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

Abstract

Magic spot nucleotides (p)ppGpp are important signaling molecules in bacteria and plants. In the latter, RelA-SpoT homologue (RSH) enzymes are responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is more difficult in plants than in bacteria due to lower concentrations and more severe matrix effects. Here, we report that capillary electrophoresis mass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundance and identity in Arabidopsis thaliana. This goal is achieved by combining a titanium dioxide extraction protocol and pre-spiking with chemically synthesized stable isotope-labeled internal reference compounds. The high sensitivity and separation efficiency of CE-MS enables monitoring of changes in (p)ppGpp levels in A. thaliana upon infection with the pathogen Pseudomonas syringae pv. tomato (PstDC3000). We observed a significant increase of ppGpp post infection that is also stimulated by the flagellin peptide flg22 only. This increase depends on functional flg22 receptor FLS2 and its interacting kinase BAK1 indicating that pathogen-associated molecular pattern (PAMP) receptor-mediated signaling controls ppGpp levels. 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 activity display no ppGpp accumulation upon infection and flg22 treatment, supporting the involvement of these synthases in PAMP-triggered innate immune responses to pathogens within the chloroplast.

Figure 1

(A) Structures of (p)ppGpp and different RelA/SpoT homologues (RSH enzymes) found in Arabidopsis thaliana that are responsible for (p)ppGpp metabolism. (B) Overview of the domain structures of the RSH enzymes in A. thaliana. The colored boxes represent domains, and their location within proteins is roughly indicated. Domains written in gray indicate dysfunctionality of the domain. All homologues have an N-terminal chloroplast transit peptide (cTP). AtCRSH lacks the ThrRS, GTPase, and SpoT (TGS) domain but contains a Ca²⁺ binding EF hand motif (EFh).

Figure 2

Synthesis of magic spot nucleotide [¹⁵N]₅ - isotopologues. (a) P₂Cl₄O₃ (20 equiv), 0 °C, 3 h. (b) NaHCO₃ – buffer (1 M) (c) RNase T2, pH = 7.5, 37 °C, 12 h. (d) 4 (3 equiv), ETT (5 equiv), DMF, rt, 15 min. (e) mCPBA (3 equiv), −20 °C, 15 min. (f) DBU, rt, 30 min. (g) RNase T2, pH = 5.5, 37 °C, 12 h. Abbreviations: ETT 5-(ethylthio)–1H-tetrazole; mCPBA meta-chloroperbenzoic acid; DBU 1,8-diazabicyclo(5.4.0)undec-7-ene; rt room temperature; Fm fluorenylmethyl.

Scheme 1. (A) Flow-Chart of Sample Preparation for CE-ESI-MS Analysis. Different Treatments are Discussed in the Section on Plant Pathogen Interactions Below. (B) CE-MS Measurement of a Reference Nucleotide Mixture (5 μM Stock Solutions, Injection Volume 10 nL), Magic Spot Nucleotides Highlighted in Red, the Red Box Is Additionally Magnified on the Upper Right

Figure 3

(A) Photographs of A. thaliana seedlings taken after several days of infection with P. syringaepv. tomato (PstDC3000). (B) Quantitative analysis of ppGpp levels in A. thaliana seedlings 1, 2, or 3 days post infection (dpi) indicated as pmol per mg of fresh weight (FW). 150 mg of plant material was extracted under light. Data are presented as means ± SD (n = 7). (C) Examples of electropherograms obtained from CE-ESI-MS analysis with a QqQ analyzer in MRM mode. The recorded transition was 607.0 → 508.7 in negative ionization mode. Internal heavy references were spiked at 10 μM in either uninfected (mock) or infected samples (1 dpi). (D) Levels of ppGpp in wildtype (Col-0) versus single mutants (rsh1, rsh2, rsh3, crsh-2) or a double mutant (rsh2 rsh3) 1 day without infection (mock) or 1 dpi with P. syringae(PstDC3000). Data are presented as means ± SD (n = 7–14); ns = not significant; ****P < 0.0001, Student’s t-test.

Figure 4

(A) Domain structure of flagellin and sequences of derived peptides (flg22 and flg^Atum). (B) Quantitative analysis of ppGpp levels in A. thaliana seedlings 1 h after treatment with peptides (10 μM) or mock control indicated as pmol mg^–1 of fresh weight (FW). Data are presented as means ± SD (n ≥ 5). (C) qPCR analysis of gene expression 1 day after infection with PstDC3000 (upper panel) or treatment for 1h with 10 μM flg22 (lower panel). Data are presented as means ± SD (n = 4); ns = not significant; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test.