Gα subunits [11]. Deviation of the conserved catalytic motifs of some Gα subunits is found in Dictyostelium (slime moulds), Naegleria, and plants, suggesting reduced levels or complete loss of nucleotide-dependent activity [1,[12][13][14]. Some of these Gα subunits also lack the N-terminal residues required for tethering to the plasma membrane, which potentially influences their sub-cellular localization and function.…”

“…Plants have two types of Gα subunits: canonical Gα subunits, hereafter GPAs, and atypical, extra-large Gα subunits, hereafter XLGs. While GPAs are conserved, and their ancestry is clearly related to the common Gα ancestor [15], XLGs are structurally distinct and unique to plants [11,14,16]. XLG homologs are present in all major land plant phyla, including the most primitive group of existing land plants, mosses (Bryophyta), placing the origin of XLGs at the dawn of land plants diversification [11,[17][18][19].…”

EXTRA LARGE G-PROTEIN 2 (XLG2) mediates cell death and hyperimmunity via a novel, apoplastic ROS-independent pathway inArabidopsis thaliana

“…XLG2 has been shown in multiple studies to facilitate PAMP-induced ROS bursts together with XLG3 (Maruta et al, 2015;Liang et al, 2016;Liang et al, 2018;Maruta et al, 2021). Therefore, we investigated if the role of XLG2 in ROS control is important for the formation of the cerk1-4 phenotype.…”

“…XLG proteins harbour a Gα domain and weak GTP binding and GTPase activity have been demonstrated for XLG2 in vitro (Heo et al, 2012;Lou et al, 2020). To investigate if XLG2 GTPase activity plays any role in formation of the cerk1-4 cell death phenotype, we mutated its GTP binding site (T476N) as described previously (Heo et al, 2012;Maruta et al, 2021) (Fig. 1e).…”