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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

Cercosporin resistance of Neurospora crassa 71cR-transformed strains.White bars: 71cR transformed strains; patterned bars: wild type N. crassa (WT = 71cR control; wt = 24cF control); grey bars: 24cF-transformed strains. Data are the result of two independent experiments with 5 replications each. Strains marked with *** have significantly greater resistance than wild type (P < 0.05). Error bars represent 95% confidence intervals.
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pone.0140676.g002: Cercosporin resistance of Neurospora crassa 71cR-transformed strains.White bars: 71cR transformed strains; patterned bars: wild type N. crassa (WT = 71cR control; wt = 24cF control); grey bars: 24cF-transformed strains. Data are the result of two independent experiments with 5 replications each. Strains marked with *** have significantly greater resistance than wild type (P < 0.05). Error bars represent 95% confidence intervals.

Mentions: The cercosporin-sensitive fungus N. crassa was transformed with 24cF and 71cR to test the ability of these genes to impart cercosporin resistance. N. crassa was chosen for this assay as it is highly sensitive to cercosporin, easily transformable, and has been shown in previous studies as an excellent system for screening genes that impart cercosporin resistance [13, 17]. Hyg-resistant colonies after transformation were screened for the presence of the genes by PCR. Out of a total of 84 and 93 hyg-resistant colonies, respectively, of 71cR and 24cF transformants, 60 and 12, respectively, were confirmed to contain the intact transgene by PCR screening (data not shown). All 12 of the 24cF and 27 of the 71cR PCR-positive strains were assayed for resistance to cercosporin by measuring radial growth on cercosporin-containing medium relative to growth on control medium. Cercosporin at 10 μM inhibits radial growth of N. crassa, resulting in approximately 30% of the radial growth on medium lacking cercosporin (Fig 2 and S1 Fig). Of the randomly chosen 27 PCR-positive 71cR transformants tested, eleven were found to be significantly more resistant to cercosporin than wt (P < 0.05) (Fig 2). However, none of the 12 PCR-positive 24cF transformants tested had significantly greater resistance to cercosporin than wt (P < 0.05) (Fig 2).


Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

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

Cercosporin resistance of Neurospora crassa 71cR-transformed strains.White bars: 71cR transformed strains; patterned bars: wild type N. crassa (WT = 71cR control; wt = 24cF control); grey bars: 24cF-transformed strains. Data are the result of two independent experiments with 5 replications each. Strains marked with *** have significantly greater resistance than wild type (P < 0.05). Error bars represent 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140676.g002: Cercosporin resistance of Neurospora crassa 71cR-transformed strains.White bars: 71cR transformed strains; patterned bars: wild type N. crassa (WT = 71cR control; wt = 24cF control); grey bars: 24cF-transformed strains. Data are the result of two independent experiments with 5 replications each. Strains marked with *** have significantly greater resistance than wild type (P < 0.05). Error bars represent 95% confidence intervals.
Mentions: The cercosporin-sensitive fungus N. crassa was transformed with 24cF and 71cR to test the ability of these genes to impart cercosporin resistance. N. crassa was chosen for this assay as it is highly sensitive to cercosporin, easily transformable, and has been shown in previous studies as an excellent system for screening genes that impart cercosporin resistance [13, 17]. Hyg-resistant colonies after transformation were screened for the presence of the genes by PCR. Out of a total of 84 and 93 hyg-resistant colonies, respectively, of 71cR and 24cF transformants, 60 and 12, respectively, were confirmed to contain the intact transgene by PCR screening (data not shown). All 12 of the 24cF and 27 of the 71cR PCR-positive strains were assayed for resistance to cercosporin by measuring radial growth on cercosporin-containing medium relative to growth on control medium. Cercosporin at 10 μM inhibits radial growth of N. crassa, resulting in approximately 30% of the radial growth on medium lacking cercosporin (Fig 2 and S1 Fig). Of the randomly chosen 27 PCR-positive 71cR transformants tested, eleven were found to be significantly more resistant to cercosporin than wt (P < 0.05) (Fig 2). However, none of the 12 PCR-positive 24cF transformants tested had significantly greater resistance to cercosporin than wt (P < 0.05) (Fig 2).

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