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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

Disruption of 71cR.A. The gDNA of wt C. nicotianae is shown as a black bar with the start and stop codons of 71cR indicated. In disrupted transformants the hyg cassette is predicted to integrate in the location specified by dotted lines. The homologous regions of each split marker are represented as double lines. Location of primers used in PCR screening is also shown. B. Gel image of screening putative 71cR disruptants with primers that span the hyg cassette integration site (71cR-Rev3 and 71cR-inv5’; red arrows in panel A). Integration results in a 2 kb band; wt band is 400bp (indication of non-disruptant genotype). Wild type C. nicotianae and transformant #15 showed only the wild type band; transformant #23 has both bands indicating an ectopic integration of the split markers. C. Bands represent the 1.7 kb amplification of the 5’ integration site by using a 71cR locus-specific forward primer outside the split marker 1 sequence (71cR-Forw10) and a reverse primer specific to the hyg cassette sequence (Hyg-F2) (blue arrows in panel A). D. Bands represent the 715 bp amplification of the 3’ integration site (Hyg-R and 71cR-DisR; green arrows in panel A).
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pone.0140676.g004: Disruption of 71cR.A. The gDNA of wt C. nicotianae is shown as a black bar with the start and stop codons of 71cR indicated. In disrupted transformants the hyg cassette is predicted to integrate in the location specified by dotted lines. The homologous regions of each split marker are represented as double lines. Location of primers used in PCR screening is also shown. B. Gel image of screening putative 71cR disruptants with primers that span the hyg cassette integration site (71cR-Rev3 and 71cR-inv5’; red arrows in panel A). Integration results in a 2 kb band; wt band is 400bp (indication of non-disruptant genotype). Wild type C. nicotianae and transformant #15 showed only the wild type band; transformant #23 has both bands indicating an ectopic integration of the split markers. C. Bands represent the 1.7 kb amplification of the 5’ integration site by using a 71cR locus-specific forward primer outside the split marker 1 sequence (71cR-Forw10) and a reverse primer specific to the hyg cassette sequence (Hyg-F2) (blue arrows in panel A). D. Bands represent the 715 bp amplification of the 3’ integration site (Hyg-R and 71cR-DisR; green arrows in panel A).

Mentions: A split marker strategy was used to generate C. nicotianae disruption mutants by homologous recombination. A total of 14 hyg-resistant colonies were screened to confirm 71cR disruption by PCR analysis using primer sequences shown in Table 2. Primers 71cR-Rev3 and 71cR-inv5’ that span the hyg-resistance cassette were used to screen the disruptants; with these primers the wt band is 400 bp and the band resulting from disruption is 2 kb (Fig 4A and 4B). A total of 12 transformants were confirmed as lacking the 400 bp wild type band. One had an ectopic integration of the split markers (#23) and one lacked evidence for integration of the split marker (#15) (Fig 4B). Eleven of the disruptants and the strain with ectopic integration of the split markers (#23) were then screened using a 71cR locus-specific primer for the 5’ end outside the split marker sequence (71cR-Forw10) and a primer specific to hyg cassette sequence (Hyg-F2). Presence of the 1.7 kb fragment confirmed the correct location of split marker integration for 10 of the putative disruptants, and no band was seen on amplification of strain #23 (transformant with ectopic integration of the split markers) (Fig 4C). Finally, we screened transformants using a 71cR locus-specific primer for the 3’ end outside the split marker sequence (71cR-DisR) and a second primer specific to the hyg cassette sequence (Hyg-R) (Fig 4D); all of the transformants were also confirmed to have a 715 bp band again confirming the correct location of the split marker integration. Through this analysis, 10 transformants (# 10, 13, 14, 16, 17, 18, 19, 20, 21, 24) were confirmed as being disrupted for 71cR.


Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

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

Disruption of 71cR.A. The gDNA of wt C. nicotianae is shown as a black bar with the start and stop codons of 71cR indicated. In disrupted transformants the hyg cassette is predicted to integrate in the location specified by dotted lines. The homologous regions of each split marker are represented as double lines. Location of primers used in PCR screening is also shown. B. Gel image of screening putative 71cR disruptants with primers that span the hyg cassette integration site (71cR-Rev3 and 71cR-inv5’; red arrows in panel A). Integration results in a 2 kb band; wt band is 400bp (indication of non-disruptant genotype). Wild type C. nicotianae and transformant #15 showed only the wild type band; transformant #23 has both bands indicating an ectopic integration of the split markers. C. Bands represent the 1.7 kb amplification of the 5’ integration site by using a 71cR locus-specific forward primer outside the split marker 1 sequence (71cR-Forw10) and a reverse primer specific to the hyg cassette sequence (Hyg-F2) (blue arrows in panel A). D. Bands represent the 715 bp amplification of the 3’ integration site (Hyg-R and 71cR-DisR; green arrows in panel A).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608573&req=5

pone.0140676.g004: Disruption of 71cR.A. The gDNA of wt C. nicotianae is shown as a black bar with the start and stop codons of 71cR indicated. In disrupted transformants the hyg cassette is predicted to integrate in the location specified by dotted lines. The homologous regions of each split marker are represented as double lines. Location of primers used in PCR screening is also shown. B. Gel image of screening putative 71cR disruptants with primers that span the hyg cassette integration site (71cR-Rev3 and 71cR-inv5’; red arrows in panel A). Integration results in a 2 kb band; wt band is 400bp (indication of non-disruptant genotype). Wild type C. nicotianae and transformant #15 showed only the wild type band; transformant #23 has both bands indicating an ectopic integration of the split markers. C. Bands represent the 1.7 kb amplification of the 5’ integration site by using a 71cR locus-specific forward primer outside the split marker 1 sequence (71cR-Forw10) and a reverse primer specific to the hyg cassette sequence (Hyg-F2) (blue arrows in panel A). D. Bands represent the 715 bp amplification of the 3’ integration site (Hyg-R and 71cR-DisR; green arrows in panel A).
Mentions: A split marker strategy was used to generate C. nicotianae disruption mutants by homologous recombination. A total of 14 hyg-resistant colonies were screened to confirm 71cR disruption by PCR analysis using primer sequences shown in Table 2. Primers 71cR-Rev3 and 71cR-inv5’ that span the hyg-resistance cassette were used to screen the disruptants; with these primers the wt band is 400 bp and the band resulting from disruption is 2 kb (Fig 4A and 4B). A total of 12 transformants were confirmed as lacking the 400 bp wild type band. One had an ectopic integration of the split markers (#23) and one lacked evidence for integration of the split marker (#15) (Fig 4B). Eleven of the disruptants and the strain with ectopic integration of the split markers (#23) were then screened using a 71cR locus-specific primer for the 5’ end outside the split marker sequence (71cR-Forw10) and a primer specific to hyg cassette sequence (Hyg-F2). Presence of the 1.7 kb fragment confirmed the correct location of split marker integration for 10 of the putative disruptants, and no band was seen on amplification of strain #23 (transformant with ectopic integration of the split markers) (Fig 4C). Finally, we screened transformants using a 71cR locus-specific primer for the 3’ end outside the split marker sequence (71cR-DisR) and a second primer specific to the hyg cassette sequence (Hyg-R) (Fig 4D); all of the transformants were also confirmed to have a 715 bp band again confirming the correct location of the split marker integration. Through this analysis, 10 transformants (# 10, 13, 14, 16, 17, 18, 19, 20, 21, 24) were confirmed as being disrupted for 71cR.

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