Publications by authors named "Margaret E Daub"

32 Publications

Phytopathogenic Cercosporoid Fungi-From Taxonomy to Modern Biochemistry and Molecular Biology.

Int J Mol Sci 2020 Nov 13;21(22). Epub 2020 Nov 13.

Department of Biochemistry and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19 Street, 20-033 Lublin, Poland.

Phytopathogenic cercosporoid fungi have been investigated comprehensively due to their important role in causing plant diseases. A significant amount of research has been focused on the biology, morphology, systematics, and taxonomy of this group, with less of a focus on molecular or biochemical issues. Early and extensive research on these fungi focused on taxonomy and their classification based on in vivo features. Lately, investigations have mainly addressed a combination of characteristics such as morphological traits, host specificity, and molecular analyses initiated at the end of the 20th century. Some species that are important from an economic point of view have been more intensively investigated by means of genetic and biochemical methods to better understand the pathogenesis processes. Cercosporin, a photoactivated toxin playing an important role in diseases, has been extensively studied. Understanding cercosporin toxicity in relation to reactive oxygen species (ROS) production facilitated the discovery and regulation of the cercosporin biosynthesis pathway, including the gene cluster encoding pathway enzymes. Furthermore, these fungi may be a source of other biotechnologically important compounds, e.g., industrially relevant enzymes. This paper reviews methods and important results of investigations of this group of fungi addressed at different levels over the years.
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http://dx.doi.org/10.3390/ijms21228555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697478PMC
November 2020

Engineering Cercospora disease resistance via expression of Cercospora nicotianae cercosporin-resistance genes and silencing of cercosporin production in tobacco.

PLoS One 2020 16;15(3):e0230362. Epub 2020 Mar 16.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, United States of America.

Fungi in the genus Cercospora cause crop losses world-wide on many crop species. The wide host range and success of these pathogens has been attributed to the production of a photoactivated toxin, cercosporin. We engineered tobacco for resistance to Cercospora nicotianae utilizing two strategies: 1) transformation with cercosporin autoresistance genes isolated from the fungus, and 2) transformation with constructs to silence the production of cercosporin during disease development. Three C. nicotianae cercosporin autoresistance genes were tested: ATR1 and CFP, encoding an ABC and an MFS transporter, respectively, and 71cR, which encodes a hypothetical protein. Resistance to the pathogen was identified in transgenic lines expressing ATR1 and 71cR, but not in lines transformed with CFP. Silencing of the CTB1 polyketide synthase and to a lesser extent the CTB8 pathway regulator in the cercosporin biosynthetic pathway also led to the recovery of resistant lines. All lines tested expressed the transgenes, and a direct correlation between the level of transgene expression and disease resistance was not identified in any line. Resistance was also not correlated with the degree of silencing in the CTB1 and CTB8 silenced lines. We conclude that expression of fungal cercosporin autoresistance genes as well as silencing of the cercosporin pathway are both effective strategies for engineering resistance to Cercospora diseases where cercosporin plays a critical role.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230362PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075572PMC
June 2020

A polyketide synthase gene cluster associated with the sexual reproductive cycle of the banana pathogen, Pseudocercospora fijiensis.

PLoS One 2019 25;14(7):e0220319. Epub 2019 Jul 25.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, United States of America.

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220319PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657885PMC
March 2020

A novel polyketide synthase gene cluster in the plant pathogenic fungus Pseudocercospora fijiensis.

PLoS One 2019 8;14(2):e0212229. Epub 2019 Feb 8.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, United States of America.

Pseudocercospora fijiensis, causal agent of black Sigatoka of banana, produces polyketide synthase (PKS) pathways shown to be important in disease development by related Dothideomycete fungi. Genome analysis of the P. fijiensis PKS8-1 gene identified it as part of a gene cluster including genes encoding two transcription factors, a regulatory protein, a glyoxylase/beta-lactamase-like protein, an MFS transporter, a cytochrome P450, two aldo/keto reductases, a dehydrogenase, and a decarboxylase. Genome analysis of the related pathogens Pseudocercospora musae, Pseudocercospora eumusae, and Pseudocercospora pini-densiflorae, identified orthologous clusters containing a nearly identical combination of genes. Phylogenetic analysis of PKS8-1 identified homology to PKS proteins in the monodictyphenone and cladofulvin pathways in Aspergillus nidulans and Cladosporium fulvum, respectively. Analysis of clustered genes showed that the PKS8-1 cluster shares genes for enzymes involved in the production of the emodin intermediate in the monodictyphenone and cladofulvin pathways, but differs in many genes, suggesting production of a different metabolic product. Time course analysis of gene expression in infected banana showed up-regulation of PKS8-1 and four of eight clustered genes as early as 2 weeks post-inoculation and remaining high through 9 weeks. Overexpression of the pathway through constitutive expression of an aflR-like transcription factor gene in the cluster resulted in increased expression in culture of PKS8-1 as well as the four clustered genes that are up-regulated in infected plants. No differences were seen in timing or severity of disease symptoms with the overexpression strains relative to controls, however gene expression analysis showed no difference in expression in planta by an overexpression strain relative to controls. Thus constitutive expression of the aflR-like gene is not sufficient to upregulate the pathway above normal expression in planta. Pathway expression during all phases of disease development and conservation of the pathway in related Pseudocercospora species support a role for this pathway in disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368318PMC
November 2019

Transcriptome sequencing of Mycosphaerella fijiensis during association with Musa acuminata reveals candidate pathogenicity genes.

BMC Genomics 2016 08 30;17:690. Epub 2016 Aug 30.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695-7612, USA.

Background: Mycosphaerella fijiensis, causative agent of the black Sigatoka disease of banana, is considered the most economically damaging banana disease. Despite its importance, the genetics of pathogenicity are poorly understood. Previous studies have characterized polyketide pathways with possible roles in pathogenicity. To identify additional candidate pathogenicity genes, we compared the transcriptome of this fungus during the necrotrophic phase of infection with that during saprophytic growth in medium.

Results: Transcriptome analysis was conducted, and the functions of differentially expressed genes were predicted by identifying conserved domains, Gene Ontology (GO) annotation and GO enrichment analysis, Carbohydrate-Active EnZymes (CAZy) annotation, and identification of genes encoding effector-like proteins. The analysis showed that genes commonly involved in secondary metabolism have higher expression in infected leaf tissue, including genes encoding cytochrome P450s, short-chain dehydrogenases, and oxidoreductases in the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily. Other pathogenicity-related genes with higher expression in infected leaf tissue include genes encoding salicylate hydroxylase-like proteins, hydrophobic surface binding proteins, CFEM domain-containing proteins, and genes encoding secreted cysteine-rich proteins characteristic of effectors. More genes encoding amino acid transporters, oligopeptide transporters, peptidases, proteases, proteinases, sugar transporters, and proteins containing Domain of Unknown Function (DUF) 3328 had higher expression in infected leaf tissue, while more genes encoding inhibitors of peptidases and proteinases had higher expression in medium. Sixteen gene clusters with higher expression in leaf tissue were identified including clusters for the synthesis of a non-ribosomal peptide. A cluster encoding a novel fusicoccane was also identified. Two putative dispensable scaffolds were identified with a large proportion of genes with higher expression in infected leaf tissue, suggesting that they may play a role in pathogenicity. For two other scaffolds, no transcripts were detected in either condition, and PCR assays support the hypothesis that at least one of these scaffolds corresponds to a dispensable chromosome that is not required for survival or pathogenicity.

Conclusions: Our study revealed major changes in the transcriptome of Mycosphaerella fijiensis, when associating with its host compared to during saprophytic growth in medium. This analysis identified putative pathogenicity genes and also provides support for the existence of dispensable chromosomes in this fungus.
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http://dx.doi.org/10.1186/s12864-016-3031-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006380PMC
August 2016

Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis.

PLoS One 2016 7;11(7):e0158471. Epub 2016 Jul 7.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, 27695-7612, United States of America.

Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity) for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity) to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that they may encode polyketides important in pathogenicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158471PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936691PMC
July 2017

Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

PLoS One 2015 16;10(10):e0140676. Epub 2015 Oct 16.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140676PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608573PMC
June 2016

Membrane transporters in self resistance of Cercospora nicotianae to the photoactivated toxin cercosporin.

Curr Genet 2015 Nov 11;61(4):601-20. Epub 2015 Apr 11.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA.

The goal of this work is to characterize membrane transporter genes in Cercospora fungi required for autoresistance to the photoactivated, active-oxygen-generating toxin cercosporin they produce for infection of host plants. Previous studies implicated a role for diverse membrane transporters in cercosporin resistance. In this study, transporters identified in a subtractive cDNA library between a Cercospora nicotianae wild type and a cercosporin-sensitive mutant were characterized, including two ABC transporters (CnATR2, CnATR3), an MFS transporter (CnMFS2), a uracil transporter, and a zinc transport protein. Phylogenetic analysis showed that only CnATR3 clustered with transporters previously characterized to be involved in cercosporin resistance. Quantitative RT-PCR analysis of gene expression under conditions of cercosporin toxicity, however, showed that only CnATR2 was upregulated, thus this gene was selected for further characterization. Transformation and expression of CnATR2 in the cercosporin-sensitive fungus Neurospora crassa significantly increased cercosporin resistance. Targeted gene disruption of CnATR2 in the wild type C. nicotianae, however, did not decrease resistance. Expression analysis of other transporters in the cnatr2 mutant under conditions of cercosporin toxicity showed significant upregulation of the cercosporin facilitator protein gene (CFP), encoding an MFS transporter previously characterized as playing an important role in cercosporin autoresistance in Cercospora species. We conclude that cercosporin autoresistance in Cercospora is mediated by multiple genes, and that the fungus compensates for mutations by up-regulation of other resistance genes. CnATR2 may be a useful gene, alone or in addition to other known resistance genes, for engineering Cercospora resistance in crop plants.
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http://dx.doi.org/10.1007/s00294-015-0486-xDOI Listing
November 2015

The role of Cercospora zeae-maydis homologs of Rhodobacter sphaeroides 1O2-resistance genes in resistance to the photoactivated toxin cercosporin.

FEMS Microbiol Lett 2015 Jan 4;362(2):1-7. Epub 2014 Dec 4.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA

The photosynthetic bacterium Rhodobacter sphaeroides and plant pathogenic fungus Cercospora nicotianae have been used as models for understanding resistance to singlet oxygen ((1)O(2)), a highly toxic reactive oxygen species. In Rhodobacter and Cercospora, (1)O(2) is derived, respectively, from photosynthesis and from the (1)O(2)-generating toxin cercosporin which the fungus produces to parasitize plants. We identified common genes recovered in transcriptome studies of putative (1)O(2)-resistance genes in these two systems, suggesting common (1)O(2)-resistance mechanisms. To determine if the Cercospora homologs of R. sphaeroides (1)O(2)-resistance genes are involved in resistance to cercosporin, we expressed the genes in the cercosporin-sensitive fungus Neurospora crassa and assayed for increases in cercosporin resistance. Neurospora crassa transformants expressing genes encoding aldo/keto reductase, succinyl-CoA ligase, O-acetylhomoserine (thiol) lyase, peptide methionine sulphoxide reductase and glutathione S-transferase did not have elevated levels of cercosporin resistance. Several transformants expressing aldehyde dehydrogenase were significantly more resistant to cercosporin. Expression of the transgene and enzyme activity did not correlate with resistance, however. We conclude that although the genes tested in this study are important in (1)O(2) resistance in R. sphaeroides, their Cercospora homologs are not involved in resistance to (1)O(2) generated from cercosporin.
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http://dx.doi.org/10.1093/femsle/fnu036DOI Listing
January 2015

The SOS4 pyridoxal kinase is required for maintenance of vitamin B6-mediated processes in chloroplasts.

Plant Physiol Biochem 2013 Feb 22;63:281-91. Epub 2012 Dec 22.

Department of Plant Biology, North Carolina State University, Raleigh, NC 27695-7612, USA.

Vitamin B(6) (pyridoxal 5'-phosphate and its vitamers) is an important cofactor in numerous enzymatic reactions. In spite of its importance, the consequences of altering vitamin B(6) content on plant growth and development are not well understood. This study compares two mutants for vitamin B(6)-metabolizing enzymes in Arabidopsis thaliana: a pdx1.3 mutant in the de novo synthesis pathway and a salvage pathway sos4 mutant that accumulates more vitamin B(6). We show that despite a difference in total B(6) content in leaf tissue, both mutants share similar phenotypes, including chlorosis, decreased size, altered chloroplast ultrastructure, and root sensitivity to sucrose. Assay of B(6) vitamer content from isolated chloroplasts showed that, despite differing B(6) vitamer content in whole leaf tissue, both mutants share a common deficiency in total and phosphorylated vitamers in chloroplasts. One of the splice variants of the SOS4 proteins was shown to be located in the chloroplast. Our data indicate that some of the phenotypic consequences shared between the pdx1.3 and sos4 mutants are due to B(6) deficiency in chloroplasts, and show that SOS4 is required for maintenance of phosphorylated B(6) vitamer concentrations in chloroplasts. Further, our data are consistent with a diffusion model for transport of vitamin B(6) into chloroplasts.
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http://dx.doi.org/10.1016/j.plaphy.2012.12.003DOI Listing
February 2013

Reactive oxygen species in plant pathogenesis: the role of perylenequinone photosensitizers.

Antioxid Redox Signal 2013 Sep 15;19(9):970-89. Epub 2013 Feb 15.

Department of Plant Biology, North Carolina State University, Raleigh, NC 27695, USA.

Significance: Reactive oxygen species (ROS) play multiple roles in interactions between plants and microbes, both as host defense mechanisms and as mediators of pathogenic and symbiotic associations. One source of ROS in these interactions are photoactivated, ROS-generating perylenequinone pigments produced via polyketide metabolic pathways in plant-associated fungi. These natural products, including cercosporin, elsinochromes, hypocrellins, and calphostin C, are being utilized as medicinal agents, enzyme inhibitors, and in tumor therapy, but in nature, they play a role in the establishment of pathogenic associations between fungi and their plant hosts.

Recent Advances: Photoactivated perylenequinones are photosensitizers that use light energy to form singlet oxygen (¹O₂) and free radical oxygen species which damage cellular components based on localization of the perylenequinone molecule. Production of perylenequinones during infection commonly results in lipid peroxidation and membrane damage, leading to leakage of nutrients from cells into the intercellular spaces colonized by the pathogen. Perylenequinones show almost universal toxicity against organisms, including plants, mice, bacteria, and most fungi. The producing fungi are resistant, however, and serve as models for understanding resistance mechanisms.

Critical Issues: Studies of resistance mechanisms by perylenequinone-producing fungi such as Cercospora species are leading to an understanding of cellular resistance to ¹O₂ and oxidative stress. Recent studies show commonalities between resistance mechanisms in these fungi with extensive studies of ¹O₂ and oxidative stress responses in photosynthetic organisms.

Future Directions: Such studies hold promise both for improved medical use and for engineering crop plants for disease resistance.
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http://dx.doi.org/10.1089/ars.2012.5080DOI Listing
September 2013

Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis.

Plant Mol Biol 2011 May 1;76(1-2):157-69. Epub 2011 May 1.

Department of Plant Biology, North Carolina State University, Raleigh, NC 27695-7612, USA.

Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.
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http://dx.doi.org/10.1007/s11103-011-9777-xDOI Listing
May 2011

The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

Fungal Genet Biol 2009 Feb 6;46(2):146-58. Epub 2008 Dec 6.

Department of Plant Biology, North Carolina State University, Raleigh, North Carolina 27695, USA.

The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.
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http://dx.doi.org/10.1016/j.fgb.2008.11.007DOI Listing
February 2009

Mannitol biosynthesis is required for plant pathogenicity by Alternaria alternata.

FEMS Microbiol Lett 2008 Aug 28;285(1):122-9. Epub 2008 Jun 28.

Department of Plant Pathology, NC State University, Raleigh, NC 27695, USA.

Mannitol has been hypothesized to play a role in antioxidant defense. In previous work, we confirmed the presence of the two mannitol biosynthetic enzymes, mannitol dehydrogenase (MtDH) and mannitol 1-phosphate 5-dehydrogenase (MPDH), in the fungus Alternaria alternata and created disruption mutants for both enzymes. These mutants were used to investigate the role of mannitol in pathogenicity of A. alternata on its host, tobacco. Conidia of all mutants were viable and germinated normally. GC-MS analysis demonstrated elevated levels of trehalose in the mutants, suggesting that trehalose may substitute for mannitol as a storage compound for germination. Tobacco inoculation showed no reduction in lesion severity caused by the MtDH mutant as compared with wild type; however, the MPDH mutant and a mutant in both enzymes caused significantly less disease. Microscopy analysis indicated that the double mutant was unaffected in the ability to germinate and produce appressoria on tobacco leaves and elicited a defense response from the host, indicating that it was able to penetrate and infect the host. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for spore germination either in vitro or in planta or for initial infection.
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http://dx.doi.org/10.1111/j.1574-6968.2008.01224.xDOI Listing
August 2008

Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway.

Plant Physiol 2007 Nov 14;145(3):985-96. Epub 2007 Sep 14.

Department of Plant Biology, North Carolina State University, Raleigh, North Carolina 27695-7612, USA.

PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.
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http://dx.doi.org/10.1104/pp.107.105189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2048783PMC
November 2007

Identification of genes differentially expressed in the phytopathogenic fungus Cercospora nicotianae between cercosporin toxin-resistant and -susceptible strains.

FEMS Microbiol Lett 2007 Oct 11;275(2):326-37. Epub 2007 Sep 11.

Department of Plant Biology, North Carolina State University, Raleigh, NC, USA.

Plant pathogens from the genus Cercospora produce cercosporin, a photoactivated fungal toxin that generates toxic reactive oxygen species. Mechanisms governing toxin auto-resistance in Cercospora spp. are poorly understood. In this work, suppressive subtractive hybridization was used to identify genes differentially expressed between the cercosporin-resistant wild-type (WT) Cercospora nicotianae and a sensitive strain lacking a transcription factor (CRG1) that regulates resistance. Out of 338 sequences recovered, 185 unique expressed sequence tags (ESTs) were obtained and classified into functional categories. The majority of genes showed predicted expression differences, and 38.5% were differentially expressed at least twofold between the WT and mutant strain. ESTs were recovered with homology to genes involved in detoxification of noxious compounds, multidrug membrane transporters and antioxidant and polyketide biosynthetic enzymes as well as to ATPases and ATP synthases. The findings suggest that CRG1 regulates genes involved in pH responses in addition to those involved in toxin resistance and biosynthesis.
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http://dx.doi.org/10.1111/j.1574-6968.2007.00903.xDOI Listing
October 2007

Regulation of the Arabidopsis thaliana vitamin B6 biosynthesis genes by abiotic stress.

Plant Physiol Biochem 2007 Feb 20;45(2):152-61. Epub 2007 Jan 20.

Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616, USA.

Vitamin B(6) (pyridoxine and its vitamers) plays an essential role as a co-factor for enzymatic reactions and has also recently been implicated in defense against cellular oxidative stress. The biosynthetic pathway was thoroughly characterized in Escherichia coli, however most organisms, including plants, utilize an alternate pathway involving two genes, PDX1 and PDX2. Arabidopsis thaliana contains one copy of PDX2, but three full-length copies of PDX1, one each on chromosomes 2, 3, and 5 (referred to as PDX1.1, PDX1.2, and PDX1.3, respectively). Phylogenetic analysis of the PDX1 homologues in A. thaliana showed that PDX1.1 and PDX1.3 clustered with the homologues from the other dicots, whereas PDX1.2 was more divergent, and did not cluster with either the dicots or monocots. Expression analysis using quantitative PCR showed that PDX1.1 and PDX1.3 were highly expressed in A. thaliana rosettes, while PDX1.2 showed only low level expression. All three PDX1 genes and PDX2 were responsive to abiotic stressors including high light, chilling, drought, and ozone, however, the response of PDX1.2 was disparate from that of the other PDX genes, showing a lessened response to high light, chilling, and drought, but an increased response to ozone. Green fluorescent protein fusion studies demonstrated that PDX2 localizes in the nucleus and membranes of cells, consistent with recent published data for PDX1. Insight into regulation of the biosynthetic genes during abiotic stress could have important applications in the development of stress-tolerant crops.
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http://dx.doi.org/10.1016/j.plaphy.2007.01.007DOI Listing
February 2007

Mannitol metabolism in the phytopathogenic fungus Alternaria alternata.

Fungal Genet Biol 2007 Apr 7;44(4):258-68. Epub 2006 Nov 7.

Department of Plant Pathology, NC State University, Raleigh, NC 27695, USA.

Mannitol metabolism in fungi is thought to occur through a mannitol cycle first described in 1978. In this cycle, mannitol 1-phosphate 5-dehydrogenase (EC 1.1.1.17) was proposed to reduce fructose 6-phosphate into mannitol 1-phosphate, followed by dephosphorylation by a mannitol 1-phosphatase (EC 3.1.3.22) resulting in inorganic phosphate and mannitol. Mannitol would be converted back to fructose by the enzyme mannitol dehydrogenase (EC 1.1.1.138). Although mannitol 1-phosphate 5-dehydrogenase was proposed as the major biosynthetic enzyme and mannitol dehydrogenase as a degradative enzyme, both enzymes catalyze their respective reverse reactions. To date the cycle has not been confirmed through genetic analysis. We conducted enzyme assays that confirmed the presence of these enzymes in a tobacco isolate of Alternaria alternata. Using a degenerate primer strategy, we isolated the genes encoding the enzymes and used targeted gene disruption to create mutants deficient in mannitol 1-phosphate 5-dehydrogenase, mannitol dehydrogenase, or both. PCR analysis confirmed gene disruption in the mutants, and enzyme assays demonstrated a lack of enzymatic activity for each enzyme. GC-MS experiments showed that a mutant deficient in both enzymes did not produce mannitol. Mutants deficient in mannitol 1-phosphate 5-dehydrogenase or mannitol dehydrogenase alone produced 11.5 and 65.7 %, respectively, of wild type levels. All mutants grew on mannitol as a sole carbon source, however, the double mutant and mutant deficient in mannitol 1-phosphate 5-dehydrogenase grew poorly. Our data demonstrate that mannitol 1-phosphate 5-dehydrogenase and mannitol dehydrogenase are essential enzymes in mannitol metabolism in A. alternata, but do not support mannitol metabolism operating as a cycle.
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http://dx.doi.org/10.1016/j.fgb.2006.09.008DOI Listing
April 2007

An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae.

Appl Environ Microbiol 2006 Sep;72(9):6070-8

Department of Plant Pathology, 2214 Gardner Hall, North Carolina State University, Raleigh, NC 27695, USA.

The polyketide toxin cercosporin plays a key role in pathogenesis by fungal species of the genus Cercospora. The bacterium Xanthomonas campestris pv. zinniae is able to rapidly degrade this toxin. Growth of X. campestris pv. zinniae strains in cercosporin-containing medium leads to the breakdown of cercosporin and to the formation of xanosporic acid, a nontoxic breakdown product. Five non-cercosporin-degrading mutants of a strain that rapidly degrades cercosporin (XCZ-3) were generated by ethyl methanesulfonate mutagenesis and were then transformed with a genomic library from the wild-type strain. All five mutants were complemented with the same genomic clone, which encoded a putative transcriptional regulator and an oxidoreductase. Simultaneous expression of these two genes was necessary to complement the mutant phenotype. Sequence analysis of the mutants showed that all five mutants had point mutations in the oxidoreductase gene and no mutations in the regulator. Quantitative reverse transcription-PCR (RT-PCR) showed that the expression of both of these genes in the wild-type strain is upregulated after exposure to cercosporin. Both the oxidoreductase and transcriptional regulator genes were transformed into three non-cercosporin-degrading bacteria to determine if they are sufficient for cercosporin degradation. Quantitative RT-PCR analysis confirmed that the oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to degrade cercosporin, suggesting that additional factors are required for cercosporin degradation. Further study of cercosporin degradation in X. campestris pv. zinniae may allow for the engineering of Cercospora-resistant plants by using a suite of genes.
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http://dx.doi.org/10.1128/AEM.00483-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1563685PMC
September 2006

Photoactivated perylenequinone toxins in fungal pathogenesis of plants.

FEMS Microbiol Lett 2005 Nov 6;252(2):197-206. Epub 2005 Sep 6.

Department of Botany, North Carolina State University, Raleigh, 27695-7612, USA.

Several genera of plant pathogenic fungi produce photoactivated perylenequinone toxins involved in pathogenesis of their hosts. These toxins are photosensitizers, absorbing light energy and generating reactive oxygen species that damage the membranes of the host cells. Studies with toxin-deficient mutants and on the involvement of light in symptom development have documented the importance of these toxins in successful pathogenesis of plants. This review focuses on the well studied perylenequinone toxin, cercosporin, produced by species in the genus Cercospora. Significant progress has been made recently on the biosynthetic pathway of cercosporin, with the characterization of genes encoding a polyketide synthase and a major facilitator superfamily transporter, representing the first and last steps of the biosynthetic pathway, as well as important regulatory genes. In addition, the resistance of Cercospora fungi to cercosporin and to the singlet oxygen that it generates has led to the use of these fungi as models for understanding cellular resistance to photosensitizers and singlet oxygen. These studies have shown that resistance is complex, and have documented a role for transporters, transient reductive detoxification, and quenchers in cercosporin resistance.
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http://dx.doi.org/10.1016/j.femsle.2005.08.033DOI Listing
November 2005

The CTB1 gene encoding a fungal polyketide synthase is required for cercosporin biosynthesis and fungal virulence of Cercospora nicotianae.

Mol Plant Microbe Interact 2005 May;18(5):468-76

Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, 700 Experiment Station Road, Lake Alfred 33850, USA.

Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp.
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http://dx.doi.org/10.1094/MPMI-18-0468DOI Listing
May 2005

Functional complementation between the PDX1 vitamin B6 biosynthetic gene of Cercospora nicotianae and pdxJ of Escherichia coli.

FEBS Lett 2004 Apr;564(1-2):143-6

Department of Botany, North Carolina State University, Raleigh, NC 27695-7612, USA.

The pathway for de novo vitamin B(6) biosynthesis has been characterized in Escherichia coli, however plants, fungi, archaebacteria, and most bacteria utilize an alternative pathway. Two unique genes of the alternative pathway, PDX1 and PDX2, have been described. PDX2 encodes a glutaminase, however the enzymatic function of the product encoded by PDX1 is not known. We conducted reciprocal transformation experiments to determine if there was functional homology between the E. coli pdxA and pdxJ genes and PDX1 of Cercospora nicotianae. Although expression of pdxJ and pdxA in C. nicotianae pdx1 mutants, either separately or together, failed to complement the pyridoxine mutation in this fungus, expression of PDX1 restored pyridoxine prototrophy to the E. coli pdxJ mutant. Expression of PDX1 in the E. coli pdxA mutant restored very limited ability to grow on medium lacking pyridoxine. We conclude that the PDX1 gene of the alternative B(6) pathway encodes a protein responsible for synthesis of the pyridoxine ring.
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http://dx.doi.org/10.1016/S0014-5793(04)00329-1DOI Listing
April 2004

Expression of the cercosporin toxin resistance gene ( CRG1) as a dicistronic mRNA in the filamentous fungus Cercospora nicotianae.

Curr Genet 2003 Sep 11;43(6):415-24. Epub 2003 Jun 11.

Citrus Research and Education Center and Department of Plant Pathology, Institute of Food and Agricultural Sciences, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USA.

The CRG1 gene in Cercospora nicotianae encodes a transcription factor and is required for cercosporin toxin resistance and production. Cloning and sequencing of the downstream region of the CRG1 gene led to the discovery of an adjacent gene ( PUT1) encoding a putative uracil transporter. Expression of CRG1 and PUT1 as assessed by Northern analysis indicated that, in addition to the expected monocistronic mRNAs (2.6 kb and 2.0 kb, respectively), a common 4.5-kb mRNA could be identified, using either a CRG1 or a PUT1 gene probe. The 2.6-kb transcript identified only by the CRG1 probe was expressed constitutively, whereas the 2.0-kb transcript identified only by the PUT1 probe was differentially expressed in various media. Four cDNA clones containing CRG1, PUT1, and the CRG1- PUT1 intergenic region were identified as part of the products from the 4.5-kb transcript. Both the 4.5-kb and 2.6-kb transcripts were not detectable in three crg1-disrupted mutants, using the CRG1 probe. The 2.0-kb transcript, but not the 4.5-kb one was detected using the PUT1 probe in the three crg1-disrupted mutants. Taken together, we conclude that the 4.5-kb transcript is a dicistronic mRNA of both CRG1 and PUT1 in the fungus C. nicotianae. This is the first example of a dicistronic mRNA identified in filamentous fungi.
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http://dx.doi.org/10.1007/s00294-003-0414-3DOI Listing
September 2003

Xanosporic acid, an intermediate in bacterial degradation of the fungal phototoxin cercosporin.

Phytochemistry 2003 Mar;62(5):723-32

Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695, USA.

The red fungal perylenequinone phototoxin cercosporin is oxidized by Xanthomonas campestris pv zinniae to a non-toxic, unstable green metabolite xanosporic acid, identified via its lactone as 1,12-bis(2'R-hydroxypropyl)-4,9-dihydroxy-6,7-methylenedioxy-11-methoxy-3-oxaperylen-10H-10-one-2-carboxylic acid. Xanosporolactone was isolated in approximately 2:1 ratio of M:P atropisomers.
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http://dx.doi.org/10.1016/s0031-9422(02)00517-4DOI Listing
March 2003

The CRG1 gene required for resistance to the singlet oxygen-generating cercosporin toxin in Cercospora nicotianae encodes a putative fungal transcription factor.

Biochem Biophys Res Commun 2003 Mar;302(2):302-10

Citrus Research and Education Center and Department of Plant Pathology, IFAS, University of Florida, Lake Alfred, FL 33850, USA.

The Cercospora nicotianae CRG1 gene is involved in cellular resistance to the perylenequinone toxin, cercosporin, that generates highly toxic singlet oxygen upon exposure to light. The entire open reading frame (ORF) of CRG1 was isolated and sequenced. The gene contains an ORF of 1950bp including a 65-bp intron. The predicted 650 amino acid CRG1 protein contains a Cys(6)Zn(2) binuclear cluster DNA-binding motif with homology to various fungal regulatory proteins, indicating that CRG1 may act functionally as a transcription activator. Targeted gene disruption of CRG1 resulted in mutants that are partially sensitive to cercosporin and reduced in cercosporin production. Genetic complementation revealed that CRG1 fully restored cercosporin resistance, but only slightly restored cercosporin production in a UV-derived mutant (CS10) containing a single nucleotide substitution in crg1. Complementation of a crg1-null mutant, however, yielded strains that are similar to the wild-type in both phenotypes. These results indicate that the transcription regulator CRG1 is involved in the activation of genes associated with cercosporin resistance and production in the fungus Cercospora nicotianae.
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http://dx.doi.org/10.1016/s0006-291x(03)00171-2DOI Listing
March 2003

Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata.

Plant J 2002 Oct;32(1):41-9

Department of Horticultural Science, North Carolina State University, Raleigh NC 27695, USA.

Our previous observation that host plant extracts induce production and secretion of mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher mannitol as a means of suppressing reactive oxygen-mediated plant defenses. The concurrent discovery that pathogen attack induced mannitol dehydrogenase (MTD) expression in the non-mannitol-containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were produced and evaluated for potential changes in resistance to both mannitol- and non-mannitol-secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non-mannitol-secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to mannitol-secreting fungal plant pathogens.
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http://dx.doi.org/10.1046/j.1365-313x.2001.01399.xDOI Listing
October 2002

Biodegradation of the polyketide toxin cercosporin.

Appl Environ Microbiol 2002 Sep;68(9):4173-81

Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695-7616, USA.

Cercosporin is a non-host-specific polyketide toxin produced by many species of plant pathogens belonging to the genus Cercospora. This red-pigmented, light-activated toxin is an important pathogenicity determinant for Cercospora species. In this study, we screened 244 bacterial isolates representing 12 different genera for the ability to degrade cercosporin. Cercosporin degradation was determined by screening for the presence of cleared zones surrounding colonies on cercosporin-containing culture medium and was confirmed by assaying the kinetics of degradation in liquid medium. Bacteria belonging to four different genera exhibited the cercosporin-degrading phenotype. The isolates with the greatest cercosporin-degrading activity belonged to Xanthomonas campestris pv. zinniae and X. campestris pv. pruni. Isolates of these pathovars removed over 90% of the cercosporin from culture medium within 48 h. Bacterial degradation of red cercosporin was accompanied by a shift in the color of the growth medium to brown and then green. The disappearance of cercosporin was accompanied by the appearance of a transient green product, designated xanosporic acid. Xanosporic acid and its more stable lactone derivative, xanosporolactone, are nontoxic to cercosporin-sensitive fungi and to plant tissue and are labile in the presence of light. Detailed spectroscopic analysis (to be reported in a separate publication) of xanosporolactone revealed that cercosporin loses one methoxyl group and gains one oxygen atom in the bacterial conversion. The resulting chromophore (4,9-dihydroxy-3-oxaperlylen-10H-10-one) has never been reported before but is biosynthetically plausible via oxygen insertion by a cytochrome P-450 enzyme.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124086PMC
http://dx.doi.org/10.1128/aem.68.9.4173-4181.2002DOI Listing
September 2002

Direct detection of singlet oxygen via its phosphorescence from cellular and fungal cultures.

Methods Enzymol 2002 ;352:41-52

Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

In summary, we have developed methods to detect 1O2 spectrally from viable keratinocytes and fungal mycelial cultures. Both time-resolved and steady-state spectrophotometers were used, providing complementary information on optimal conditions for the unambiguous detection of 1O2 phosphorescence. By using our techniques, we were able to confirm that photosensitizers in contact with living cells indeed generate 1O2. The model systems and the procedures described can be adopted for other studies involving 1O2 and oxidative stress in living cells.
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http://dx.doi.org/10.1016/s0076-6879(02)52005-xDOI Listing
August 2002

Functional expression and cellular localization of cercosporin-resistance proteins fused with the GFP in Cercospora nicotianae.

Curr Genet 2002 Jun 31;41(3):159-67. Epub 2002 May 31.

Department of Plant Pathology, North Carolina State University, Raleigh 27695-7616, USA.

The Cercospora nicotianae pdx1 and crg1 genes were previously identified as genes required for resistance to the singlet oxygen ((1)O(2))-generating toxin cercosporin. The pdx1 gene has subsequently been shown to be required for pyridoxine biosynthesis, but both the precise biochemical function of the PDX1 protein and the function of the CRG1 protein remain undefined, as both sequences lack defined enzymatic domains or cofactor-binding sites. The gfp gene encoding green fluorescent protein was translationally fused with pdx1 and crg1. Transformation of these constructs into strains mutant in these respective genes resulted in green-fluorescent transformants complemented for the mutant phenotype. Microscopic studies revealed that in transformants transformed with gfp alone, fluorescence was distributed evenly throughout the cytoplasm and excluded from the vacuoles. Expression of PDX1::GFP either under the constitutive Aspergillus nidulans gpdA promoter or its own native promoter was visualized as distinct fluorescent circular structures in the cytoplasm, suggesting that PDX1::GFP was probably localized in the intracellular vesicles. Expression of CRG1 fused with GFP at either its N- or C-terminus resulted in low green fluorescence, compared with that of GFP alone or PDX1::GFP. The green fluorescence of either of the CRG1::GFP fusion proteins was barely observable in transformants and was generally seen as a few scattered regions of fluorescence in the hyphae. Southern blot analysis indicated multiple copies of the constructs were integrated into the fungal genome. Northern analysis revealed that pdx1:: gfp and crg1:: gfp were each expressed as an intact transcriptional unit. Cell fractionation followed by immunoblotting against a GFP antibody showed that GFP alone and PDX1::GFP were detected exclusively in the cytoplasmic fraction. The two CRG1::GFP proteins were barely detected in the cytoplasmic fraction and not at all from the membrane fraction, a result inconsistent with microscopic observation and computer sequence analysis, which suggests that CRG1 is a membrane protein.
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http://dx.doi.org/10.1007/s00294-002-0289-8DOI Listing
June 2002

The Photoactivated Cercospora Toxin Cercosporin: Contributions to Plant Disease and Fundamental Biology.

Annu Rev Phytopathol 2000 Sep;38:461-490

Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695-7616; e-mail:

Plant pathogenic fungi in eight genera produce light-activated perylenequinone toxins that are toxic to plants via the generation of activated oxygen species, particularly singlet oxygen. Studies on the cercosporin toxin produced by Cercospora species have documented an important role for this toxin in pathogenesis of host plants. Cercosporin-generated active oxygen species destroy the membranes of host plants, providing nutrients to support the growth of these intercellular pathogens. Resistance of Cercospora species to the toxic effects of their own toxin has allowed these organisms to be used as a model for understanding the cellular basis of resistance to singlet oxygen and to general oxidative stress. In particular, the recent discovery that pyridoxine (vitamin B6) quenches singlet oxygen has led to the understanding of a novel role for this vitamin in cells as well as the discovery of a novel pathway of biosynthesis.
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http://dx.doi.org/10.1146/annurev.phyto.38.1.461DOI Listing
September 2000