Research ArticlePLANT DEFENSES

An engineered pathway for N-hydroxy-pipecolic acid synthesis enhances systemic acquired resistance in tomato

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Science Signaling  22 Oct 2019:
Vol. 12, Issue 604, eaay3066
DOI: 10.1126/scisignal.aay3066
  • Fig. 1 Expression of Arabidopsis NHP biosynthesis enzymes in N. benthamiana.

    (A) Canonical pathway for biosynthesis of N-hydroxy-pipecolic acid (NHP) from l-lysine (l-Lys). PLP, pyridoxal phosphate; Pip, pipecolic acid. (B) Extracted ion abundance (EIC) of Pip (m/z = 156) and NHP (m/z = 172), as determined by GC-MS, in N. benthamiana leaves transduced with Agrobacterium strains harboring the indicated combinations of GFP and Arabidopsis ALD1, SARD4, and FMO1 (filled boxes). Bars represent the means ± SD. n = 3 independent biological replicates. Amounts reported as zero indicate no detection of metabolites.

  • Fig. 2 Local NHP application induces systemic defense in tomato leaves.

    (A) Diagram showing design of tomato (S. lycopersicum cultivar VF36) leaf treatment for SAR experiments. Two bottom leaflets of a tomato leaf were treated with 1 mM NHP or vehicle only (mock) for 24 hours, and then, the three top leaflets of the same leaf were inoculated with Pseudomonas syringae pathovar tomato strain DC3000 (Pst). (B) Titer of Pst in top leaflets at 4 days post-infection (dpi). Bars represent means ± SD. n = 3 independent biological replicates per condition per trial. Trials 1 and 2 indicate batches of plants grown months apart. Asterisks denote the significant differences between indicated samples using a one-tailed t test (**P < 0.01). (C) Disease symptoms of top leaflets infected with Pst at 4 dpi. For each treatment, panels show symptoms of a whole leaflet (left) and an enlarged region of the respective leaflet (right). Scale bars, 1 cm. Images are representative of n = 3 independent biological replicates per condition per trial. Trials 1 and 2 indicate batches of plants grown months apart.

  • Fig. 3 Engineering the NHP biosynthesis pathway in tomato leaves.

    (A) Diagram showing conditions for metabolite analysis. Two bottom leaflets of a tomato leaf were incubated with Agrobacteria carrying the gene encoding GFP or a combination of Agrobacteria carrying GFP, Arabidopsis ALD1, and Arabidopsis FMO1 (GAF) for 48 hours, and then, the two bottom treated leaflets and the three top untreated leaflets were harvested and analyzed by GC-MS. (B) EIC of Pip (m/z = 156) and NHP (m/z = 172), as determined by GC-MS, in leaflets from (A). Bars for bottom leaves represent means ± SD (one to two leaflets each from n = 3 independent plants). Bars for top leaves represent means ± SD (three leaflets each from n = 3 independent plants). Amounts reported as zero indicate no detection of metabolites. (C) Diagram showing conditions for the bacterial growth assay. Two bottom leaflets of a tomato leaf were treated with Agrobacteria strains as in (A). At 48 hpi, the three top untreated leaflets were inoculated with Pst. (D) The titer of Pst in the top leaflets was determined 4 days later. Bars represent means ± SD (three leaflets each from n = 3 independent plants). Asterisks denote the significant differences between indicated samples using a one-tailed t test (*P < 0.05 and ***P < 0.001). Trials 1 and 2 indicate batches of plants grown months apart.

  • Fig. 4 Evidence for NHP production across the plant kingdom.

    (A) Phylogenetic tree of sequenced plant genomes created using the PhyloT tree generator (https://phylot.biobyte.de). Circles are scaled by the percent amino acid identity of the best BLAST (blastp) hit between the Arabidopsis thaliana NHP biosynthetic proteins (yellow) and the ALD1, SARD4, and FMO1 homologs of the respective plant proteome (black). Solid circles signify that the homolog returned the respective A. thaliana protein in a reverse BLAST into the A. thaliana proteome (best reciprocal BLAST). (B) Detection of endogenous NHP production and FMO1 homolog activity in six plant species. Dashed lines label the species and common name, respectively, for plants examined: N. benthamiana, tobacco relative; S. lycopersicum, tomato; A. thaliana, thale cress; B. rapa, mustard; G. max, soybean; Zea mays, corn. To measure endogenous NHP, seedlings were grown hydroponically and then mock-treated (−Pst; gray) or treated with Pseudomonas syringae pathovar tomato strain DC3000 to induce SAR (+Pst; red). NHP was measured 48 hours later by GC-MS. Bars indicate EIC for NHP (m/z = 172). Asterisks indicate a significant NHP increase in Pst-treated plants (one-tailed t test; *P < 0.05). Pip and NHP-Glc abundances for this experiment were also measured (fig. S7). To measure enzyme activity, the FMO1 homologs from each plant with highest identity with Arabidopsis FMO1 were transiently expressed in N. benthamiana leaves in the presence of 1 mM Pip. Bars (blue) indicate EIC of NHP measured in leaves expressing the respective FMO1 homolog. Pip amounts were also measured, and an Arabidopsis FMO1 mutant (10) was included as a control (fig. S8). Bars represent the means ± SD (n = 3 independent biological replicates). Values reported as zero indicate no detection of metabolites.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/604/eaay3066/DC1

    Fig. S1. Abundances of unknown compounds during transient expression of Arabidopsis NHP biosynthesis genes in N. benthamiana.

    Fig. S2. Production of Pip and NHP in tomato plants in response to Pst.

    Fig. S3. Local NHP treatment induces systemic defense in pepper plants.

    Fig. S4. Production of NHP during transient expression in tomato leaves.

    Fig. S5. Engineering NHP biosynthesis in tomato leaves.

    Fig. S6. BLAST analysis of plant defense metabolites.

    Fig. S7. Production of Pip, NHP, and NHP-Glc in seedlings in response to Pst.

    Fig. S8. Production of NHP catalyzed by FMO1 homologs transiently expressed in N. benthamiana leaves.

    Table S1. Percent amino acid identity of best BLAST hits to A. thaliana proteins.

    Table S2. Primers used in this study.

  • This PDF file includes:

    • Fig. S1. Abundances of unknown compounds during transient expression of Arabidopsis NHP biosynthesis genes in N. benthamiana.
    • Fig. S2. Production of Pip and NHP in tomato plants in response to Pst.
    • Fig. S3. Local NHP treatment induces systemic defense in pepper plants.
    • Fig. S4. Production of NHP during transient expression in tomato leaves.
    • Fig. S5. Engineering NHP biosynthesis in tomato leaves.
    • Fig. S6. BLAST analysis of plant defense metabolites.
    • Fig. S7. Production of Pip, NHP, and NHP-Glc in seedlings in response to Pst.
    • Fig. S8. Production of NHP catalyzed by FMO1 homologs transiently expressed in N. benthamiana leaves.
    • Table S1. Percent amino acid identity of best BLAST hits to A. thaliana proteins.
    • Table S2. Primers used in this study.

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