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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 the 71cR gene in selected Neurospora crassa transformants.Each sample was normalized against the tubulin reference gene, and fold-change relative to expression of the lowest expressing Neurospora crassa transformant was calculated according to the 2(-ΔΔC(T)) method. Error bars represent 95% confidence intervals.
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pone.0140676.g003: Quantitative RT-PCR analysis of gene expression of the 71cR gene in selected Neurospora crassa transformants.Each sample was normalized against the tubulin reference gene, and fold-change relative to expression of the lowest expressing Neurospora crassa transformant was calculated according to the 2(-ΔΔC(T)) method. Error bars represent 95% confidence intervals.

Mentions: Expression of the transgenes was assayed in selected transformants screened for resistance. RT-qPCR analysis of 71cR expression in the two resistant 71cR transformants assayed (#349 and 350) showed high levels of expression (15,973- and 9,006- fold increase respectively), compared to the lowest expresser, a non-resistant 71cR transformant (#344) (Fig 3). Expression of the other non-resistant 71cR transformant was much lower than the two resistant 71cR transformants (199-fold change increase relative to #344). Thus 71cR expression correlated with cercosporin resistance. For 24cF, all four transformants tested had similar CT values as the high-expressing 71cR transformants, indicating expression of 24cF, and there was little difference in expression between the four 24cF transformants (data not shown). From these results we concluded that the 71cR protein can provide resistance to cercosporin toxicity, but that 24cF cannot.


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 the 71cR gene in selected Neurospora crassa transformants.Each sample was normalized against the tubulin reference gene, and fold-change relative to expression of the lowest expressing Neurospora crassa transformant was calculated according to the 2(-ΔΔC(T)) method. Error bars represent 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140676.g003: Quantitative RT-PCR analysis of gene expression of the 71cR gene in selected Neurospora crassa transformants.Each sample was normalized against the tubulin reference gene, and fold-change relative to expression of the lowest expressing Neurospora crassa transformant was calculated according to the 2(-ΔΔC(T)) method. Error bars represent 95% confidence intervals.
Mentions: Expression of the transgenes was assayed in selected transformants screened for resistance. RT-qPCR analysis of 71cR expression in the two resistant 71cR transformants assayed (#349 and 350) showed high levels of expression (15,973- and 9,006- fold increase respectively), compared to the lowest expresser, a non-resistant 71cR transformant (#344) (Fig 3). Expression of the other non-resistant 71cR transformant was much lower than the two resistant 71cR transformants (199-fold change increase relative to #344). Thus 71cR expression correlated with cercosporin resistance. For 24cF, all four transformants tested had similar CT values as the high-expressing 71cR transformants, indicating expression of 24cF, and there was little difference in expression between the four 24cF transformants (data not shown). From these results we concluded that the 71cR protein can provide resistance to cercosporin toxicity, but that 24cF cannot.

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