@misc{daub_herrero_chung_2005, title={Photoactivated perylenequinone toxins in fungal pathogenesis of plants}, volume={252}, ISSN={["0378-1097"]}, DOI={10.1016/j.femsle.2005.08.033}, abstractNote={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.}, number={2}, journal={FEMS MICROBIOLOGY LETTERS}, author={Daub, ME and Herrero, S and Chung, KR}, year={2005}, month={Nov}, pages={197–206} }
 @article{chung_ehrenshaft_wetzel_daub_2003, title={Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae}, volume={270}, ISSN={["1617-4623"]}, DOI={10.1007/s00438-003-0902-7}, abstractNote={We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.}, number={2}, journal={MOLECULAR GENETICS AND GENOMICS}, author={Chung, KR and Ehrenshaft, M and Wetzel, DK and Daub, ME}, year={2003}, month={Oct}, pages={103–113} }
 @article{chung_daub_ehrenshaft_2003, title={Expression of the cercosporin toxin resistance gene (CRG1) as a dicistronic mRNA in the filamentous fungus Cercospora nicotianae}, volume={43}, ISSN={["1432-0983"]}, DOI={10.1007/s00294-003-0414-3}, abstractNote={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.}, number={6}, journal={CURRENT GENETICS}, author={Chung, KR and Daub, ME and Ehrenshaft, M}, year={2003}, month={Sep}, pages={415–424} }
 @article{chung_ehrenshaft_daub_2002, title={Functional expression and cellular localization of cercosporin-resistance proteins fused with the GFP in Cercospora nicotianae}, volume={41}, ISSN={["0172-8083"]}, DOI={10.1007/s00294-002-0289-8}, abstractNote={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.}, number={3}, journal={CURRENT GENETICS}, author={Chung, KR and Ehrenshaft, M and Daub, ME}, year={2002}, month={Jun}, pages={159–167} }
 @article{chung_jenns_ehrenshaft_daub_1999, title={A novel gene required for cercosporin toxin resistance in the fungus Cercospora nicotianae}, volume={262}, DOI={10.1007/pl00008642}, abstractNote={Cercosporin, a photosensitizing perylenequinone toxin produced by the plant pathogenic Cercospora fungi, generates the highly toxic singlet oxygen (1O2) upon exposure to light. Cercosporin shows broad toxicity against a wide range of organisms, including bacteria, fungi, plants, and animals; however, Cercospora fungi are resistant to its effects. A novel gene, crg1 (cercosporin-resistance gene) was isolated from a wild-type strain of C. nicotianae by genetic complementation of a C. nicotianae mutant (CS10) which is cercosporin sensitive and down-regulated in cercosporin production. Sequence analysis indicated that crg1 encodes a putative protein of 550 amino acids with four putative transmembrane helical regions, however CRG1 shows no strong similarity to any other protein in sequence databases. Northern analysis identified two transcripts (4.5 and 2.6 kb) that are unaffected by the presence of light or cercosporin. Southern analysis demonstrated that crg1 is present in a single copy in the C. nicotianae genome and can be detected only in Cercospora species. Targeted disruption of crg1 resulted in mutants that, like CS10, are sensitive to cercosporin. However, unlike CS10, crg1 disruption mutants are not down-regulated in toxin production. Both CS10 and the crg1 disruption mutants are unaffected in their response to other 1O2-generating photosensitizers, suggesting that CRG1 functions specifically against cercosporin, rather than against 1O2.}, number={2}, journal={Molecular and General Genetics}, author={Chung, K. R. and Jenns, A. E. and Ehrenshaft, M. and Daub, M. E.}, year={1999}, pages={382–389} }
 @article{ehrenshaft_chung_jenns_daub_1999, title={Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae}, volume={34}, ISSN={["0172-8083"]}, DOI={10.1007/s002940050423}, abstractNote={The Cercospora nicotianae SOR1 gene is required for resistance to singlet oxygen-generating photosensitizers. SOR1 was characterized in the wild-type and in five photosensitizer-sensitive mutant strains which are complemented to photosensitizer resistance by transformation with SOR1. Sequence analysis determined that three of the mutants contain SOR1 copies with mutations encoding substitutions in the protein-coding sequence; however, two other mutants had wild-type SOR1 protein and promoter sequences. All five mutants accumulate SOR1 mRNA at levels comparable to that of the wild-type strain. In the wild-type strain, SOR1 accumulation is enhanced two-fold by light, but is unaffected by the presence of cercosporin, the photosensitizer synthesized by C. nicotianae. Southern analysis indicates that SOR1 is present in other fungi that synthesize structurally related perylenequinone photosensitizers.}, number={6}, journal={CURRENT GENETICS}, author={Ehrenshaft, M and Chung, KR and Jenns, AE and Daub, ME}, year={1999}, month={Jan}, pages={478–485} }
 @article{daub_ehrenshaft_jenns_chung_1998, title={Active oxygen in fungal pathogenesis of plants: the role of cercosporin in Cercospora diseases}, volume={32}, number={1998}, journal={Recent Advances in Phytochemistry}, author={Daub, M. E. and Ehrenshaft, M. and Jenns, A. E. and Chung, K. R.}, year={1998}, pages={31–56} }
 @article{ehrenshaft_jenns_chung_daub_1998, title={SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms}, volume={1}, ISSN={["1097-2765"]}, DOI={10.1016/S1097-2765(00)80060-X}, abstractNote={Filamentous Cercospora fungi are resistant to photosensitizing compounds that generate singlet oxygen. C. nicotianae photosensitizer-sensitive mutants were restored to full resistance by transformation with SOR1 (Singlet Oxygen Resistance 1), a gene recovered from a wild-type genomic library. SOR1 null mutants generated via targeted gene replacement confirmed the requirement for SOR1 in photosensitizer resistance. SOR1 RNA is present throughout the growth cycle. Although resistance to singlet oxygen is rare in biological systems, SOR1, a gene with demonstrated activity against singlet-oxygen-generating photosensitizers, is highly conserved in organisms from widely diverse taxa. The characterization of SOR1 provides an additional phenotype to this large group of evolutionarily conserved genes.}, number={4}, journal={MOLECULAR CELL}, author={Ehrenshaft, M and Jenns, AE and Chung, KR and Daub, ME}, year={1998}, month={Mar}, pages={603–609} }
 @article{chung_schardl_1997, title={Vegetative compatibility between and within Epichloe species}, volume={89}, ISSN={["0027-5514"]}, DOI={10.2307/3760992}, abstractNote={Epichloë species (Clavicipitaceae, Ascomycota) are biotrophic symbionts of cool-season grasses and grow slowly on defined media. Nine Epichloë biological species (mating populations) have been identified. Vegetative compatibility within or between Epichloë species has not yet been investigated. We selected chlorate-resistant strains that were nitrate non-utilizing (nit) mutants from ten isolates representing four biological species. Each mutant grew appressed on a defined medium with nitrate as the sole nitrogen source, in contrast to the abundant aerial mycelia produced by the prototrophic wild type isolates on the same medium. Each mutant was putatively identified as nitl (nitrate reductase structural gene mutant), nit3 (mutant in the pathway-specific regulatory locus or nitrite reductase gene), or NitM (mutant affecting molybdenum cofactor production) by growth phenotypes on medium with nitrite, hypoxanthine, uric acid or ammonium as the sole nitrogen source. When appropriate nit mutants were paired, heterokaryon formation and complementation were indicated by a zone of prototrophic growth. Complementation occurred between complementary mutants of most isolates, even from different species; however, complementation did not occur in all replications. Interspecific heterokaryon formation was confirmed by recovery of parental phenotypes and cosegregation of β-tubulin gene polymorphisms among single-conidiospore isolates from the complementation zone. There was no indication that mating type interactions affected heterokaryon formation, and no discrete vegetative compatibility groups were identified.}, number={4}, journal={MYCOLOGIA}, author={Chung, KR and Schardl, CL}, year={1997}, pages={558–565} }