Aberrant synthesis of indole-3-acetic acid in Saccharomyces cerevisiae triggers morphogenic transition, a virulence trait of pathogenic fungi

Abstract

Many plant-associated microbes synthesize the auxin indole-3-acetic acid (IAA), and several IAA biosynthetic pathways have been identified in microbes and plants. Saccharomyces cerevisiae has previously been shown to respond to IAA by inducing pseudohyphal growth. We observed that IAA also induced hyphal growth in the human pathogen Candida albicans and thus may function as a secondary metabolite signal that regulates virulence traits such as hyphal transition in pathogenic fungi. Aldehyde dehydrogenase (Ald) is required for IAA synthesis from a tryptophan (Trp) precursor in Ustilago maydis. Mutant S. cerevisiae with deletions in two ALD genes are unable to convert radiolabeled Trp to IAA, yet produce IAA in the absence of exogenous Trp and at levels higher than wild type. These data suggest that yeast may have multiple pathways for IAA synthesis, one of which is not dependent on Trp.

Figure 1.—

The IAA biosynthetic pathway identified in this study (in boldface type) and the analogous pathway identified in U. maydis (right, underlined) where the homologs of Ald2 and Ald3 have been shown to catalyze the conversion of indole-3-acetaldehyde to indole-3-acetic acid.

Figure 2.—

(A) S. cerevisiae produces a molecule that comigrates with commercially available IAA. Wild-type yeast cells were incubated with [¹⁴C]Trp, and products of the conditioned media were resolved by thin layer chromatography (TLC). Commercially available IAA and [¹⁴C]IAA were used as controls. The position (marked with the asterisk) of the nonradiolabeled IAA control was determined by UV shadowing. (B) Total ion chromatogram (TIC, left) and full scan spectrum (right) of authentic methyl-IAA. (C) Top, methyl-IAA molecular ion m/z 189 and fragment ion m/z 130 (the site of fragmentation to form the fragment ion is indicated by dashed lines). Bottom, methyl-[¹³C₆]IAA molecular ion m/z 195 and fragment ion m/z 136. For each compound, the derivatization moiety (the methyl group) is shown in red. (D) TIC (left) and corresponding full-scan spectrum (right) of IAA (methylated prior to GC-MS analysis) that was purified from the culture medium of wild-type yeast that had been grown in the presence of Trp. The TIC shows four selected ions; m/z 130 and m/z 189 are the fragment ion and the molecular ion, respectively, of endogenous IAA (methylated prior to GC-MS analysis). Ions with m/z 136 and 195 are the fragment ion and the molecular ion, respectively, of [¹³C₆]IAA (methylated prior to GC-MS analysis) that was added to the yeast culture medium supernatant prior to extraction of IAA. The large peak in the TIC (left) with a retention time of ∼7.15 min was determined to be tryptophol by full-scan spectra analysis (not shown). (E) The CM taken from a high-density culture contained a much greater concentration of IAA than CM from a low-density culture as determined by TLC (bottom) and densitometry of the autoradiograph (top).

Figure 3.—

Products of the CM of ald single deletion mutants and ald2Δald3Δ double deletion incubated with [¹⁴C]Trp were resolved by TLC and compared to the isogenic wild-type strain. Each experiment was performed a minimum of three times. Three and two independent transformants were tested for the single and double mutants, respectively. One representative transformant for each mutant is shown.

Figure 4.—

(A) A representative diploid ald2Δ/Δ ald3Δ/Δ colony was grown on filamentation-inducing media and photographed after 3 days of growth (bar, 1 μm). (B) Haploid ald2Δald3Δ strains were spotted onto SC media and washed. Before wash, unwashed plates; after wash, the plates after washing. (C) A filter disk saturated with IAA (right) was placed on a lawn of ald2Δald3Δ mutant cells (bottom) and compared to the wild-type cells (top). Control disks (left) do not contain IAA. Plates were incubated for 3 days in the dark. The clear area around the IAA-containing filter disks indicates a zone of growth inhibition.

Figure 5.—

The human pathogen Candida albicans was exposed to IAA [experimental plates (E–H) contain 50 μm IAA and control plates (A–D) contain no IAA; bar in A, 10 μm]. A–H show the edge of a patch of C. albicans (A and E, cph1efg1; B and F, cph1; C and G, efg1; and D and H, isogenic wild-type control strains) growing on synthetic low ammonium media with xylose as a carbon source. Plates were incubated in the dark to prevent photodegradation of IAA. I and J show the IAA sensitivity profile of a cap1 homozygous deletion mutant as compared to an isogenic wild-type and a heterozygous mutant [experimental plates (I) contain 120 μm IAA, and control plates (J) contain no IAA].