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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

Quantitative RT-PCR analysis of gene expression of hypothetical protein genes in the C. nicotianae atr1 mutant treated with cercosporin in the light.Each sample was normalized against the actin reference gene, and fold-change relative to no-cercosporin was calculated according to the 2(-ΔΔC(T)) method [21]. Data represent the mean of two independent experiments with three replications each. Error bars represent 95% confidence intervals.
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pone.0140676.g001: Quantitative RT-PCR analysis of gene expression of hypothetical protein genes in the C. nicotianae atr1 mutant treated with cercosporin in the light.Each sample was normalized against the actin reference gene, and fold-change relative to no-cercosporin was calculated according to the 2(-ΔΔC(T)) method [21]. Data represent the mean of two independent experiments with three replications each. Error bars represent 95% confidence intervals.

Mentions: In order to define a possible role in cercosporin resistance, the 13 genes encoding hypothetical proteins were assayed for changes in expression under conditions of cercosporin toxicity. The cercosporin-sensitive C. nicotianae atr1 mutant, deficient in the ATR1 ABC transporter involved in cercosporin resistance [12], was treated with cercosporin under high-light conditions to induce toxicity, and gene expression was assayed by RT-qPCR. This mutant was used because it is highly sensitive to cercosporin and has previously been shown to be useful for identifying putative cercosporin-resistance genes that are upregulated under cercosporin toxicity conditions [13]. Genes 24cF and 71cR were upregulated 9.4- and 8.7-fold, respectively, three hours after cercosporin toxicity was induced (Fig 1). None of the other 11 genes were upregulated under these conditions. Based on these gene expression results, 24cF and 71cR were chosen for further characterization.


Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

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

Quantitative RT-PCR analysis of gene expression of hypothetical protein genes in the C. nicotianae atr1 mutant treated with cercosporin in the light.Each sample was normalized against the actin reference gene, and fold-change relative to no-cercosporin was calculated according to the 2(-ΔΔC(T)) method [21]. Data represent the mean of two independent experiments with three replications each. Error bars represent 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608573&req=5

pone.0140676.g001: Quantitative RT-PCR analysis of gene expression of hypothetical protein genes in the C. nicotianae atr1 mutant treated with cercosporin in the light.Each sample was normalized against the actin reference gene, and fold-change relative to no-cercosporin was calculated according to the 2(-ΔΔC(T)) method [21]. Data represent the mean of two independent experiments with three replications each. Error bars represent 95% confidence intervals.
Mentions: In order to define a possible role in cercosporin resistance, the 13 genes encoding hypothetical proteins were assayed for changes in expression under conditions of cercosporin toxicity. The cercosporin-sensitive C. nicotianae atr1 mutant, deficient in the ATR1 ABC transporter involved in cercosporin resistance [12], was treated with cercosporin under high-light conditions to induce toxicity, and gene expression was assayed by RT-qPCR. This mutant was used because it is highly sensitive to cercosporin and has previously been shown to be useful for identifying putative cercosporin-resistance genes that are upregulated under cercosporin toxicity conditions [13]. Genes 24cF and 71cR were upregulated 9.4- and 8.7-fold, respectively, three hours after cercosporin toxicity was induced (Fig 1). None of the other 11 genes were upregulated under these conditions. Based on these gene expression results, 24cF and 71cR were chosen for further characterization.

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