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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 genes in the C. nicotianae 71cR mutant treated with cercosporin in the light.Two 71cR disrupted strains, 71cR-16 and 71cR-18, were tested. Each sample was normalized against the C. nicotianae-specific actin reference gene, and fold-change relative to the no-cercosporin control was calculated according to the 2(-ΔΔC(T)) method [21]. Data represent the mean of two independent experiments. Error bars represent 95% confidence intervals. A. Expression of 3 transporters in 71cR disruptants #16 and #18. B. Expression of CTB genes in 71cR disruptants #16 and #18.
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pone.0140676.g006: Quantitative RT-PCR analysis of gene expression of genes in the C. nicotianae 71cR mutant treated with cercosporin in the light.Two 71cR disrupted strains, 71cR-16 and 71cR-18, were tested. Each sample was normalized against the C. nicotianae-specific actin reference gene, and fold-change relative to the no-cercosporin control was calculated according to the 2(-ΔΔC(T)) method [21]. Data represent the mean of two independent experiments. Error bars represent 95% confidence intervals. A. Expression of 3 transporters in 71cR disruptants #16 and #18. B. Expression of CTB genes in 71cR disruptants #16 and #18.

Mentions: Expression of 71cR in N. crassa demonstrated that 71cR can provide cercosporin resistance, however, 71cR disruption mutants of C. nicotianae were not more sensitive to cercosporin or produce less cercosporin than wt. Previous studies have suggested that C. nicotianae upregulates other resistance genes to compensate for resistance gene mutations [13]. We thus assayed for expression of additional library genes previously shown to impart cercosporin resistance (CnATR1, CnATR2 and CnCFP) to determine if they are up-regulated in the 71cR mutant background (Fig 6A). Two 71cR disruptants (disruptant #16 and 18) were tested. Expression of CnATR1 and CnATR2 was not increased significantly in either of the disruptants. By contrast, CnCFP expression was strongly increased: 285 and 20 fold at 1 hour, and 40- and 3- fold at 3 hours in disruptants #16 and 18, respectively.


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 genes in the C. nicotianae 71cR mutant treated with cercosporin in the light.Two 71cR disrupted strains, 71cR-16 and 71cR-18, were tested. Each sample was normalized against the C. nicotianae-specific actin reference gene, and fold-change relative to the no-cercosporin control was calculated according to the 2(-ΔΔC(T)) method [21]. Data represent the mean of two independent experiments. Error bars represent 95% confidence intervals. A. Expression of 3 transporters in 71cR disruptants #16 and #18. B. Expression of CTB genes in 71cR disruptants #16 and #18.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140676.g006: Quantitative RT-PCR analysis of gene expression of genes in the C. nicotianae 71cR mutant treated with cercosporin in the light.Two 71cR disrupted strains, 71cR-16 and 71cR-18, were tested. Each sample was normalized against the C. nicotianae-specific actin reference gene, and fold-change relative to the no-cercosporin control was calculated according to the 2(-ΔΔC(T)) method [21]. Data represent the mean of two independent experiments. Error bars represent 95% confidence intervals. A. Expression of 3 transporters in 71cR disruptants #16 and #18. B. Expression of CTB genes in 71cR disruptants #16 and #18.
Mentions: Expression of 71cR in N. crassa demonstrated that 71cR can provide cercosporin resistance, however, 71cR disruption mutants of C. nicotianae were not more sensitive to cercosporin or produce less cercosporin than wt. Previous studies have suggested that C. nicotianae upregulates other resistance genes to compensate for resistance gene mutations [13]. We thus assayed for expression of additional library genes previously shown to impart cercosporin resistance (CnATR1, CnATR2 and CnCFP) to determine if they are up-regulated in the 71cR mutant background (Fig 6A). Two 71cR disruptants (disruptant #16 and 18) were tested. Expression of CnATR1 and CnATR2 was not increased significantly in either of the disruptants. By contrast, CnCFP expression was strongly increased: 285 and 20 fold at 1 hour, and 40- and 3- fold at 3 hours in disruptants #16 and 18, respectively.

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