Pipecolic acid, an endogenous mediator of defense amplification and priming, is a critical regulator of inducible plant immunity

Abstract

Metabolic signals orchestrate plant defenses against microbial pathogen invasion. Here, we report the identification of the non-protein amino acid pipecolic acid (Pip), a common Lys catabolite in plants and animals, as a critical regulator of inducible plant immunity. Following pathogen recognition, Pip accumulates in inoculated Arabidopsis thaliana leaves, in leaves distal from the site of inoculation, and, most specifically, in petiole exudates from inoculated leaves. Defects of mutants in AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) in systemic acquired resistance (SAR) and in basal, specific, and β-aminobutyric acid-induced resistance to bacterial infection are associated with a lack of Pip production. Exogenous Pip complements these resistance defects and increases pathogen resistance of wild-type plants. We conclude that Pip accumulation is critical for SAR and local resistance to bacterial pathogens. Our data indicate that biologically induced SAR conditions plants to more effectively synthesize the phytoalexin camalexin, Pip, and salicylic acid and primes plants for early defense gene expression. Biological priming is absent in the pipecolate-deficient ald1 mutants. Exogenous pipecolate induces SAR-related defense priming and partly restores priming responses in ald1. We conclude that Pip orchestrates defense amplification, positive regulation of salicylic acid biosynthesis, and priming to guarantee effective local resistance induction and the establishment of SAR.

Figure 1.

Changes in the Levels of Free Amino Acids in Arabidopsis Col-0 Plants upon Leaf Inoculation with SAR-Inducing Psm. Left column: Inoculated, lower (1°) leaves at 2 DAI. Central column: Nontreated, upper (distal, systemic, 2°) leaves at 2 DAI. Right column: Petiole exudates from inoculated leaves collected between 6 to 48 h after inoculation. Mean values for leaf samples are given in µg g⁻¹ fresh weight (FW) ± sd and for leaf exudate samples in ng mL⁻¹ exudate solution leaf⁻¹ ± sd from at least four replicate samples. Mock treatments were performed by infiltration of leaves with a 10 mM MgCl₂ solution. Asterisks denote statistically significant differences between Psm (P) and MgCl₂ (M) samples (two-tailed t test; ***P < 0.001, **P < 0.01, and *P < 0.05).

Figure 2.

Pip and Aad Accumulation in P. syringae–Inoculated and PAMP-Treated Arabidopsis Leaves. (A) and (B) Time course of Pip (A) and Aad (B) accumulation in leaves inoculated with compatible Psm and incompatible Psm avrRpm1. FW, fresh weight; hpi, hours postinoculation. (C) and (D) Accumulation of Pip in Psm-inoculated leaves of wild-type Col-0 and selected defense mutant plants at 1 DAI (C) and 2 DAI (D). (E) Accumulation of Aad in Psm-inoculated leaves of wild-type Col-0 and selected defense mutant plants at 2 DAI. (F) and (G) Leaf levels of Pip (F) and Aad (G) 2 d after leaf treatment with 10 mM MgCl₂ (c), 200 nM flg22, and 100 µg mL⁻¹ LPS purified from E. coli. Data represent the mean ± sd of at least four replicate samples. In (A) and (B), asterisks denote statistically significant differences between P. syringae and MgCl₂ samples and in (F) and (G) between control and PAMP samples (***P < 0.001, **P < 0.01, and *P < 0.05; two-tailed t test). In (C) and (D), open (closed) circles indicate statistically significant differences between an MgCl₂ (Psm) mutant and the MgCl₂ (Psm) wild-type sample (two-tailed t test).

Figure 3.

Pip and SA Accumulation in Distal (2°) Leaves and Pip Levels in Leaf Petiole Exudates. (A) and (B) Time course of Pip (A) and free SA (B) accumulation in upper (2°) leaves following inoculation of lower (1°) leaves with Psm. FW, fresh weight; hpi, hours postinoculation. (C) Systemic Pip accumulation at 2 DAI in Col-0 and different defense mutant plants. (D) Pip levels in petiole exudates of leaves collected between 6 to 48 h after Psm or MgCl₂ treatment. Bars represent the mean ± sd of at least four replicate samples. In (A) to (C), asterisks denote statistically significant differences between P. syringae and MgCl₂ samples. In (D), open (closed) circles indicate statistically significant differences between an MgCl₂ (Psm) mutant and the MgCl₂ (Psm) wild-type sample.

Figure 4.

Pathogen-Inducible Pip and Aad Accumulation Is Dependent on ALD1 and LKR, Respectively. (A) and (B) Psm-induced ALD1 and LKR expression in Col-0 plants. Transcript levels were assessed by quantitative real-time PCR analysis, are given as means ± sd of three replicate samples, and are expressed relative to the respective mock control value. Asterisks denote statistically significant differences between Psm and MgCl₂ samples. (A) Relative expression in Psm-inoculated leaves (1 DAI). (B) Relative expression in upper (2°) leaves upon Psm inoculation of lower (1°) leaves (2 DAI). (C) to (E) Accumulation of Pip (C), Aad (D), and Lys (E) in Psm-inoculated leaves of Col-0, ald1, and lkr plants at 2 DAI. Data represent the mean ± sd of at least four replicate samples. Open (closed) circles indicate statistically significant differences between an MgCl₂ (Psm) mutant and the MgCl₂ (Psm) wild-type sample. FW, fresh weight.

Figure 5.

Exogenous Pip Enhances Disease Resistance of Wild-Type Arabidopsis and Overrides ald1 Defects in PTI and ETI. (A) SAR assay in Col-0, ald1, and lkr plants. Lower (1°) leaves were infiltrated with either 10 mM MgCl₂ or Psm (OD 0.005), and 2 d later, three upper leaves (2°) were challenge infected with Psm (OD 0.001). Bacterial growth in upper leaves was assessed 3 d after 2° leaf inoculation. (B) and (D) Bacterial numbers of compatible Psm (applied in titers of OD 0.001) (B) and incompatible Psm avrRpm1 (applied in titers of OD 0.002) (D) in Arabidopsis Col-0 and ald1 leaves at 3 DAI. Plant pots were supplied with 10 mL of water or 10 mL of 1 mM (≡ 10 µmol) Pip 1 d prior to inoculation. (C) Disease symptoms of Psm-infected Col-0 and ald1 plants in the absence and presence of exogenous Pip. Arrowheads denote inoculated leaves. Bars represent the mean ± sd of at least seven replicate samples. Asterisks denote statistically significant differences between indicated samples (***P < 0.001 and **P < 0.01; ns, not significant; two-tailed t test).

Figure 6.

Exogenous Pip Overrides ald1 Defects in SAR and BABA-Induced Resistance. (A) SAR assay in Col-0, ald1, and fmo1. Water or 10 µmol Pip was applied through the soil (s) of each plant pot, and 1 d later a 1° leaf infiltration with either 10 mM MgCl₂ or Psm (OD 0.005) in three lower leaves was performed. Another 2 d later, three upper leaves (2°) were challenge infected with Psm (OD 0.001). Bacterial growth in upper leaves was assessed 3 d after 2° leaf inoculation. ns, not significant. (B) BABA-induced resistance assay in Col-0, ald1, and fmo1. Water, BABA (10 µmol), Pip (10 µmol), or a combination of both BABA and Pip (10 µmol each) were applied through the soil, and resistance toward Psm was assessed. Bars represent mean values (± sd) of colony-forming units (cfu) per cm² from at least seven replicate samples, each consisting of three leaf disks. Asterisks denote statistically significant differences between indicated samples. No significant differences in initial bacterial numbers (1 h after inoculation) were detected. (C) Pip-induced resistance in wild-type Col-0 and different mutant lines to Psm infection. Colony-forming units of Psm (applied in titers of OD 0.001) at 3 DAI. Plant pots were supplied with 10 mL of water or 10 mL of 1 mM (=10 µmol) Pip 1 d prior to inoculation.

Figure 7.

Biological SAR Confers Defense Priming on the Metabolite and Gene Expression Levels in an ALD1-Dependent Manner. Double inoculation experiment to assess defense priming during SAR in Col-0 and ald1. Plants were treated in lower (1°) leaves with MgCl₂ or Psm (OD 0.005), and 2 d later, upper (2°) leaves were infiltrated with MgCl₂ or Psm. Upper leaves were then scored for defense metabolite accumulation or defense gene expression at 10 h after inoculation. This yielded four distinguishable cases corresponding to a control situation (1° MgCl₂/2° MgCl₂), a systemic pathogen stimulus (1° Psm/2° MgCl₂), a local pathogen stimulus (1° MgCl₂/2° Psm), and a combination of both the systemic and the local stimuli (1° Psm/2° Psm). FW, fresh weight. (A) to (D) SAR priming of defense metabolite accumulation. Free SA (A), total SA (sum of free SA and conjugated SA) (B), Pip (C), and camalexin (D) accumulation at 10 h after 2° treatment. Bars represent the mean ± sd of at least four replicate samples. Different letters above the bars denote statistically significant differences between pairwise compared samples (P < 0.05, two-tailed t test). (E) to (G) SAR priming of defense gene expression. Relative ALD1 (E), FMO1 (F), and PR-1 (G) expression at 10 h after 2° treatment. Transcript levels were assessed by quantitative real-time PCR analysis, are given as means ± sd of three replicate samples, and are expressed relative to the respective mock control value. Statistical differences in transcript abundance upon 2° Psm infection of SAR-noninduced versus SAR-induced plants were assessed. Closed (open) circles indicate whether statistically significant differences in Col-0 (ald1) between the 1° MgCl₂/2° Psm and the 1° Psm/2° Psm treatments exist (two-tailed t test).

Figure 8.

Exogenous Pip Confers SAR-Related Defense Priming on the Metabolite and Gene Expression Level and Restores Systemic SA Accumulation in ald1. Water or 10 µmol Pip were applied to plants through the soil. Leaves were infiltrated 1 d later with Psm or MgCl₂. FW, fresh weight. (A) and (C) to (F) Local defense responses were scored 10 h after infiltration. At the same time, a set of samples consisting of untreated leaves was assessed. (B) Total SA accumulation in upper (2°) leaves upon Psm or MgCl₂ infiltration of lower (1°) leaves was assessed at 2 DAI for Col-0 and ald1. (A) and (C) Accumulation of total SA (A) and camalexin (C) in Col-0 and ald1 plants. Bars represent the mean ± sd of at least four replicate samples. Different letters above the bars denote statistically significant differences between pairwise compared samples (P < 0.05, two-tailed t test). (D) to (F) Relative expression of ALD1 (D), FMO1 (E), and PR-1 (F). Transcript levels were assessed by quantitative real-time PCR analysis, are given as means ± sd of three replicate samples, and are expressed relative to the respective mock control value. Asterisks denote statistically significant differences between indicated samples (***P < 0.001; two-tailed t test).

Figure 9.

Proposed Model for the Role of Pip during Activation of Local Resistance, SAR, and Defense Priming. 1°, pathogen-inoculated leaf; 2°, distal leaf. Pathogen-induced changes in free amino acids, activation of Lys catabolism, including Pip formation, the roles for Pip in defense amplification and priming, and the proposed feedback amplification cycle in 2° leaves enabling SAR establishment that involves ALD1, Pip, FMO1, ICS1, and SA are illustrated. Dotted lines represent still hypothetical events. A possible role for Pip in long-distance signaling from 1° to 2° leaves remains to be clarified.