The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants

Abstract

Innate immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs). Here, we show that elongation factor Tu (EF-Tu), the most abundant bacterial protein, acts as a PAMP in Arabidopsis thaliana and other Brassicaceae. EF-Tu is highly conserved in all bacteria and is known to be N-acetylated in Escherichia coli. Arabidopsis plants specifically recognize the N terminus of the protein, and an N-acetylated peptide comprising the first 18 amino acids, termed elf18, is fully active as inducer of defense responses. The shorter peptide, elf12, comprising the acetyl group and the first 12 N-terminal amino acids, is inactive as elicitor but acts as a specific antagonist for EF-Tu-related elicitors. In leaves of Arabidopsis plants, elf18 induces an oxidative burst and biosynthesis of ethylene, and it triggers resistance to subsequent infection with pathogenic bacteria.

Figure 1.

Induction of Extracellular Alkalinization by Bacteria and Bacterial Extracts. (A) Extracellular pH in Arabidopsis cells after treatment with crude cell-free extracts from E. coli strain GI826 (FliC⁻), R. solanacearum, and S. meliloti. At t = 0 min, cells were either treated with 10 μL/mL of bacterial extracts or bacterial extracts that were preincubated with endoproteinase Glu-C (50 μg/mL for 6 h at 25°C). (B) Response to treatment with a suspension of living E. coli FliC⁻ cells or the cell-free supernatant of this suspension, either without further treatment or after heating (95°C, 10 min) or digestion with pronase (100 μg/mL, 15 min, 25°C).

Figure 2.

Identification of the Elicitor-Active Protein as EF-Tu. (A) Alkalinization-inducing activity in extract from E. coli strain GI826 was prepurified on MonoQ-ion exchange chromatography and separated by SDS-PAGE. The dried Coomassie blue–stained gel was cut in slices, and the eluates of these slices were assayed for alkalinization-inducing activity by measuring extracellular pH in Arabidopsis cells after 20 min of treatment. (B) Amino acid sequence of mature EF-Tu protein from E. coli (Laursen et al., 1981). Eluate with highest elicitor activity was digested with trypsin, and peptide masses were compared with the masses calculated for the proteome of E. coli. Underlined sequences indicate peptides with masses matching the ones calculated for EF-Tu. With the exception of the amino acids indicated with a shaded background, EF-Tu is highly conserved with identical amino acids in >90% of the sequences from different bacteria (n > 100 sequences in the database). (C) Activity of EF-Tu and of EF-Tu digested with endoproteinase Glu-C or CNBr. Different doses of purified intact EF-Tu (closed circles), EF-Tu after digestion with endoprotease Glu-C (open triangles) and EF-Tu after cleavage with CNBr (open diamonds) were assayed for induction of alkalinization in Arabidopsis cells. Extracellular pH was measured after 20 min of treatment. Data points and bars represent mean and standard deviation of three replicates.

Figure 3.

Identification of the CNBr Fragment Carrying Elicitor Activity. (A) The CNBr digest of EF-Tu was separated on a C8 reverse-phase column. Fractions containing activity were rerun on C8 using a more shallow gradient, and eluate was assayed for UV absorption (OD₂₁₄ nm) and elicitor activity (bars). (B) Masses found in peak II with nanospray analysis. (C) Peptide masses observed after trypsin digestion of peptides in peak II that map to the CNBr fragment of EF-Tu 1-91. (D) Structure of whole unmodified EF-Tu (Song et al., 1999) completed with a tentative computer-assisted prediction (Geno3D; Combet et al., 2002) for the eight N-terminal amino acid residues. Ribbon model with the N-terminal part shown as ball and stick (drawn with WebLab ViewerLite; Molecular Simulations, Cambridge, UK).

Figure 4.

Elicitor Activity of Peptides Representing the N Terminus of EF-Tu. Different doses of synthetic peptides representing the amino acids 1 to 26 of EF-Tu, either with the N-terminal NH₂-group left free (1-26) or coupled to an extra Met residue (M-1-26), an acetyl group (ac-1-26), or Fmoc used as protective group in the peptide synthesis (Fmoc-1-26), were assayed for induction of alkalinization in Arabidopsis cells. Extracellular pH was measured after 20 min of treatment; pH at the beginning of the experiment was 4.8.

Figure 5.

Alkalinization-Inducing Activity of EF-Tu N-Terminal Peptides. Summary of EC₅₀ values determined from dose–response curves with the different peptides. Peptide sequences and N-terminal acetylation (ac∼) are indicated at the left. Bars and error bars in the right part represent EC₅₀ values and their standard errors on a logarithmic scale. Hatched bars indicate activity of peptides that act as partial agonists, inducing 50% of the pH amplitude observed for full agonists at the concentrations indicated, but fail to induce a full pH change even at the highest concentrations of 30 μM tested. No activity could be detected with peptides denoted with asterisks (EC₅₀ >10⁴ nM).

Figure 6.

Antagonistic Activity of elf12 for EF-Tu–Related Elicitors. (A) Alkalinization induced by 1 nM flg22 or 0.5 nM elf18 when applied alone or together with 30 μM elf12. (B) Effect of 30 μM elf12 on the alkalinization induced by the cell-free supernatant from living E. coli FliC⁻ or crude bacterial extracts from R. solanacearum and A. tumefaciens.

Figure 7.

Induction of Elicitor Responses in Leaf Tissues of Different Plant Species. (A) Induction of ethylene biosynthesis in leaf tissue. Leaf pieces from various plant species were mock treated (controls) or treated with 1 μM elf26, and ethylene was measured after 2 h. Results, represented as fold-induction over control, show mean and standard deviation of n = 4 replicates. (B) Oxidative burst in leaf tissues of Arabidopsis accessions Ws-0 (left panel) and Col-0 (right panel). Luminescence (relative light units [RLU]) of leaf slices in a solution with peroxidase and luminol was measured after addition of EF-Tu protein or the peptides indicated. Light emission during the first seconds of the measurements was because of phosphorescence of the green plant tissue.

Figure 8.

Induction of Defense Responses in Arabidopsis. (A) Induction of GUS activity in lines of Ws-0 and Col-0 transgenic for SIRKp:GUS. Leaves of both lines were pressure infiltrated with 1 μM flg22, 1 μM elf26, crude preparations of E. coli FliC⁻ and R. solanacearum (diluted 1:100 in 10 mM MgCl₂), or 10 mM MgCl₂ (control). After 24 h of treatment, leaves were detached from the plants and stained for GUS activity. (B) Arabidopsis wild-type Landsberg erecta-0 (Ler-0) and fls2-17 plants were pretreated for 24 h with 1 μM flg22, 1 μM elf26, or water as a control. These leaves were subsequently infected with 10⁵ colony-forming units (cfu)/mL Pst DC3000, and bacterial growth was assessed 2 d postinfection (dpi). Results show average and standard error of values obtained from four plants with two leaves analyzed each (n = 8). The solid and dashed lines indicate mean and standard deviation of cfu extractable from leaves at 0 dpi (n = 12).