@article{graham_ehrenshaft_hausner_reid_2004, title={A  highly conserved gene for vitamin B-6 biosynthesis may have consequences for stress and hormone responses in plants}, volume={121}, number={1}, journal={Physiologia Plantarum}, author={Graham, C. M. and Ehrenshaft, M. and Hausner, G. and Reid, D. M.}, year={2004}, pages={14-} }
 @article{wetzel_ehrenshaft_denslow_daub_2004, title={Functional complementation between the PDX1 vitamin B-6 biosynthetic gene of Cercospora nicotianae and pdxJ of Escherichia coli}, volume={564}, ISSN={["1873-3468"]}, DOI={10.1016/S0014-5793(04)00329-1}, abstractNote={The pathway for de novo vitamin B6 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 B6 pathway encodes a protein responsible for synthesis of the pyridoxine ring.}, number={1-2}, journal={FEBS LETTERS}, author={Wetzel, DK and Ehrenshaft, M and Denslow, SA and Daub, ME}, year={2004}, month={Apr}, pages={143–146} }
 @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{williamson_jennings_guo_pharr_ehrenshaft_2002, title={Sugar alcohols, salt stress, and fungal resistance: Polyols - Multifunctional plant protection?}, volume={127}, number={4}, journal={Journal of the American Society for Horticultural Science}, author={Williamson, J. D. and Jennings, D. B. and Guo, W. W. and Pharr, D. M. and Ehrenshaft, M.}, year={2002}, pages={467–473} }
 @article{ehrenshaft_daub_2001, title={Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria}, volume={183}, ISSN={["0021-9193"]}, DOI={10.1128/JB.183.11.3383-3390.2001}, abstractNote={ABSTRACT}, number={11}, journal={JOURNAL OF BACTERIOLOGY}, author={Ehrenshaft, M and Daub, ME}, year={2001}, month={Jun}, pages={3383–3390} }
 @misc{upchurch_callahan_ehrenshaft_2000, title={Fungal gene encoding resistance to the phytotoxin cercosporin}, volume={6,077,995}, number={2000 June 20}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Upchurch, R. G. and Callahan, T. M. and Ehrenshaft, M.}, year={2000} }
 @misc{daub_ehrenshaft_jenns_2000, title={Isolated genes and proteins encoding resistance to photosensitizers}, volume={6,063,987}, number={2000 May 16}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Daub, M. E. and Ehrenshaft, M. and Jenns, A. E.}, year={2000} }
 @misc{daub_ehrenshaft_2000, title={The photoactivated Cercospora toxin cercosporin: Contributions to plant disease and fundamental biology}, volume={38}, ISSN={["1545-2107"]}, DOI={10.1146/annurev.phyto.38.1.461}, abstractNote={ 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. }, journal={ANNUAL REVIEW OF PHYTOPATHOLOGY}, author={Daub, ME and Ehrenshaft, M}, year={2000}, pages={461-+} }
 @article{bilski_li_ehrenshaft_daub_chignell_2000, title={Vitamin B-6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants}, volume={71}, ISSN={["1751-1097"]}, DOI={10.1562/0031-8655(2000)071<0129:SIPVBP>2.0.CO;2}, abstractNote={Abstract Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1–4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 × 107 M−1 s−1 for 1; 7.5 × 107 M−1 s−1 for 2, 6.2 ×107 M−1 s−1 for 3 and 7.5 × 107 M−1 s−1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased ∼10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased ∼10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1–4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.}, number={2}, journal={PHOTOCHEMISTRY AND PHOTOBIOLOGY}, author={Bilski, P and Li, MY and Ehrenshaft, M and Daub, ME and Chignell, CF}, year={2000}, month={Feb}, pages={129–134} }
 @article{ehrenshaft_bilski_li_chignell_daub_1999, title={A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis}, volume={96}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.96.16.9374}, abstractNote={TheCercospora nicotianae SOR1(singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygen-generating phototoxins. Although homologues toSOR1occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show thatSOR1is essential in pyridoxine (vitamin B6) synthesis inC. nicotianaeandAspergillus flavus,although it shows no homology to previously identified pyridoxine synthesis genes identified inEscherichia coli. Sequence database analysis demonstrated that organisms encode eitherSOR1orE. colipyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways forde novopyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance.}, number={16}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Ehrenshaft, M and Bilski, P and Li, MY and Chignell, CF and Daub, ME}, year={1999}, month={Aug}, pages={9374–9378} }
 @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{callahan_rose_meade_ehrenshaft_upchurch_1999, title={CFP, the putative cercosporin transporter of Cercospora kikuchii, is required for wild type cercosporin production, resistance, and virulence on soybean}, volume={12}, ISSN={["1943-7706"]}, DOI={10.1094/MPMI.1999.12.10.901}, abstractNote={ Many species of the fungal genus Cercospora, including the soybean pathogen C. kikuchii, produce the phytotoxic polyketide cercosporin. Cercosporin production is induced by light. Previously, we identified several cDNA clones of mRNA transcripts that exhibited light-enhanced accumulation in C. kikuchii. Targeted disruption of the genomic copy of one of these, now designated CFP (cercosporin facilitator protein), results in a drastic reduction in cercosporin production, greatly reduced virulence of the fungus to soybean, and increased sensitivity to exogenous cercosporin. Sequence analysis of CFP reveals an 1,821-bp open reading frame encoding a 65.4-kDa protein similar to several members of the major facilitator superfamily (MFS) of integral membrane transporter proteins known to confer resistance to various antibiotics and toxins in fungi and bacteria. We propose that CFP encodes a cercosporin transporter that contributes resistance to cercosporin by actively exporting cercosporin, thus maintaining low cellular concentrations of the toxin. }, number={10}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={Callahan, TM and Rose, MS and Meade, MJ and Ehrenshaft, M and Upchurch, RG}, year={1999}, month={Oct}, pages={901–910} }
 @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{jennings_ehrenshaft_pharr_williamson_1998, title={Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense}, volume={95}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.95.25.15129}, abstractNote={Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defenses. Here we show induction of mannitol production and secretion in the phytopathogenic fungusAlternaria alternatain the presence of host-plant extracts. Conversely, we show that the catabolic enzyme mannitol dehydrogenase is induced in a non-mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing mannitol of fungal origin.}, number={25}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Jennings, DB and Ehrenshaft, M and Pharr, DM and Williamson, JD}, year={1998}, month={Dec}, pages={15129–15133} }
 @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{ehrenshaft_jenns_daub_1995, title={TARGETED GENE DISRUPTION OF CAROTENOID BIOSYNTHESIS IN CERCOSPORA-NICOTIANAE REVEALS NO ROLE FOR CAROTENOIDS IN PHOTOSENSITIZER RESISTANCE}, volume={8}, ISSN={["1943-7706"]}, DOI={10.1094/MPMI-8-0569}, number={4}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={EHRENSHAFT, M and JENNS, AE and DAUB, ME}, year={1995}, pages={569–575} }
 @article{ehrenshaft_upchurch_1993, title={HOST PROTEIN(S) INDUCES ACCUMULATION OF THE TOXIN CERCOSPORIN AND MESSENGER-RNA IN A PHYTOPATHOGENIC STRAIN OF CERCOSPORA-KIKUCHII}, volume={43}, ISSN={["0885-5765"]}, DOI={10.1006/pmpp.1993.1043}, abstractNote={Cercospora kikuchii strain S2 produces the important pathogenicity factor cercosporin in infected soybeans and in potato dextrose broth. In contrast, S2 accumulates virtually no cercosporin in a complete medium (CM) broth, a medium containing salts, yeast extract and casamino acids. In this study we show that CM cultures supplemented with aqueous extracts of soybean leaves or meal accumulated between 5 to 65 fold higher levels of cercosporin than unsupplemented cultures. Autoclaving or treating extracts with proteinase K severely attenuated their stimulatory properties, indicating soybean protein was necessary for cercosporin production in CM. Other proteins tested failed to stimulate cercosporin synthesis. The inducing effect of leaf extracts increased as plants underwent reproductive development, but fell sharply at seed set. Conditioning experiments demonstrated that stimulation of cercosporin accumulation was not due to alteration of the medium by the extract. Accumulation of a transcript whose expression is strongly correlated with cercosporin synthesis was enhanced in the presence of native soybean meal extract, but decreased when extracts were autoclaved or digested. Accumulation of other cercosporin-related transcripts also increased in the presence of native extracts, but were more strongly induced by autoclaved or digested extracts. Our data suggest that C. kikuchii responds to soybean protein(s) with increased cercosporin production and changes in transcript accumulation.}, number={2}, journal={PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY}, author={EHRENSHAFT, M and UPCHURCH, RG}, year={1993}, month={Aug}, pages={95–107} }
 @article{ehrenshaft_upchurch_1991, title={Isolation of light-enhanced cDNAs of Cercospora kikuchii}, volume={57}, number={9}, journal={Applied and Environmental Microbiology}, author={Ehrenshaft, M. and Upchurch, R. G.}, year={1991}, pages={2671} }