A receptor-like kinase mediated phosphorylation of Gα protein affects signaling during nodulation

Abstract

1 Heterotrimeric G-proteins, comprised of Gα, Gβ and Gγ subunits regulate signaling in 2 eukaryotes. In metazoans, G-proteins are activated by GPCR-mediated GDP to GTP 3 exchange on Gα; however, the role of receptors in regulating plant G-protein signaling 4 remains equivocal. Mounting evidence points to the involvement of receptor-like kinases 5 (RLKs) in regulating plant G-protein signaling pathways, but their mechanistic details 6 remain limited. We have previously shown that during soybean nodulation, the nod factor 7 receptor 1 (NFR1) interacts with G-protein components and indirectly controls signaling. 8  We explored the direct regulation of G-protein signaling by RLKs using protein-protein 9 interactions, receptor-mediated phosphorylation and the effects of such phosphorylations 10 on soybean nodule formation. 11 Results presented in this study demonstrate a direct, phosphorylation-based regulation of 12 Gα by symbiosis receptor kinase (SymRK). SymRKs interact with and phosphorylate Gα 13 at multiple residues, including two in its active site, which abolishes GTP binding. In 14 addition, phospho-mimetic Gα fails to interact with Gβγ, potentially allowing for 15 constitutive signaling by the freed Gβγ. 16 These results uncover a novel mechanism of G-protein cycle regulation in plants where 17 receptor-mediated phosphorylation of Gα not only affects its activity, but also influences 18 the availability of its signaling partners, thereby exerting a two-pronged control on 19 signaling. 20 100 Intriguingly, two of the phosphorylated amino acids are in the conserved GTP-binding domain of 101 the Gα protein. Phosphorylation of the serine residues in this site abolishes the ability of Gα to 102 bind GTP, essentially making the protein biochemically inactive. Furthermore, the phospho-103 mimetic Gα is unable to bind Gβγ proteins, suggesting an altered dynamics and/or availability of 104 the active proteins for downstream signaling. Our results thus uncover a novel G-protein signaling 105 mechanism where not only the activity but also the availability of the signaling proteins is 106 dependent on receptor-mediated phosphorylation of the Gα protein. 107 MATERIAL AND METHODS 108 Plant material and hairy root transformation 109 Soybean (Glycine max) wild type ('Williams 82') seeds were grown on (BM 7 35%) in the 110 greenhouse (16 h light/8 h dark) for 12 days at 25°C. Hairy root transformation was performed as Rhizobium strain (USDA136) cultured in Vincent's rich medium was used for bacterial infection. 113 Nodules were counted at 32 days after rhizobium infection. Three biological replicates were used 114 for each construct. At least 35 to 40 transgenic hairy roots were used in individual experiment for 115 each construct. The data were averaged, and statistically significant values were determined by 116 Dunn's multiple comparisons test. 117 Generation of constructs 118 The gene fragment for RNAi construct of SymRK was cloned into the pCR8/GW vector 119 (Invitrogen) and subsequently transferred into binar...