AtObgC-AtRSH1 interaction may play a vital role in stress response signal transduction in Arabidopsis

Abstract

The interaction of Obg (Spo0B-associated GTP-binding protein) GTPase and SpoT, which is a bifunctional ppGpp (guanosine 3',5'-bispyrophosphate) hydrolase/synthetase, is vital for the modulation of intracellular ppGpp levels during bacterial responses to environmental cues. It has been recently reported that the ppGpp level is also inducible by various stresses in the chloroplasts of plant cells. However, the function of the Obg-SpoT interaction in plants remains elusive. The results from the present and previous studies suggest that AtRSH1 is a putative bacterial SpoT homolog in Arabidopsis and that its transcription levels are responsive to wounding and salt stresses. In this study, we used a yeast two-hybrid analysis to map the regions required for the AtObgC-AtRSH1 interaction. Moreover, protein-protein docking simulations revealed reasonable geometric and electrostatic complementarity in the binding surfaces of the two proteins. The data support our experimental results, which suggest that the conserved domains in AtObgC and the N terminus of AtRSH1 containing the TGS domain contribute to their interaction. In addition, quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AtObgC and AtRSH1 exhibit a similar inhibition pattern under wounding and salt-stress conditions, but the inhibition pattern was not greatly influenced by the presence or absence of light. Based on in vivo analyses, we further confirmed that the AtRSH1 and AtObgC proteins similarly localize in chloroplasts. Based on these results, we propose that the AtObgC-AtRSH1 interaction plays a vital role in ppGpp-mediated stress responses in chloroplasts.

Keywords: ACT; Arabidopsis thaliana; At; AtObgC; AtRSH1; BiFC; C-terminal domain; CTD; Chloroplast; DS 3.0 software; ESP; MEGA 5.05; N-terminal domain; NTD; ONPG; Obg; ObgC; RD29A; RSH; RT-PCR; RelA/SpoT homolog; Spo0B-associated GTP-binding protein; Stress response; TGS; TP; VDW; VSP2; aspartate kinase-chorismate mutase-TyrA; bimolecular fluorescence complementation; chloroplast-targeting Obg GTPase; discovery studio version 3.0 software; electrostatic potentials; guanosine 3′,5′-bispyrophosphate; molecular evolutionary genetics analysis; o-nitrophenyl-β-D-galactopyranoside; ppGpp; qRT-PCR; quantitative real-time PCR; responsive to desiccation 29A; reverse transcription PCR; threonyl-tRNA synthetase-GTPase-SpoT proteins; transit peptide; van der Waals; vegetative storage protein 2.