Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 25:12:731425.
doi: 10.3389/fmicb.2021.731425. eCollection 2021.

Verification of TRI3 Acetylation of Trichodermol to Trichodermin in the Plant Endophyte Trichoderma taxi

Affiliations

Verification of TRI3 Acetylation of Trichodermol to Trichodermin in the Plant Endophyte Trichoderma taxi

Haijiang Chen et al. Front Microbiol. .

Abstract

Trichodermin, a trichothecene first isolated in Trichoderma species, is a sesquiterpenoid antibiotic that exhibits significant inhibitory activity to the growth of many pathogenic fungi such as Candida albicans, Rhizoctonia solani, and Botrytis cinerea by inhibiting the peptidyl transferase involved in eukaryotic protein synthesis. Trichodermin has also been shown to selectively induce cell apoptosis in several cancer cell lines and thus can act as a potential lead compound for developing anticancer therapeutics. The biosynthetic pathway of trichodermin in Trichoderma has been identified, and most of the involved genes have been functionally characterized. An exception is TRI3, which encodes a putative acetyltransferase. Here, we report the identification of a gene cluster that contains seven genes expectedly involved in trichodermin biosynthesis (TRI3, TRI4, TRI6, TRI10, TRI11, TRI12, and TRI14) in the trichodermin-producing endophytic fungus Trichoderma taxi. As in Trichoderma brevicompactum, TRI5 is not included in the cluster. Functional analysis provides evidence that TRI3 acetylates trichodermol, the immediate precursor, to trichodermin. Disruption of TRI3 gene eliminated the inhibition to R. solani by T. taxi culture filtrates and significantly reduced the production of trichodermin but not of trichodermol. Both the inhibitory activity and the trichodermin production were restored when native TRI3 gene was reintroduced into the disruption mutant. Furthermore, a His-tag-purified TRI3 protein, expressed in Escherichia coli, was able to convert trichodermol to trichodermin in the presence of acetyl-CoA. The disruption of TRI3 also resulted in lowered expression of both the upstream biosynthesis TRI genes and the regulator genes. Our data demonstrate that T. taxi TRI3 encodes an acetyltransferase that catalyzes the esterification of the C-4 oxygen atom on trichodermol and thus plays an essential role in trichodermin biosynthesis in this fungus.

Keywords: TRI3; Trichoderma; antifungal activity; trichodermin biosynthesis; trichothecene.

PubMed Disclaimer

Conflict of interest statement

HC and JL was employed by China Tobacco Guizhou Industrial Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The TRI gene cluster in Trichoderma taxi ZJUF0986, Trichoderma brevicompactum, and Trichoderma arundinaceum. The orientations of arrows represented the direction of transcription, and the relative position of genes was proportionally scaled.
FIGURE 2
FIGURE 2
Phylogenetic tree based on alignment of TRI3 from Trichoderma, Stachybotrys, Beauveria, Fusarium, and Aureobasidium. The species were chosen as representatives of hits from a BLASTp search. Sequence alignment and tree building were performed by MEGA5.0 using the neighbor-joining method, and phylogeny was tested by 500 bootstrap replications. The numbers on branches were calculated bootstrap values. The accession numbers for TRI3 genes were as follows: TaTRI3 (CAY87361), TbTRI3 (CBY93780), Stachybotrys chartarum TRI3 (KEY64037), Stachybotrys chlorohalonata TRI3 (KFA68878), FgTRI3 (BAC22114), FpTRI3 (XP_009263555), FaTRI3 (AAM48923), FsTRI3 (AAK33072), BbTRI3 (XP_008602023), and ApTRI3 (KEQ86789).
FIGURE 3
FIGURE 3
PCR confirmation of insertion of the HPH cassette in TRI3 gene. The relative positions of primers ATMT-F/R (A) and ATMT2-F/R (B) are shown. (C) Two transformants were confirmed by PCR. Primer pair ATMT-F/R was used for lane 1a, 2a, and 3a; while ATMT2-F/R was used for lane 1b, 2b, and 3b. 1a, 1b and 2a, 2b were designated for the two transformants. Wild-type strain was used in 3a and 3b.
FIGURE 4
FIGURE 4
Disruption of TRI3 relieved growth inhibition to Rhizoctonia solani. From left to right: 0% (v/v), 10% (v/v), or 20% (v/v) of harvested fermentation broth from wild-type strain or TtTRI3 disruption strain, or complementation strain was added in potato dextrose agar (PDA). Photos were taken 3 days after incubation.
FIGURE 5
FIGURE 5
Trichodermin and trichodermol production in strain ZJUF0986, ΔTtTRI3, and ΔTtTRI3-TRI3. Amount of trichodermin (A) and trichodermol (B) in fermentation broth was detected by gas chromatography (GC)–MS. Peaks for trichodermin and trichodermol were determined by the corresponding reference chemicals. The concentration of reference chemicals in the prepared samples was 188 ppm (part per million) for trichodermin and 42 ppm for trichodermol.
FIGURE 6
FIGURE 6
In vitro conversion of trichodermol to trichodermin catalyzed by bacterial expressed TtTRI3. (A) The structure of the constructed plasmid for expression of TtTRI3. The 6xHis tag was engineered to fuse immediately upstream of the TtTRI3 coding region. (B) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) shows the purification of Escherichia coli-expressed TtTRI3. The black arrow indicates the band of the target protein. The molecular weight (MW) of this band on the gel is consistent with the calculated theoretical MW, 60 kDa. L: cell lysate; FT: flowthrough from Ni-NTA column after cell lysate was loaded; E: eluate from TtTRI3-bound column; M: protein marker. (C) GC-MS analysis results showed that addition of TtTRI3 resulted in the appearance of trichodermin in reaction mixture. The upper two curves show the respective retention time of trichodermin and trichodermol. The two curves at the bottom represent curves obtained from ethyl acetate extract from trichodermol reaction mixture, with combination of either TtTRI3 (sample M15) or cell lysate prepared from Escherichia coli cell bearing empty vector pQE30 (sample CK).
FIGURE 7
FIGURE 7
Comparison of TRI genes expression level between wild-type strain and ΔTtTRI3. Expression level for TRI genes was determined by qRT-PCR. Expression level of genes in ZJUF0986 was normalized as 1. Calculation was based on data obtained from three independent experiments.
FIGURE 8
FIGURE 8
The proposed trichodermin biosynthetic pathway in Trichoderma taxi.

Similar articles

Cited by

References

    1. Alexander N. J., Hohn T. M., McCormick S. P. (1998). The TRI11 gene of Fusarium sporotrichiodes encodes a cytochrome P-450 monooxygenase required for C-15 hydroxylation in trichothecene biosynthesis. Appl. Environ. Microbiol. 64 221–225. 10.1128/aem.64.1.221-225.1998 - DOI - PMC - PubMed
    1. Alexander N. J., McCormick S. P., Hohn T. M. (1999). TRI12, a trichothecene efflux pump from Fusarium sporotrichioides: gene isolation and expression in yeast. Mol. Gen. Genet. 261 977–984. 10.1007/s004380051046 - DOI - PubMed
    1. Alexander N. J., McCormick S. P., Larson T. M., Jurgenson J. E. (2004). Expression of TRI15 in Fusarium sporotrichioides. Curr. Genet. 45 157–162. 10.1007/s00294-003-0467-3 - DOI - PubMed
    1. Brown D. W., Dyer R. B., McCormick S. P., Kendra D. F., Plattner R. D. (2004). Functional demarcation of the Fusarium. Fungal. Genet. Biol. 41 454–462. 10.1016/j.fgb.2003.12.002 core trichothecene gene cluster - DOI - PubMed
    1. Cardoza R. E., Malmierca M. G., Hermosa M. R., Alexander N. J., McCormick S. P., Proctor R. H., et al. (2011). Identification of loci and functional characterization of trichothecene biosynthetic genes in the filamentous fungus Trichoderma. Appl. Environ. Microbiol. 77 4867–4877. 10.1128/aem.00595-11 - DOI - PMC - PubMed