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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

Alignment of 71cR amino acid sequence with the 130 amino acid sequences in S1 Table.Chimera 1.10.1 was used to display the degree of conservation for each residue within the alignment of the conserved domain [38]. 71cR C. nicotianae sequence is shown along with the consensus sequence from the whole dataset. The numbers above the amino acid residues indicate the positions within the sequence alignment. Color of each amino acid was determined according to the ClustalX [42] color scheme. The bars show the conservation between 71cR and all of the 130 sequences in S1 Table. Stars on top of the amino acids indicate the complete conservation of that sequence except the outgroup (E on position 135 is completely conserved throughout every single one including the outgroup). The red boxes around the letters show the ligand binding sites predicted by Raptor software.
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pone.0140676.g009: Alignment of 71cR amino acid sequence with the 130 amino acid sequences in S1 Table.Chimera 1.10.1 was used to display the degree of conservation for each residue within the alignment of the conserved domain [38]. 71cR C. nicotianae sequence is shown along with the consensus sequence from the whole dataset. The numbers above the amino acid residues indicate the positions within the sequence alignment. Color of each amino acid was determined according to the ClustalX [42] color scheme. The bars show the conservation between 71cR and all of the 130 sequences in S1 Table. Stars on top of the amino acids indicate the complete conservation of that sequence except the outgroup (E on position 135 is completely conserved throughout every single one including the outgroup). The red boxes around the letters show the ligand binding sites predicted by Raptor software.

Mentions: As the phylogenetic analyses did not enable us to make conclusions about function, we analyzed secondary and tertiary structure of the 71cR amino acid sequence as these structures are important for their biological functions. The secondary and tertiary structure of the 71cR amino acid sequence was analyzed using Raptor software [35]. The secondary structure of 71cR is predicted to have 7 beta sheets and 4 alpha helices (Fig 8A). For the tertiary structure predictions, Raptor software predicted the best template to be Protein PFL_3262 from Pseudomonas fluorescens (2imj:A). The predicted model has a p-value of 2.96e-11. The predicted structure of 71cR protein is a half barrel structure with 7 beta sheets enclosing the alpha helix (Fig 8B). The two other alpha helices are positioned near beta sheets #4, 5, and 6 (Fig 8B). The predicted model also states that this protein forms a dimer. The Raptor server predicted that 71cR binds steroids, and identified 12 ligand binding sites for steroid binding (A19, W23, N43, K61, Y68, F86, Y88, Y90, C102, E106, R119, M121). The pocket multiplicity value predicted for steroid binding is 74, and we compared it to the pocket multiplicity of a known steroid delta-isomerase (Accession number: P07445); its pocket multiplicity was also 74, providing high support for 71cR binding to steroids. To determine whether the predicted ligand binding sites are conserved in 71cR, 130 of its closest orthologs (S1 Table) were aligned to identify conserved residues within the sequence (Fig 9). The 71cR sequence was mostly conserved throughout its length. All of the ligand binding sites found in the Raptor analysis except Y90 and C102 were shown to be conserved.


Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

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

Alignment of 71cR amino acid sequence with the 130 amino acid sequences in S1 Table.Chimera 1.10.1 was used to display the degree of conservation for each residue within the alignment of the conserved domain [38]. 71cR C. nicotianae sequence is shown along with the consensus sequence from the whole dataset. The numbers above the amino acid residues indicate the positions within the sequence alignment. Color of each amino acid was determined according to the ClustalX [42] color scheme. The bars show the conservation between 71cR and all of the 130 sequences in S1 Table. Stars on top of the amino acids indicate the complete conservation of that sequence except the outgroup (E on position 135 is completely conserved throughout every single one including the outgroup). The red boxes around the letters show the ligand binding sites predicted by Raptor software.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140676.g009: Alignment of 71cR amino acid sequence with the 130 amino acid sequences in S1 Table.Chimera 1.10.1 was used to display the degree of conservation for each residue within the alignment of the conserved domain [38]. 71cR C. nicotianae sequence is shown along with the consensus sequence from the whole dataset. The numbers above the amino acid residues indicate the positions within the sequence alignment. Color of each amino acid was determined according to the ClustalX [42] color scheme. The bars show the conservation between 71cR and all of the 130 sequences in S1 Table. Stars on top of the amino acids indicate the complete conservation of that sequence except the outgroup (E on position 135 is completely conserved throughout every single one including the outgroup). The red boxes around the letters show the ligand binding sites predicted by Raptor software.
Mentions: As the phylogenetic analyses did not enable us to make conclusions about function, we analyzed secondary and tertiary structure of the 71cR amino acid sequence as these structures are important for their biological functions. The secondary and tertiary structure of the 71cR amino acid sequence was analyzed using Raptor software [35]. The secondary structure of 71cR is predicted to have 7 beta sheets and 4 alpha helices (Fig 8A). For the tertiary structure predictions, Raptor software predicted the best template to be Protein PFL_3262 from Pseudomonas fluorescens (2imj:A). The predicted model has a p-value of 2.96e-11. The predicted structure of 71cR protein is a half barrel structure with 7 beta sheets enclosing the alpha helix (Fig 8B). The two other alpha helices are positioned near beta sheets #4, 5, and 6 (Fig 8B). The predicted model also states that this protein forms a dimer. The Raptor server predicted that 71cR binds steroids, and identified 12 ligand binding sites for steroid binding (A19, W23, N43, K61, Y68, F86, Y88, Y90, C102, E106, R119, M121). The pocket multiplicity value predicted for steroid binding is 74, and we compared it to the pocket multiplicity of a known steroid delta-isomerase (Accession number: P07445); its pocket multiplicity was also 74, providing high support for 71cR binding to steroids. To determine whether the predicted ligand binding sites are conserved in 71cR, 130 of its closest orthologs (S1 Table) were aligned to identify conserved residues within the sequence (Fig 9). The 71cR sequence was mostly conserved throughout its length. All of the ligand binding sites found in the Raptor analysis except Y90 and C102 were shown to be conserved.

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