The Gibberella fujikuroi niaD Gene Encoding Nitrate Reductase: Isolation, Sequence, Homologous Transformation and Electrophoretic Karyotype Location

Tudzynski, Bettina; Mende, Katrin; Weltring, Klaus-Michael; Kinghorn, James R.; Unkles, Shiela E.

doi:10.1099/13500872-142-3-533

Cited by 64 publications

(46 citation statements)

References 29 publications

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“…For complementation of the mutant strain SG139 with the intact P450-1 gene, protoplasts of SG139 were transformed with 10 g of the circular complementation vector P450-1-GC. For complementation of the restriction enzyme-meditated integration-deletion mutant Gib1 (18) carrying an additional nia-D mutation, a cotransformation was performed by using 10 g each of the vector P450-1-GK and the vector pJ1, containing the intact G. fujikuroi niaD gene (19).…”

Section: Methodsmentioning

confidence: 99%

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

Rojas

Hedden

Gaskin

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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125

106

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Recent studies have shown that the genes of the gibberellin (GA) biosynthesis pathway in the fungus Gibberella fujikuroi are organized in a cluster of at least seven genes. P450 -1 is one of four cytochrome P450 monooxygenase genes in this cluster. Disruption of the P450 -1 gene in the GA-producing wild-type strain IMI 58289 led to total loss of GA production. Analysis of the P450 -1-disrupted mutants indicated that GA biosynthesis was blocked immediately after ent-kaurenoic acid. The function of the P450 -1 gene product was investigated further by inserting the gene into mutants of G. fujikuroi that lack the entire GA gene cluster; the gene was highly expressed under GA production conditions in the absence of the other GA-biosynthesis genes. Cultures of transformants containing P450 -1 converted ent- cytochrome P450 ͉ ent-kaurenoid metabolism ͉ GA14 synthase

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Section: Methodsmentioning

confidence: 99%

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

Rojas

Hedden

Gaskin

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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125

106

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“…Protoplasts were prepared from F. fujikuroi IMI58289. Transformation was carried out as previously described (47). About 10 7 protoplasts were transformed with ϳ10 g of the amplified replacement cassettes for the knockouts.…”

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confidence: 99%

Two Histone Deacetylases, FfHda1 and FfHda2, Are Important for Fusarium fujikuroi Secondary Metabolism and Virulence

Studt

Schmidt

Jahn

et al. 2013

Appl Environ Microbiol

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Histone modifications are crucial for the regulation of secondary metabolism in various filamentous fungi. Here we studied the involvement of histone deacetylases (HDACs) in secondary metabolism in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites, including phytohormones, pigments, and mycotoxins. Deletion of three Zn 2؉ -dependent HDAC-encoding genes, ffhda1, ffhda2, and ffhda4, indicated that FfHda1 and FfHda2 regulate secondary metabolism, whereas FfHda4 is involved in developmental processes but is dispensable for secondary-metabolite production in F. fujikuroi. Single deletions of ffhda1 and ffhda2 resulted not only in an increase or decrease but also in derepression of metabolite biosynthesis under normally repressing conditions. Moreover, double deletion of both the ffhda1 and ffhda2 genes showed additive but also distinct phenotypes with regard to secondary-metabolite biosynthesis, and both genes are required for gibberellic acid (GA)-induced bakanae disease on the preferred host plant rice, as ⌬ffhda1 ⌬ffhda2 mutants resemble the uninfected control plant. Microarray analysis with a ⌬ffhda1 mutant that has lost the major HDAC revealed differential expression of secondarymetabolite gene clusters, which was subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are involved not only in gene silencing but also in the activation of some genes. Chromatin immunoprecipitation with the ⌬ffhda1 mutant revealed significant alterations in the acetylation state of secondary-metabolite gene clusters compared to the wild type, thereby providing insights into the regulatory mechanism at the chromatin level. Altogether, manipulation of HDAC-encoding genes constitutes a powerful tool to control secondary metabolism in filamentous fungi.

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“…Transformation of G. fujikuroi-The preparation of protoplasts and the transformation procedure were as previously described (46). For gene replacement, 10 7 protoplasts (50 l) of strain IMI58289 were transformed with 10 g of the KpnI/SacI fragment of the gene replacement vector p⌬cpr.…”

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confidence: 99%

The NADPH-cytochrome P450 Reductase Gene from Gibberella fujikuroi Is Essential for Gibberellin Biosynthesis

Malonek

Rojas

Hedden

et al. 2004

Journal of Biological Chemistry

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136

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The fungus Gibberella fujikuroi is used for the commercial production of gibberellins (GAs), which it produces in very large quantities. Four of the seven GA biosynthetic genes in this species encode cytochrome P450 monooxygenases, which function in association with NADPH-cytochrome P450 reductases (CPRs) that mediate the transfer of electrons from NADPH to the P450 monooxygenases. Only one cpr gene (cpr-Gf) was found in G. fujikuroi and cloned by a PCR approach. The encoded protein contains the conserved CPR functional domains, including the FAD, FMN , and NADPH binding motifs. cpr-Gf disruption mutants were viable but showed a reduced growth rate. Furthermore, disruption resulted in total loss of GA 3 , GA 4 , and GA 7 production, but low levels of non-hydroxylated C 20 -GAs (GA 15 and GA 24 ) were still detected. In addition, the knock-out mutants were much more sensitive to benzoate than the wild type due to loss of activity of another P450 monooxygenase, the detoxifying enzyme, benzoate p-hydroxylase. The UV-induced mutant of G. fujikuroi, SG138, which was shown to be blocked at most of the GA biosynthetic steps catalyzed by P450 monooxygenases, displayed the same phenotype. Sequence analysis of the mutant cpr allele in SG138 revealed a nonsense mutation at amino acid position 627. The mutant was complemented with the cpr-Gf and the Aspergillus niger cprA genes, both genes fully restoring the ability to produce GAs. Northern blot analysis revealed co-regulated expression of the cpr-Gf gene and the GA biosynthetic genes P450-1, P450-2, P450-4 under GA production conditions (nitrogen starvation). In addition, expression of cpr-Gf is induced by benzoate. These results indicate that CPR-Gf is the main but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism.

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The Gibberella fujikuroi niaD Gene Encoding Nitrate Reductase: Isolation, Sequence, Homologous Transformation and Electrophoretic Karyotype Location

Cited by 64 publications

References 29 publications

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

Two Histone Deacetylases, FfHda1 and FfHda2, Are Important for Fusarium fujikuroi Secondary Metabolism and Virulence

The NADPH-cytochrome P450 Reductase Gene from Gibberella fujikuroi Is Essential for Gibberellin Biosynthesis

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