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, 81 (20), 7003-11

Biosynthesis and Secretion of Indole-3-Acetic Acid and Its Morphological Effects on Tricholoma vaccinum-Spruce Ectomycorrhiza

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Biosynthesis and Secretion of Indole-3-Acetic Acid and Its Morphological Effects on Tricholoma vaccinum-Spruce Ectomycorrhiza

Katrin Krause et al. Appl Environ Microbiol.

Abstract

Fungus-derived indole-3-acetic acid (IAA), which is involved in development of ectomycorrhiza, affects both partners, i.e., the tree and the fungus. The biosynthesis pathway, excretion from fungal hyphae, the induction of branching in fungal cultures, and enhanced Hartig net formation in mycorrhiza were shown. Gene expression studies, incorporation of labeled compounds into IAA, heterologous expression of a transporter, and bioinformatics were applied to study the effect of IAA on fungal morphogenesis and on ectomycorrhiza. Tricholoma vaccinum produces IAA from tryptophan via indole-3-pyruvate, with the last step of this biosynthetic pathway being catalyzed by an aldehyde dehydrogenase. The gene ald1 was found to be highly expressed in ectomycorrhiza and induced by indole-3-acetaldehyde. The export of IAA from fungal cells is supported by the multidrug and toxic extrusion (MATE) transporter Mte1 found in T. vaccinum. The addition of IAA and its precursors induced elongated cells and hyphal ramification of mycorrhizal fungi; in contrast, in saprobic fungi such as Schizophyllum commune, IAA did not induce morphogenetic changes. Mycorrhiza responded by increasing its Hartig net formation. The IAA of fungal origin acts as a diffusible signal, influencing root colonization and increasing Hartig net formation in ectomycorrhiza.

Figures

FIG 1
FIG 1
GC-MS quantification of IAA from T. vaccinum cultures supplemented with labeled tryptophan. IAA was purified weekly over a period of 4 weeks and analyzed by GC-MS. IAA peaks were obtained by tracing [13C6]IAA. The IAA amounts shown represent IAA only from labeled tryptophan. Bars denote standard errors, and letters indicate significant differences in IAA amounts (P < 0.05). dpi, days postinoculation.
FIG 2
FIG 2
Feeding cells l-[2H5]tryptophan resulted in increased IAA concentrations, as documented by mass spectrometry after 4 weeks. Fragment ions of pure substances of [2H5]IAA (m/z 77, 107, 108, 134, 135, and 194) and of l-[2H5]tryptophan (m/z 77, 91, 165, 181, 197, 209, and 210) were used for identification.
FIG 3
FIG 3
Tryptophan-dependent biosynthesis pathways for IAA production (59). The biosynthetic pathway used by T. vaccinum (boldface) was confirmed by feeding cells labeled precursors (for which structural formulas are given). Genes coding for the enzymes involved were identified from T. vaccinum genomic sequences: tam1 (coding for a tryptophan aminotransferase; GenBank accession no. KP096350), ipd1 (coding for an indole-3-pyruvic acid decarboxylase; GenBank accession no. KP096351), ald1 (coding for an aldehyde dehydrogenase; GenBank accession no. HM363121), and iah1 (coding for an indole-3-acetamide hydrolase; GenBank accession number KP096352). Conversion of IAN to IAA has not yet been fully clarified; thus this potential route is indicated as a dotted line.
FIG 4
FIG 4
Effect of IAA precursors on ald1 gene expression. Expression was tested in three biological replicates by qPCR and normalized relative to a standard curve. Bars denote standard errors.
FIG 5
FIG 5
T. vaccinum cell length and ramification in response to feeding cells external IAA or precursors at different concentrations. A total of 100 cells were counted (n = 3 replicates). Bars denote standard errors.
FIG 6
FIG 6
Expression of ald1 in T. vaccinum wild-type (T.v.-WT) and the ald1-overexpressing strain T. vaccinum 1.16 (T.v.-116). The regulation was evaluated in three biological replicates by qPCR and normalized to three reference genes (act1, cis1, and tef1). Bars denote standard errors, and letters indicate significantly different expression levels (P < 0.05).
FIG 7
FIG 7
Effect of IAA on ectomycorrhiza development in microtome sections of 4-month-old spruce ectomycorrhiza with T. vaccinum in hydroponic culture. The ald1-overexpressing T. vaccinum strain 1.16 (a), the transformation recipient (b), and a nonmycorrhizal short root of P. abies (c) are shown. Scale bar, 100 μm.
FIG 8
FIG 8
Drop test of Saccharomyces cerevisiae expressing the empty vector or mte1 showing the response to IAA applied at two concentrations (0.025 and 0.05 mM).

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