Plant RelA/SpoT Homolog Confers Salt Tolerance in Escherichia coli and Saccharomyces cerevisiae

Abstract

;To analyze the mechanisms of salt tolerance in the halophyte Suaeda japonica, Escherichia coli was used as a host organism to undertake functional screening of cDNAs encoding proteins that may play an important role for the salt-tolerance mechanisms. A transformant expressing RelA/SpoT homolog (Sj-RSH) was found to have enhanced salt tolerance. In E. coli, RelA/SpoT controlled the amount of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effectors of the bacterial stringent response. Complementation analysis using the relA mutant of E. coli showed that Sj-RSH conferred the phenotype associated with (p)ppGpp synthesis. Furthermore, expression of Sj-RSH driven by the GAL1 promoter also gave rise to enhanced salt tolerance in yeast. Northern blot analyses of the yeast transformant revealed that the transcriptional levels of stress responsive genes including GPD1, VMA6, BMH1, HYP1 and HOG1 were clearly enhanced in the Sj-RSH transformant when compared with an empty vector transformant under stress-free and 1.5 M NaCl stress conditions. These results suggest that (p)ppGpp synthesis mediated by plant RelA/SpoT homologs plays a critical role for the transcriptional induction of several stress responsive genes, directly or indirectly in yeast, and that the conserved stress-resistance system may exist in higher plants.

Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp

“…Suaeda japonica is a halophyte that can grow in the presence of 0.7 M NaCl and has been used to study salt tolerance mechanisms in plants (Yamada et al, 2003). In the present study, we demonstrated that introducing a relA-spoT homologue (Sj-RSH) isolated from Suaeda japonica into the yeast S. cerevisiae results in ectopic expression of RSH protein, leading to the accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli.…”

“…Increases in cellular salt tolerance associated with expression of the Sj-RSH gene in E. coli and S. cerevisiae have been reported previously (Yamada et al, 2003). Working with strains KO-938 (expressing Sj-RSH) and KO-937 (vector control) as used in the previous work, we further studied the effects of Sj-RSH expression on cellular tolerance to various stressful conditions.…”

“…To express Sj-RSH in yeast, the full-length Sj-RSH cDNA fragment (Yamada et al, 2003) was digested with SmaI and XhoI and cloned into the yeast expression plasmid pAUR123 that had been treated with the same enzymes, generating pAUR-SjRSH. To construct the Sj-RSH-EGFP fusion plasmid, the enhanced GFP (EGFP) gene fragment was excised from pEGFP (Clontech) using SmaI and StuI and inserted into the HpaI site of pAUR-SjRSH, thus generating pTN1010.…”

“…The PCR fragment was digested with Acc65I and XbaI, and cloned into pYC2/CT that had been treated with the same enzymes, thus generating pTN1036. pYES2-SjRSH, which harbours the Sj-RSH gene under the control of the GAL1 promoter, was constructed as described previously (Yamada et al, 2003). All plasmid sequences were checked by DNA sequencing.…”

“…Despite the essential lack of a relA-spoT homologue and ppGpp in the yeast Saccharomyces cerevisiae, Yamada et al (2003) found that heterologous expression of RSH isolated from the plant Suaeda japonica (named Sj-RSH; highly homologous with At-RSH2 and At-RSH3 of Arabidopsis thaliana) conferred salt tolerance upon S. cerevisiae. Although no attempts were made to detect ppGpp, the results raise the possibility that eukaryotic cells, including yeasts, although not capable of synthesizing ppGpp by themselves, may have latent systems to respond to ppGpp.…”