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. 2018 Feb 8;13(2):e0192293.
doi: 10.1371/journal.pone.0192293. eCollection 2018.

Characterization of indole-3-pyruvic Acid Pathway-Mediated Biosynthesis of Auxin in Neurospora Crassa

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Free PMC article

Characterization of indole-3-pyruvic Acid Pathway-Mediated Biosynthesis of Auxin in Neurospora Crassa

Puspendu Sardar et al. PLoS One. .
Free PMC article

Abstract

Plants, bacteria and some fungi are known to produce indole-3-acetic acid (IAA) by employing various pathways. Among these pathways, the indole-3-pyruvic acid (IPA) pathway is the best studied in green plants and plant-associated beneficial microbes. While IAA production circuitry in plants has been studied for decades, little is known regarding the IAA biosynthesis pathway in fungal species. Here, we present the first data for IAA-producing genes and the associated biosynthesis pathway in a non-pathogenic fungus, Neurospora crassa. For this purpose, we used a computational approach to determine the genes and outlined the IAA production circuitry in N. crassa. We then validated these data with experimental evidence. Here, we describe the homologous genes that are present in the IPA pathway of IAA production in N. crassa. High-performance liquid chromatography and thin-layer chromatography unambiguously identified IAA, indole-3-lactic acid (ILA) and tryptophol (TOL) from cultures supplemented with tryptophan. Deletion of the gene (cfp) that encodes the enzyme indole-3-pyruvate decarboxylase, which converts IPA to indole-3-acetaldehyde (IAAld), results in an accumulation of higher levels of ILA in the N. crassa culture medium. A double knock-out strain (Δcbs-3;Δahd-2) for the enzyme IAAld dehydrogenase, which converts IAAld to IAA, shows a many fold decrease in IAA production compared with the wild type strain. The Δcbs-3;Δahd-2 strain also displays slower conidiation and produces many fewer conidiospores than the wild type strain.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Multiple sequence alignment of tryptophan aminotransferase homologs from different fungal species.
Residues with 100% identity are shown in yellow font with a black background, residues with 75% identity are shown in black font with a red background, and residues with 50% identity are shown in black font with a green background. PLP binding residues are marked with a pink star at the top. Sequences are presented in a discontinuous fashion. Blue discontinuous symbols are shown when there is a discontinuity of the sequence.
Fig 2
Fig 2. Structural alignment and characterization of ligand binding sites of predicted N. crassa enzymes.
Predicted structures and known templates are shown in blue and gray, respectively. Residues present in predicted enzymes and templates are indicated in orange and green, respectively. All structures are rendered as a ribbon. Key amino acid residues involved in ligand binding are rendered in a stick model. Residues in close vicinity are only highlighted. Ligands are presented in a ball and stick model. (A) The overall structural alignment of predicted ARO-8 structure with a known enzyme structure (4JE5). PLP is bound inside the enzymatic catalytic site. (B) Structural insight into the ligand binding site of both the predicted and 4JE5 known structure. (C) Structural alignment of the predicted structure of the pyruvate decarboxylase homolog from N. crassa with a known pyruvate decarboxylase structure (2VJY). TPP is bound inside the enzymatic catalytic site. (D) Characterization of the ligand binding site of the predicted enzyme using the 2VJY structure as a template.
Fig 3
Fig 3. IAA and related indolic compound production by N. crassa upon tryptophan supplementation.
N. crassa produces IAA and other related indolic compounds when supplemented with tryptophan. (A) TLC analysis to find the lowest concentration of tryptophan that can be used by N. crassa to produce IAA. White arrows in lane 1 and 2 indicate traces of IAA. Different tryptophan concentrations were used in lane 1 (5 μM), 2 (1 μM), 3 (100 nM), 4 (50 nM), 5 (25 nM), and 6 (without tryptophan). (B) HPLC analysis of the metabolites produced by wild type N. crassa with and without tryptophan supplementation for 72 hours. (C) Metabolite profiling of wild type as well as single knock-out N. crassa strains using HPLC. Single knock-outs for the genes specifically present in the IPA pathway of IAA biosynthesis were used. The following standards were used: tryptophan (Trp), indole-3-lactic acid (ILA), indole-3-acetic acid (IAA), tryptophol (TOL) and indole-3-acetonitrile (IAN).
Fig 4
Fig 4. Proposed tryptophan-dependent IAA biosynthetic pathway in N. crassa.
The pathway with solid bold arrows was functionally characterized by the identification of novel genes, and intermediate compounds were also identified and estimated. Pathways with dashed bold arrows were proposed accordingly based on the feeding test with IAM and TAM. Solid thin arrows denote pathways proposed in green plants but not known in fungi including N. crassa. Unidentified homologues of the genes in N. crassa for the respective biosynthetic steps are indicated by question marks. IAN, with bracketed lines, has not been identified in the N. crassa culture. All other indoles without any brackets (except IAM and TAM) were identified from the culture upon tryptophan feeding. Indole-3-pyruvic acid (IPA) and indole-3-acetaldehyde (IAAld) are very unstable compounds and spontaneously convert into indole-3-lactic acid (ILA) and indole-3-ethanol (TOL), respectively. ILA and TOL can be readily identified in culture as a proxy for IPA and IAAld, respectively. Numbers in the figure reflect different enzymes: 1—tryptophan aminotransferase, 2—pyruvate decarboxylase, 3—aldehyde dehydrogenase, 4—flavin monooxygenase, 5—L-amino acid oxidase, 6—N-acylethanolamine amidohydrolase, 7 –aromatic-L-amino acid decarboxylase and glutamate decarboxylase.
Fig 5
Fig 5. Effect of the Δcfp strain on ILA production in N. crassa.
(A) HPLC peaks (peak intensity is given in mv as in Fig 3) of indole-3-lactic acid (ILA) from the wild type and Δcfp strain are shown in different colors. (B) Amount of ILA produced in 72-hour cultures of wild type and Δcfp knock-out strains supplemented with tryptophan. * p < 0.05.
Fig 6
Fig 6. Reduction in IAA production by the synergistic effect of the Δcbs-3;Δahd-2 double knock-out in N. crassa.
Individual knock-out strains for the aldehyde dehydrogenase genes cbs-3 and ahd-2 did not have any significant effect on IAA production, but the synergistic effect of the Δcbs-3;Δahd-2 double knock-out resulted in a strong reduction of IAA production. (A) HPLC peaks of IAA for wild type along with other knock-out strains are shown in different colors as mentioned in the figure. IAA production was decreased in the Δcbs-3;Δahd-2 double knock-out strain compared with the wild-type strain. (B) Comparison of the amount of IAA produced by wild type and Δcbs-3;Δahd-2 strains in 72 hours. * p < 0.05.
Fig 7
Fig 7. Conidiospore production by the Δcbs-3;Δahd-2 double mutant.
(A) and (C) are wild type strains, and (B) and (D) are Δcbs-3;Δahd-2 double knock-out strains after growth for three and five days, respectively. (E) A numerical comparison of conidia production by the Δcbs-3;Δahd-2 double knock-out strain with wild-type and the Δcfp single knock-out strain. dai: days after inoculation. * p < 0.05.
Fig 8
Fig 8. Indole production by the wt N. crassa strain supplemented with tyrosine and other indoles.
Indole production by wt N. crassa was checked by supplementation with different concentrations of tyrosine. (A) lanes 1–5 tyrosine in 5 mM, 2.5 mM, 1.25 mM, 625 μM and 312.5 μM. (B) TLC analysis to determine the ability to produce indoles, including IAA, by various indolic compounds. Black arrows indicate traces of IAA. Indolic compounds that were used as supplements are as follows: lane 1—mM tryptophan, lane 2–2.5 mM tryptamine, 3–1 mM indole-3-acetamide, 4–1 mM indole-3-lactic acid, 5–1 mM tryptophol, 6–10 μM IAA. Following standards were used: tryptophol (TOL), tryptamine (TAM), indole-3-acetamide (IAM), indole-3-acetic acid (IAA), tryptophan (Trp), indole-3-pyruvic acid (IPA) and indole-3-lactic acid (ILA).

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Grant support

The authors received no specific funding for this work. However PS received a stipend from the Max-Planck-International-Research-School Evolutionary Biology.
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