Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts through Specific RelA/SpoT Homologues

Magic spot nucleotides (p)ppGpp are important signalingmoleculesin bacteria and plants. In the latter, RelA-SpoT homologue (RSH) enzymesare responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is moredifficult in plants than in bacteria due to lower concentrations andmore severe matrix effects. Here, we report that capillary electrophoresismass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundanceand identity in Arabidopsis thaliana. This goal is achieved by combining a titanium dioxide extractionprotocol and pre-spiking with chemically synthesized stable isotope-labeledinternal reference compounds. The high sensitivity and separationefficiency of CE-MS enables monitoring of changes in (p)ppGpp levelsin A. thaliana upon infection withthe pathogen Pseudomonas syringae pv. tomato (PstDC3000). We observed a significant increase ofppGpp post infection that is also stimulated by the flagellin peptideflg22 only. This increase depends on functional flg22 receptor FLS2and its interacting kinase BAK1 indicating that pathogen-associatedmolecular pattern (PAMP) receptor-mediated signaling controls ppGpplevels. Transcript analyses showed an upregulation of RSH2 upon flg22 treatment and both RSH2 and RSH3 after PstDC3000 infection. Arabidopsis mutants deficient in RSH2 and RSH3 activitydisplay no ppGpp accumulation upon infection and flg22 treatment,supporting the involvement of these synthases in PAMP-triggered innateimmune responses to pathogens within the chloroplast.

Automated summary

Nucleotides (p)ppGpp play important roles as signaling molecules in bacteria and plants. In this study, the researchers used CE-MS to analyze the abundance and composition of ppGpp in Arabidopsis thaliana and found a significant increase of ppGpp levels upon infection with the bacteria pathogen Pseudomonas syringae pv. tomato (PstDC3000), stimulated by the flagellin peptide flg22. This increase is dependent on the functional flg22 receptor FLS2 and its interacting kinase BAK1, indicating that PAMP receptor-mediated signaling controls ppGpp levels.