10 research outputs found

    Overview of tryptophan-dependent indole-3 acetic acid (IAA) biosynthesis pathway(s) in bacteria.

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    <p>Enzymes with demonstrated biochemical activities are indicated. Enzyme abbreviations: tryptophan 2-monooxygenase (TMO), indole acetamide hydrolase (IAH), tryptophan side chain oxidase (TSO), indole pyruvate decarboxylase (IPDC) and aldehyde dehydrogenase (ALD). Two ALD enzymes, AldA and AldB, that catalyze conversion of IAAld to IAA are described in this study. Compound abbreviations: tryptophan (Trp), indole-3-acetaldoxime (IAOx), indole-3-acetonitrile (IAN), indole-3-acetamide (IAM), indole-3-pyruvate (IPyA), indole-3-acetaldehyde (IAAld) and tryptamine (TAM).</p

    Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of <i>Pseudomonas syringae</i> strain DC3000

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    <div><p>The bacterial pathogen <i>Pseudomonas syringae</i> modulates plant hormone signaling to promote infection and disease development. <i>P</i>. <i>syringae</i> uses several strategies to manipulate auxin physiology in <i>Arabidopsis thaliana</i> to promote pathogenesis, including its synthesis of indole-3-acetic acid (IAA), the predominant form of auxin in plants, and production of virulence factors that alter auxin responses in the host; however, the role of pathogen-derived auxin in <i>P</i>. <i>syringae</i> pathogenesis is not well understood. Here we demonstrate that <i>P</i>. <i>syringae</i> strain DC3000 produces IAA via a previously uncharacterized pathway and identify a novel indole-3-acetaldehyde dehydrogenase, AldA, that functions in IAA biosynthesis by catalyzing the NAD-dependent formation of IAA from indole-3-acetaldehyde (IAAld). Biochemical analysis and solving of the 1.9 ├ů resolution x-ray crystal structure reveal key features of AldA for IAA synthesis, including the molecular basis of substrate specificity. Disruption of <i>aldA</i> and a close homolog, <i>aldB</i>, lead to reduced IAA production in culture and reduced virulence on <i>A</i>. <i>thaliana</i>. We use these mutants to explore the mechanism by which pathogen-derived auxin contributes to virulence and show that IAA produced by DC3000 suppresses salicylic acid-mediated defenses in <i>A</i>. <i>thaliana</i>. Thus, auxin is a DC3000 virulence factor that promotes pathogenicity by suppressing host defenses.</p></div

    Heterologous expression of putative DC3000 aldehyde dehydrogenases in <i>E</i>. <i>coli</i>.

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    <p>DC3000 genes encoding putative aldehyde dehydrogenase proteins were expressed in <i>E</i>. <i>coli</i> BL21(DE3) cells. A) Quantification of IAA produced by strains expressing PSPTO_0728, PSPTO_2673, and PSPTO_3644 and pET-21a as a negative control. B) Quantification of IAA produced by strains expressing PSPTO_0092, PSPTO_3064, and PSPTO_3323. PSPTO_2673 was included as a control for comparison to panel A. IAA levels were measured in supernatants 24 hrs post-induction with addition of 0.25 mM IAAld. Values are an average ┬▒ SEM (n = 3). Letters indicate significant difference between samples within a given time point (<i>p</i><0.01). Similar results were obtained from two additional independent experiments.</p

    Quantification of IAA production in DC3000 <i>ald</i> mutants.

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    <p>A) Measurement of IAA accumulation in supernatants of DC3000 <i>ald</i> single mutants grown for 48 hrs in HSC media supplemented with 0.25 mM IAAld. B) Growth of <i>ald</i> mutants in HSC media supplemented with 0.25 mM IAAld. These cultures were used for quantification of IAA shown in panel A. C) Measurement of IAA accumulation in supernatants of <i>aldA</i>, <i>aldB</i> and the <i>aldA aldB</i> double mutant grown for 48 hrs in HSC media supplemented with 0.25 mM IAAld. The <i>aldB</i> mutant in this experiment is the <i>aldB</i>::pJP5603Tet mutant. For panels A-C, values are an average of three biological replicates ┬▒ SEM (error bars too small to see in panel B). Letters indicate significant difference between samples within a given time point (<i>p</i><0.01. c* in panel A indicates that IAA levels in <i>aldB</i> are significantly different from wt at p<0.05).</p

    Overall structure of AldA.

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    <p>A) The AldA tetramer is shown as a ribbon tracing with each subunit differentially colored. Two subunits (gold and white) were in the asymmetric unit of the crystal with the other two subunits (green and rose) related by crystallographic symmetry. N-termini are labeled. B) Domain organization of the AldA monomer. The view is rotated 90┬░ relative to panel A and shows the two subunits in the asymmetric unit. The catalytic (red), cofactor binding (blue), and oligomerization (rose) domains are highlighted in one monomer. The position of NAD<sup>+</sup> (space-filling model) is indicated. C) Substrate binding sites on opposite sides of the AldA monomer. The two views of an AldA monomer are rotated 180┬░ and show the locations of the NAD(H) and IAAld/IAA binding sites on each face of the monomer. D) Ligand binding tunnel. The positions of NAD<sup>+</sup> (rose) and IAA (gold) in the tunnel (grey surface) relative to the catalytic cysteine (Cys302) are shown. The position of docked IAAld (rose), which overlaps with IAA, is indicated.</p

    <i>PR1</i> expression in plants inoculated by <i>ald</i> mutants and growth of <i>ald</i> mutants on SA-deficient <i>sid2-2</i> plants.

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    <p>A) <i>PR1</i> expression in Col-0 plants at 24 hrs following syringe infiltration (OD<sub>600</sub> = 1x10<sup>-5</sup>). Significant elevation of <i>PR1</i> expression in <i>aldA</i>-infected plants was observed in three independent experiments, and in two experiments for <i>aldB</i>-infected plants. B) Growth of <i>ald</i> single and double mutants on wild type <i>A</i>. <i>thaliana</i> (Col-0) and <i>sid2-2</i> mutant plants following syringe infiltration. Graph shows composite data from 4 independent experiments. Letters indicate significant difference between samples within a given time point (<i>p</i><0.02).</p

    Substrate and cofactor binding sites of AldA.

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    <p>A) Electron density of NAD<sup>+</sup> and IAA. The 2F<sub>o</sub>-F<sub>c</sub> omit map (1.5 ¤â) for NAD<sup>+</sup> and IAA is shown. B) NAD(H) binding site. Side-chains of residues interacting with NAD<sup>+</sup> (rose) are shown as stick-renderings. Waters interacting with the cofactor are shown as red spheres. Hydrogen bonds are indicated by dotted lines. C) IAAld/IAA binding site. NAD<sup>+</sup>, IAA, and side-chains are shown as stick-renderings with dotted lines indicating hydrogen bonds. D-F) Hydrophobicity of the substrate binding sites of AldA (panel D), AldB (panel E), and AldC (panel F). Homology models of AldB and AldC were generated based on the x-ray structure of AldA. Hydrophobicity was calculated using the Color-h script in PyMol. Darkest red indicates strongest hydrophobicity to white as the most polar. G-I) Electrostatic surface of the substrate binding sites of AldA (panel G), AldB (panel H), and AldC (panel I). Electrostatic surface charge was generated using the APBS plugin in PyMol with red = acidic and blue = basic.</p

    Growth of <i>ald</i> mutants on <i>A</i>. <i>thaliana</i>.

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    <p>A) Growth of DC3000 and <i>ald</i> mutants following syringe infiltration of <i>A</i>. <i>thaliana</i>. Graph shows composite data from 4 independent experiments. Letters indicate significant difference between samples within a given time point (<i>p</i><0.001). B) Growth of DC3000, <i>aldA</i>, <i>aldB</i> and the <i>aldA aldB</i> double mutant following syringe infiltration of <i>A</i>. <i>thaliana</i>. Letters indicate significant difference between samples within a given time point (<i>p</i><0.05).</p
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