ThpacC Acts as a Positive Regulator of Homodimericin A Biosynthesis and Antifungal Activities of <i>Trichoderma harzianum</i> 3.9236

Wu, Mengyue; Wei, Huiling; Ma, Ke; Cui, Peiqi; Zhu, Shaozhou; Lai, Daowan; Ren, Jinwei; Wang, Wenzhao; Fan, Aili; Lin, Wenhan; Su, Haijia

doi:10.1021/acs.jafc.1c04330

Cited by 5 publications

(4 citation statements)

References 50 publications

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“…Its structure was proposed based on the synthetic route, HRMS and comparison of the NMR spectra with 10 . Homodimericin B ( 11 ) was then identified and isolated from the same fungus, T. harzianum , showing consistent NMR spectra with the synthetic sample 52 . Rigorous characterization of the complicated skeleton of 11 was still lacking, so we attempted to utilize i-HMBC to unambiguously confirm the structure.…”

Section: Resultsmentioning

confidence: 88%

Unequivocal identification of two-bond heteronuclear correlations in natural products at nanomole scale by i-HMBC

et al. 2023

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HMBC is an essential NMR experiment for determining multiple bond heteronuclear correlations in small to medium-sized organic molecules, including natural products, yet its major limitation is the inability to differentiate two-bond from longer-range correlations. There have been several attempts to address this issue, but all reported approaches suffer various drawbacks, such as restricted utility and poor sensitivity. Here we present a sensitive and universal methodology to identify two-bond HMBC correlations using isotope shifts, referred to as i-HMBC (isotope shift detection HMBC). Experimental utility was demonstrated at the sub-milligram / nanomole scale with only a few hours of acquisition time required for structure elucidation of several complex proton-deficient natural products, which could not be fully elucidated by conventional 2D NMR experiments. Because i-HMBC overcomes the key limitation of HMBC without significant reduction in sensitivity or performance, i-HMBC can be used as a complement to HMBC when unambiguous identifications of two-bond correlations are needed.

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Section: Resultsmentioning

confidence: 88%

Unequivocal identification of two-bond heteronuclear correlations in natural products at nanomole scale by i-HMBC

et al. 2023

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“…The promoter regions of nine PAT cluster genes contain one or more PacC-binding consensus sequences, suggesting that PacC likely affects the biosynthesis of PAT by regulating the PAT gene cluster ( 50 ). PacC acts as a transcriptional activator to regulate gene expression by binding to the consensus sequence (5′-GCCARG-3′) ( 51 , 52 ). In the present study, we have demonstrated that the purified PacC could directly bind to the promoter regions of ACTT6 and ACTTR , which are known to be critical components in the biosynthesis of ACT.…”

Section: Discussionmentioning

confidence: 99%

The Alternaria alternata StuA transcription factor interacting with the pH-responsive regulator PacC for the biosynthesis of host-selective toxin and virulence in citrus

Chen,

Cao,

Jiao

et al. 2023

Microbiol Spectr

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The tangerine pathotype of Alternaria alternata produces a host-selective toxin termed Alternaria citri toxin (ACT). The molecular mechanisms underlying the global regulation and biosynthesis of ACT remain unknown. In the present study, the function of an APSES transcription factor StuA was investigated. StuA was shown to be required for ACT biosynthesis and fungal virulence. StuA was found, for the first time, to physically interact with a pH-responsive transcription regulator PacC using yeast two-hybrid, bimolecular fluorescence complementation, and GST pull-down assays. Functional analyses revealed that StuA and PacC regulate the expression of genes involved in toxin biosynthesis and virulence. Mutation of stuA via targeted gene deletion or silencing pacC yielded fungal strains that decreased the expression of seven toxin biosynthetic genes ( ACCT ) and toxin production. EMSA analyses revealed that PacC could bind to the promoters of ACTT6 encoding an enoyl-CoA hydratase and ACTTR encoding an ACT pathway-specific transcription factor. Site-directed mutagenesis of five potential protein kinase A (PKA) phosphorylation sites in StuA revealed that none of the sites was involved in ACT production, indicating that the function of StuA in the regulation of ACT gene expression is not dependent on phosphorylation. Overall, our results confirmed that PacC is one of the key regulators interacting with StuA for the biosynthesis of ACT. Environmental pH may play a decisive role during A. alternata pathogenesis. Our results also revealed a previously unrecognized (StuA-PacC)→ACTTR module for the biosynthesis of ACT in A. alternata . IMPORTANCE In this study, we used Alternaria alternata as a biological model to report the role of StuA in phytopathogenic fungi. Our findings indicated that StuA is required for Alternaria citri toxin (ACT) biosynthesis and fungal virulence. In addition, StuA physically interacts with PacC. Disruption of stuA or pacC led to decreased expression of seven toxin biosynthetic genes (ACCT) and toxin production. PacC could recognize and bind to the promoter regions of ACTT6 and ACTTR . Our results revealed a previously unrecognized (StuA-PacC)→ACTTR module for the biosynthesis of ACT in A. alternata , which also provides a framework for the study of StuA in other fungi.

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“…Compounds 1−3 were tested for their antifungal activities against a phytopathogen F. fujikuroi and a biocontrol fungus T. harzianum by the mycelium growth rate method. 15,16 Compounds were dissolved in dimethyl sulfoxide (DMSO) to obtain initial solutions of 6.25 and 2.5 mg/mL and diluted by PDA (10 mL) to obtain final concentrations of 25 and 10 μg/mL, respectively. The same amount of DMSO and azoxystrobin was added to PDA (10 mL) as the negative control and positive control, respectively.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Discovery of a Hybrid Molecule with Phytotoxic Activity by Genome Mining, Heterologous Expression, and OSMAC Strategy

Lu,

Li,

Dou

et al. 2024

J. Agric. Food Chem.

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Genome mining in association with the OSMAC (one strain, many compounds) approach provides a feasible strategy to extend the chemical diversity and novelty of natural products. In this study, we identified the biosynthetic gene cluster (BGC) of restricticin, a promising antifungal agent featuring a reactive primary amine, from the fungus Aspergillus sclerotiorum LZDX-33-4 by genome mining. Combining heterologous expression and the OSMAC strategy resulted in the production of a new hybrid product (1), along with N-acetyl-restricticin (2) and restricticinol (3). The structure of 1 was determined by spectroscopic data, including optical rotation and electronic circular dichroism (ECD) calculations, for configurational assignment. Compound 1 represents a fusion of restricticin and phytotoxic cichorin. The biosynthetic pathway of 1 was proposed, in which the condensation of a primary amine of restricticin with a precursor of cichorine was postulated. Compound 1 at 5 mM concentration inhibited the growth of the shoots and roots of Lolium perenne, Festuca arundinacea, and Lactuca sativa with inhibitory rates of 71.3 and 88.7% for L. perenne, 79.4 and 73.0% for F. arundinacea, and 58.2 and 52.9% for L. sativa. In addition, compound 1 at 25 μg/mL showed moderate antifungal activity against Fusarium fujikuroi and Trichoderma harzianum with inhibition rates of 22.6 and 31.6%, respectively. These results suggest that heterologous expression in conjunction with the OSMAC approach provides a promising strategy to extend the metabolite novelty due to the incorporation of endogenous metabolites from the host strain with exogenous compounds, leading to the production of more complex compounds and the acquisition of new physiological functions.

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ThpacC Acts as a Positive Regulator of Homodimericin A Biosynthesis and Antifungal Activities of Trichoderma harzianum 3.9236

Cited by 5 publications

References 50 publications

Unequivocal identification of two-bond heteronuclear correlations in natural products at nanomole scale by i-HMBC

Unequivocal identification of two-bond heteronuclear correlations in natural products at nanomole scale by i-HMBC

The Alternaria alternata StuA transcription factor interacting with the pH-responsive regulator PacC for the biosynthesis of host-selective toxin and virulence in citrus

Discovery of a Hybrid Molecule with Phytotoxic Activity by Genome Mining, Heterologous Expression, and OSMAC Strategy

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