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Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

Beseli A, Noar R, Daub ME - PLoS ONE (2015)

Bottom Line: In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species' resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant.Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production.Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta-isomerase family.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, United States of America.

ABSTRACT
The photoactivated toxin, cercosporin, produced by Cercospora species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason, Cercospora species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species' resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the Cercospora nicotianae subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two, 24cF and 71cR, were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of 24cF and 71cR in the cercosporin-sensitive fungus, Neurospora crassa, showed that 71cR provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in 24cF transformants. Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the 71cR mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta-isomerase family.

No MeSH data available.


Related in: MedlinePlus

Phenotypic characterization of the 71cR disruptants.A. Cercosporin resistance of Cercospora nicotianae wild type (black bar), 71cR-disruptant strains (grey bars) and 71cR-transformed, but non-disruptant (white bar). The cercosporin sensitive crg1 mutant had 0% growth on cercosporin relative to control growth (not shown). Data are the result of two independent experiments with 4 replications each. Error bars represent standard error. B. Cercosporin production by Cercospora nicotianae wild type (black bar), 71cR-disruptant strains (grey bars) and 71cR-transformed, but non-disruptant strain (white bar). Cercosporin production was measured by extraction of mycelium with 5N KOH and measuring absorbance at 480 nm. Data are the results of two independent experiments with 2 replications each. Error bars represent standard error.
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pone.0140676.g005: Phenotypic characterization of the 71cR disruptants.A. Cercosporin resistance of Cercospora nicotianae wild type (black bar), 71cR-disruptant strains (grey bars) and 71cR-transformed, but non-disruptant (white bar). The cercosporin sensitive crg1 mutant had 0% growth on cercosporin relative to control growth (not shown). Data are the result of two independent experiments with 4 replications each. Error bars represent standard error. B. Cercosporin production by Cercospora nicotianae wild type (black bar), 71cR-disruptant strains (grey bars) and 71cR-transformed, but non-disruptant strain (white bar). Cercosporin production was measured by extraction of mycelium with 5N KOH and measuring absorbance at 480 nm. Data are the results of two independent experiments with 2 replications each. Error bars represent standard error.

Mentions: The ten 71cR-disruption strains were tested for cercosporin sensitivity by growing them on cercosporin-containing medium in the light. The wt, a transformed but non-disrupted strain (#23), and the cercosporin-sensitive crg1 mutant [6] were used as controls. Results are shown in Fig 5A. Under the conditions of the assay, the crg1 mutant does not grow on cercosporin (0% growth relative to the control [not shown]). There was no statistically significant difference in cercosporin resistance between the wt, the non-disrupted transformant (#23), and 71cR disruptants in radial growth on cercosporin. As other resistance proteins such as ATR1 and CFP have shown a dual role in both cercosporin resistance and production [10, 12], 71cR disruptants were also assayed for cercosporin production (Fig 5B) as compared to wt and the non-disrupted transformant #23. Cercosporin production varied between the different strains. However none of the 71cR disruptants produced significantly less cercosporin than wt.


Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

Beseli A, Noar R, Daub ME - PLoS ONE (2015)

Phenotypic characterization of the 71cR disruptants.A. Cercosporin resistance of Cercospora nicotianae wild type (black bar), 71cR-disruptant strains (grey bars) and 71cR-transformed, but non-disruptant (white bar). The cercosporin sensitive crg1 mutant had 0% growth on cercosporin relative to control growth (not shown). Data are the result of two independent experiments with 4 replications each. Error bars represent standard error. B. Cercosporin production by Cercospora nicotianae wild type (black bar), 71cR-disruptant strains (grey bars) and 71cR-transformed, but non-disruptant strain (white bar). Cercosporin production was measured by extraction of mycelium with 5N KOH and measuring absorbance at 480 nm. Data are the results of two independent experiments with 2 replications each. Error bars represent standard error.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608573&req=5

pone.0140676.g005: Phenotypic characterization of the 71cR disruptants.A. Cercosporin resistance of Cercospora nicotianae wild type (black bar), 71cR-disruptant strains (grey bars) and 71cR-transformed, but non-disruptant (white bar). The cercosporin sensitive crg1 mutant had 0% growth on cercosporin relative to control growth (not shown). Data are the result of two independent experiments with 4 replications each. Error bars represent standard error. B. Cercosporin production by Cercospora nicotianae wild type (black bar), 71cR-disruptant strains (grey bars) and 71cR-transformed, but non-disruptant strain (white bar). Cercosporin production was measured by extraction of mycelium with 5N KOH and measuring absorbance at 480 nm. Data are the results of two independent experiments with 2 replications each. Error bars represent standard error.
Mentions: The ten 71cR-disruption strains were tested for cercosporin sensitivity by growing them on cercosporin-containing medium in the light. The wt, a transformed but non-disrupted strain (#23), and the cercosporin-sensitive crg1 mutant [6] were used as controls. Results are shown in Fig 5A. Under the conditions of the assay, the crg1 mutant does not grow on cercosporin (0% growth relative to the control [not shown]). There was no statistically significant difference in cercosporin resistance between the wt, the non-disrupted transformant (#23), and 71cR disruptants in radial growth on cercosporin. As other resistance proteins such as ATR1 and CFP have shown a dual role in both cercosporin resistance and production [10, 12], 71cR disruptants were also assayed for cercosporin production (Fig 5B) as compared to wt and the non-disrupted transformant #23. Cercosporin production varied between the different strains. However none of the 71cR disruptants produced significantly less cercosporin than wt.

Bottom Line: In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species' resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant.Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production.Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta-isomerase family.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, United States of America.

ABSTRACT
The photoactivated toxin, cercosporin, produced by Cercospora species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason, Cercospora species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species' resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the Cercospora nicotianae subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two, 24cF and 71cR, were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of 24cF and 71cR in the cercosporin-sensitive fungus, Neurospora crassa, showed that 71cR provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in 24cF transformants. Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the 71cR mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta-isomerase family.

No MeSH data available.


Related in: MedlinePlus