Restoration of wild-type virulence to Tri5 disruption mutants of Gibberella zeae via gene reversion and mutant complementation

Proctor, Robert H.; Höhn, Thomas; McCormick, Susan P.

doi:10.1099/00221287-143-8-2583

Cited by 89 publications

(64 citation statements)

References 31 publications

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“…Due to their significant similarities to other known genes, many of these sequences are promising candidates for future investigations. With the recent development of suitable transformation and gene inactivation systems for a number of Fusarium species (Proctor et al 1997;Ferná ndez-Martín et al 2000;Trail et al 2003; this study), the specific roles of the tagged sequences in mating and/or other aspects of the fungal life cycle can be uncovered in the near future.…”

Section: Discussionmentioning

confidence: 99%

“…Complete medium, CM (Correll et al 1987) and carrot agar, CA (Klittich and Leslie 1988) were used to compare growth and morphology of the wild type strains, FGSC 7600 and 7603 as well as the DFvMAT1-2-1 mutants. For transformation, F. verticillioides FGSC 7603 was grown on YEPD-2G medium (Proctor et al 1997) containing yeast extract (3.0 g), peptone (10.0 g), glucose (20.0 g) per litre of distilled water as shaken culture (180 rpm) for 9-10 h at 28°C. All the other incubations occurred at 23/ 24°C under a 12 h of light-12 h of darkness diurnal cycle.…”

Section: Methodsmentioning

confidence: 99%

“…This fragment was ligated into pBluescript II KS+ (Stratagene, La Jolla, California, USA). Subsequently, a 1973 bp NdeI fragment of the FvMAT1-2-1 sequence was replaced with a 3805 bp hygromycin expression cassette Proctor et al (1997). Transformants were selected on regeneration medium (0.1% yeast extract, 0.1% casein hydrolysate, 1.6% bacto agar and 0.8 M sucrose) containing 200 lg ml -1 hygromycin B (HygB, Duchefa, Haarlem, The Netherlands).…”

Section: Disruption Of Fvmat1-2-1mentioning

confidence: 99%

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Tagging target genes of the MAT1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A)

Keszthelyi

Jeney

Kerényi

et al. 2006

Antonie van Leeuwenhoek

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Mating type in filamentous ascomycetes is controlled by idiomorphic alleles, named MAT1-1 and MAT1-2, which contain 1-3 genes. Of these genes MAT1-1-1 and MAT1-2-1 encode putative transcription factors and are thus considered to be the major regulators of sexual communication and mating. Fungi with no known sexual stage may also have fully functional mating type genes and therefore it was plausible to hypothesize that the MAT products may also regulate other types of genes not involved directly in the mating process. To identify putative target genes of these transcription factors in Fusarium verticillioides, DMAT1-2-1 knock out mutants were produced and transcript profiles of mutant and wild type were compared by means of differential cDNA hybridization. Clones, either up-or down-regulated in the DMAT1-2-1 mutant were sequenced and a total of 248 sequences were blasted against the NCBI database as well as the Gibberella zeae and Gibberella moniliformis genomes. Fifty-five percent of the clones were down-regulated in the mutant, indicating that the MAT1-2-1 product positively affected these tagged sequences. On the other hand, 45% were found to be up-regulated in the mutant, suggesting that the MAT1-2-1 product also exerted a negative regulatory function on this set of genes. Sequences involved in protein synthesis and metabolism occurred more frequently among the clones up-regulated in the mutant, whereas genes belonging to cell signalling and communication were especially frequently tagged among the sequences down-regulated in the mutant.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Disruption Of Fvmat1-2-1mentioning

confidence: 99%

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Tagging target genes of the MAT1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A)

Keszthelyi

Jeney

Kerényi

et al. 2006

Antonie van Leeuwenhoek

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“…In the first approach, a revertant was generated by allowing a Tri5 disruption mutant to pass through the sexual phase of its life cycle. In one of the resulting progeny, the disrupted Tri5 had reverted to wild-type and the strain regained the ability to produce trichothecenes and to produce seedling blight on wheat plants in a growth chamber test (Proctor et al, 1997). To facilitate strain tracking during field tests, this revertant was tagged by transforming it with a vector containing the hygB gene.…”

Section: Introductionmentioning

confidence: 99%

Analysis of aberrant virulence of Gibberella zeae following transformation-mediated complementation of a trichothecene-deficient (Tri5) mutant

et al. 2000

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Gibberella zeae causes wheat ear blight and produces trichothecene toxins in infected grain. In previous studies, trichothecene production in this fungus was disabled by specific disruption of the trichodiene synthase gene (Tri5) and was restored by two methods : gene reversion and transformation-mediated mutant complementation. In previous field tests of wheat ear blight, trichothecene-nonproducing mutants were less virulent than the wild-type progenitor strain from which they were derived. Trichothecene-producing revertants also were restored to wild-type levels of virulence. In contrast, in the field test of wheat ear blight reported here, trichothecene-producing strains obtained by Tri5 mutant complementation were not restored to wildtype levels of virulence. The complemented mutants showed a slightly reduced radial growth compared to the wild-type strain, but otherwise appeared normal in morphology, pigmentation and sexual fertility. Genetic analysis indicated that the aberrant virulence of a complemented mutant was likely due to non-target effects that occurred during the process of transforming the trichothecene-nonproducing mutant with Tri5. These results confirm previous findings that trichothecenes contribute to the virulence of G. zeae, but also demonstrate that manipulating this fungus in the laboratory may cause it to undergo subtle changes that reduce its virulence.

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“…This PCR product was ligated with a 4 kb HygR cassette, obtained from pAN7-1 (Punt et al, 1987) by digestion with HindIII and BglII, to give pDCut. For transformation (Proctor et al, 1997), FKMC1995 protoplasts obtained from 2610 8 conidia were incubated with 30 mg pDCut previously linearized with NotI.…”

Section: Methodsmentioning

confidence: 99%

The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism

2014

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Survival of micro-organisms in natural habitats depends on their ability to adapt to variations in osmotic conditions. We previously described the gene cut-1 of Neurospora crassa, encoding a protein of the haloacid dehalogenase family with an unknown function in the osmotic stress response. Here we report on the functional analysis of cutA, the orthologous gene in the phytopathogenic fungus Fusarium fujikuroi. cutA mRNA levels increased transiently after exposure to 0.68 M NaCl and were reduced upon return to normal osmotic conditions; deletion of the gene resulted in a partial reduction in tolerance to osmotic stress. ΔcutA mutants contained much lower intracellular levels of glycerol than the wild-type, and did not exhibit the increase following hyper-osmotic shock expected from the high osmolarity glycerol (HOG) response. cutA is linked and divergently transcribed with the putative glycerol dehydrogenase gene gldB, which showed the same regulation by osmotic shock. The intergenic cutA/gldB regulatory region contains putative stress-response elements conserved in other fungi, and both genes shared other regulatory features, such as induction by heat shock and by illumination. Photoinduction was also observed in the HOG response gene hogA, and was lost in mutants of the white collar gene wcoA. Previous data on glycerol production in Aspergillus spp. and features of the predicted CutA protein lead us to propose that F. fujikuroi produces glycerol from dihydroxyacetone, and that CutA is the enzyme involved in the synthesis of this precursor by dephosphorylation of dihydroxyacetone-3P.

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Restoration of wild-type virulence to Tri5 disruption mutants of Gibberella zeae via gene reversion and mutant complementation

Cited by 89 publications

References 31 publications

Tagging target genes of the MAT1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A)

Tagging target genes of the MAT1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A)

Analysis of aberrant virulence of Gibberella zeae following transformation-mediated complementation of a trichothecene-deficient (Tri5) mutant

The gene cutA of Fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism

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