The identification of a key gene highlights macrocyclic ring's role in trichothecene toxicity

Abstract

Trichothecenes are toxins produced by certain species from several fungal genera, including Aspergillus, Fusarium, Isaria, Paramyrothecium, Stachybotrys, Trichoderma, and Trichothecium. These toxins are of interest because they contribute to the toxigenicity, plant pathogenicity, and/or biological control activities of some fungi. All trichothecenes have the same core (12,13-epoxytrichothec-9-ene or EPT) structure but can differ from one another by the presence or absence of a macrocyclic ring formed from polyketide and isoprenoid substituents esterified to carbon atoms 4 and 15 of EPT, respectively. Genes required for formation and some modifications of EPT have been elucidated, but almost nothing is known about genes specific to the formation of the macrocyclic ring. Therefore, we used genomic, transcriptomic, metabolomic, and gene deletion analyses to identify genes that are required specifically for the formation of the macrocyclic ring. These analyses identified one gene, TRI24, that is predicted to encode an acyltransferase and that is required for macrocyclic ring formation during biosynthesis of macrocyclic trichothecenes by the fungus Paramyrothecium roridum. In addition, a TRI24 deletion mutant of P. roridum caused less severe disease symptoms on common bean and had less antifungal activity than its wild-type progenitor strain. We propose that the reduced aggressiveness and antifungal activity of the mutant resulted from its inability to produce trichothecenes with a macrocyclic ring. To our knowledge, this is the first report of a gene required specifically for the formation of the macrocyclic ring of trichothecenes and that loss of the macrocyclic ring of trichothecenes can alter the biological activities of a fungus. KEY POINTS: • TRI24 gene is found in all known macrocyclic trichothecene-producing fungi. • A tri24-deletion mutant exhibits a reduction in antifungal and plant disease activities. • TRI24 is the first described gene specific to macrocyclic trichothecene biosynthesis.

Keywords: Paramyrothecium roridum; Gene deletion; Macrocyclic trichothecenes; Trichothecene toxicity; Trichoverrins.

Fig. 1

Chemical structures of examples of the macrocyclic trichothecenes roridins, verrucarins, and satratoxins (left) and putative intermediates of the macrocyclic trichothecene biosynthesis (right) (modified from Ueno ; Grove 2007)

Fig. 2

TRI clusters in macrocyclic trichothecene producer fungi. TRI genes are indicated by arrows with different colors, depending on their described function. TRI24 and TRI25 are indicated by white arrows. Gray arrows indicated genes presumably not involved in trichothecene biosynthesis or with unknown functions; the numbers above colored arrows indicate TRI designations (e.g., 17 indicates TRI17). MONOS, Monosporascus sp. 5C6A; SCHAR, Stachybotrys chartarum 40,293; PRORI, Paramyrothecium roridum; TBALE, Trichoderma balearicum; TCRYS, Trichoderma chrystalligenum; TOLIG, Trichoderma oligosporum; TPSYC, Trichoderma psychrophilum

Fig. 3

HPLC analysis of P. roridum wild type (upper central panel), ∆tri24 mutant and ∆tri24-T24.5 complemented transformant (lower left panels), and ∆tri25 mutant and ∆tri25_T25.2 complemented transformant (lower right panels) from 72-h broths. The main peaks were pointed by arrows, and labeled with 1–5, for further reference. Those new peaks detected in the ∆tri24 mutant were pointed with blue arrows

Fig. 4

TLC (left panel) analysis of 72-h PDB broths of wild type (wt) and ∆tri24 mutant (∆T24), and (right panel) antibiogram of the TLC plate against K. marxianus

Fig. 5

Proton nuclear magnetic resonance (A) and carbon-13 nuclear magnetic resonance (carbon-13 NMR spectroscopy) (B) of spot 4 (15-hydroxy trichodermadiene)

Fig. 6

HPLC analysis from feeding cultures of wild-type Ta37 (control) and two transformants of Ta37 expressing the P. roridum TRI24 gene (transformants Ta37_T24.1 and Ta37_T24.2). Cultures were incubated in YEPD without feeding (left panels) and fed with 15-hydroxy trichodermadiene A/B (4) (1.5 mg in 8 mL total volume) (right panels). The elution times of the two pairs of peaks appearing only in the TRI24-expressing transformants are shaded in yellow and green. A chromatogram of 4 (1/10 dilution) has been included for comparative purposes (left upper panel)

Fig. 7

Relative values of expression for genes in the P. roridum TRI genomic region. Expression values are based on the fold change values in the expression of each gene in the ∆tri24 mutant (∆T24) versus the wild-type strain (PR). Green and red (TRI5) bars corresponded to already known TRI genes (including TRI24), purple bars point to genes not yet characterized but with a similar pattern of expression to that observed for the TRI-biosynthetic genes in the ∆tri24 mutant, and gray bars indicated genes most likely not involved in trichothecene biosynthesis. Asterisks indicate those ratio values that are statistically significant (p < 0.01). In the lower part of the figure, arrows indicate genes and point in the direction of transcription. Numbers below the arrows indicate the gene name, when known, and the number assigned in the analysis in the P. roridum genome, e.g., TRI5_g10502, corresponded to the TRI5 gene with the 10,502 number

Fig. 8

A Antifungal effect against R. solani of PDB cultures harvested after 72-h of growth from the different P. roridum wild type (wt), ∆tri24 mutant, and the complemented strain ∆tri24_T24.5. Photographs were taken 10 days after the placement of the R. solani plugs. B Effect of TRI24 gene deletion on the plant disease activity. Leaves detached from tomato plants that have been grown for 4 weeks. Drops of 15 µL of spore suspensions (5 × 10⁶ spores/mL in germination buffer) from P. roridum wild type, ∆tri24 mutant, and from the complemented transformant ∆tri24_T24.5 were placed at two spots/leaf. Control leaves were treated only with the germination buffer without Paramyrothecium spores. Leaves, after pathogen inoculation, were incubated for 6 days at 21 °C, 16-h light/8-h dark, 50% humidity

Fig. 9

Proposed biosynthetic pathway for macrocyclic trichothecenes in P. roridum. Two alternative pathways are proposed. In the first alternative, 15-hydoxy trichodermadiene (4) is a precursor of trichoverrol (solid arrows), and in the second alternative, 15-hydroxy trichodermadiene is not a precursor (dashed arrows). If the second alternative is correct, then 15-hydroxy trichodermadiene would likely be a shunt product produced only in the absence of a functional Tri24 enzyme. The order of roridins E, D, and A formation was proposed by Tamm (1977)