@article{thomas_herrero_eng_gomaa_gillikin_noar_beseli_daub_2020, title={Engineering Cercospora disease resistance via expression of Cercospora nicotianae cercosporin-resistance genes and silencing of cercosporin production in tobacco}, volume={15}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0230362}, DOI={10.1371/journal.pone.0230362}, abstractNote={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.}, number={3}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Thomas, Elizabeth and Herrero, Sonia and Eng, Hayde and Gomaa, Nafisa and Gillikin, Jeff and Noar, Roslyn and Beseli, Aydin and Daub, Margaret E.}, editor={Wilson, Richard A.Editor}, year={2020}, month={Mar}, pages={e0230362} }
 @article{beseli_amnuaykanjanasin_herrero_thomas_daub_2015, title={Membrane transporters in self resistance of Cercospora nicotianae to the photoactivated toxin cercosporin}, volume={61}, ISSN={0172-8083 1432-0983}, url={http://dx.doi.org/10.1007/s00294-015-0486-x}, DOI={10.1007/s00294-015-0486-x}, abstractNote={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.}, number={4}, journal={Current Genetics}, publisher={Springer Science and Business Media LLC}, author={Beseli, Aydin and Amnuaykanjanasin, Alongkorn and Herrero, Sonia and Thomas, Elizabeth and Daub, Margaret E.}, year={2015}, month={Apr}, pages={601–620} }
 @article{coelho souza_herrero_maffia_daub_2014, title={Methods for Cercospora coffeicola protoplast isolation and genetic transformation with the green fluorescent protein}, volume={139}, ISSN={["1573-8469"]}, DOI={10.1007/s10658-013-0301-9}, abstractNote={Cercospora coffeicola is the causal agent of brown eye spot on coffee leaves. Although the disease has significant importance, few molecular studies have been done with C. coffeicola. Here we report a protocol for isolating protoplasts as well as development of a genetic transformation system using Green Fluorescent Protein. High yields of protoplasts (≈108/ml) were obtained from mycelial cultures from five isolates of C. coffeicola. One isolate was transformed with a vector encoding hygromycin resistance and Green Fluorescent Protein. Out of 43 hygromycin-resistant transformants obtained, Green Fluorescent Protein was highly expressed in one (2.3 %).}, number={2}, journal={EUROPEAN JOURNAL OF PLANT PATHOLOGY}, author={Coelho Souza, Andre Gomes and Herrero, Sonia and Maffia, Luiz Antonio and Daub, Margaret Elizabeth}, year={2014}, month={Jun}, pages={235–238} }
 @misc{daub_herrero_chung_2013, title={Reactive Oxygen Species in Plant Pathogenesis: The Role of Perylenequinone Photosensitizers}, volume={19}, ISSN={["1557-7716"]}, DOI={10.1089/ars.2012.5080}, abstractNote={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.}, number={9}, journal={ANTIOXIDANTS & REDOX SIGNALING}, author={Daub, Margaret E. and Herrero, Sonia and Chung, Kuang-Ren}, year={2013}, month={Sep}, pages={970–989} }
 @article{herrero_gonzalez_gillikin_velez_daub_2011, title={Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis}, volume={76}, ISSN={["1573-5028"]}, DOI={10.1007/s11103-011-9777-x}, abstractNote={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.}, number={1-2}, journal={PLANT MOLECULAR BIOLOGY}, author={Herrero, Sonia and Gonzalez, Eugenia and Gillikin, Jeffrey W. and Velez, Heriberto and Daub, Margaret E.}, year={2011}, month={May}, pages={157–169} }
 @article{herrero_daub_2007, title={Genetic manipulation of Vitamin B-6 biosynthesis in tobacco and fungi uncovers limitations to up-regulation of the pathway}, volume={172}, ISSN={["0168-9452"]}, DOI={10.1016/j.plantsci.2006.11.011}, abstractNote={Transgenic expression of vitamin biosynthetic genes has been investigated for over-production of these dietary supplements in microorganisms and plants. In plants, successful efforts have been reported with Vitamins A, C, E and B-9, however information is lacking for other vitamins. Vitamin B-6 is an essential cofactor for numerous enzymatic reactions, and has also been shown to be a potent antioxidant involved in protecting phytopathogenic Cercospora fungi from their own toxin, cercosporin. In this report, we transformed and expressed two Vitamin B-6 biosynthetic genes (PDX1 and PDX2) isolated from Cercospora nicotianae in cercosporin-sensitive organisms, tobacco and the fungal species Aspergillus flavus and Neurospora crassa. Our goal was to determine if Vitamin B-6 levels could be increased by constitutive expression of these genes, and if over-production confers resistance to oxidative stresses induced by cercosporin and salinity stress. Elevated Vitamin B-6 levels were observed in one tobacco line. For other lines evaluated in this work, expression of PDX1 and PDX2 in transgenic organisms did not result in a significant increase in Vitamin B-6 content over controls. Analysis of gene expression in tobacco indicated that the lack of elevated B-6 content was not due to lack of enzymatic activity, but to down-regulation of the endogenous tobacco genes compounded with limited transgene expression. The single line with elevated B-6 levels had higher expression of both the PDX1 and PDX2 transgenes compared to the other lines, and the observed increase on Vitamin B-6 was correlated with higher enzyme activity. Consistent with our inability to elevate cellular B-6 levels, only small changes were observed in the response to either cercosporin or high salt, and most transgenic individuals were as susceptible as controls. Compared to tobacco lines transformed to express either PDX1 or PDX2 alone, half of the transgenic tobacco lines expressing both genes were impaired in seed germination and initial growth. However no correlation was observed between the observed phenotype and Vitamin B-6 levels in seeds. This is the first report on genetic engineering to manipulate the Vitamin B-6 pathway in plants. Our results suggest that genetic manipulation of the Vitamin B-6 biosynthetic pathway is possible but is limited by regulation of endogenous genes.}, number={3}, journal={PLANT SCIENCE}, author={Herrero, Sonia and Daub, Margaret E.}, year={2007}, month={Mar}, pages={609–620} }
 @article{herrero_amnuaykanjanasin_daub_2007, title={Identification of genes differentially expressed in the phytopathogenic fungus Cercospora nicotianae between cercosporin toxin-resistant and -susceptible strains}, volume={275}, ISSN={["1574-6968"]}, DOI={10.1111/j.1574-6968.2007.00903.x}, abstractNote={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.}, number={2}, journal={FEMS MICROBIOLOGY LETTERS}, author={Herrero, Sonia and Amnuaykanjanasin, Alongkorn and Daub, Margaret E.}, year={2007}, month={Oct}, pages={326–337} }
 @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{herrero_rufty_daub_2001, title={Molecular determinants influencing the inheritance of transgenic virus resistance in segregating tobacco families transformed with the nucleocapsid gene of tomato spotted wilt virus}, volume={7}, ISSN={["1572-9788"]}, DOI={10.1023/A:1011381412397}, number={2}, journal={MOLECULAR BREEDING}, author={Herrero, S and Rufty, RC and Daub, ME}, year={2001}, pages={131–139} }
 @article{herrero_rufty_barker_1996, title={Evaluation of tobacco germ plasm for resistance to the tobacco cyst nematode, Globodera tabacum solanacearum}, volume={80}, ISSN={["1943-7692"]}, DOI={10.1094/PD-80-0061}, abstractNote={Twenty-four tobacco genotypes were evaluated in the greenhouse for resistance to a North Carolina isolate of the tobacco cyst nematode Globodera tabacum subsp. solanacearum. The experimental design was a split-plot with eight replications. The experiment was repeated three times. Genotypes were assigned to whole plots, and nematode infestation levels (0 or 10,000 eggs per 1,300 cm 3 of soil) were assigned to subplots. Fresh stalk and root weights of all plants were recorded. Two new variables were created based on these measurements: percent stalk weight [%SW = (SW of infected plant + SW of uninfected plant) x 100] and percent root weight [%RW = (RW of infected plant + RW of uninfected plant) x 100]. Nematode reproduction, measured as number of cysts and eggs per pot, and reproduction factor (final number of eggs + initial number of eggs), was determined for each genotype. Significant differences (P ≤ 0.05) were observed among tobacco genotypes for nematode reproduction. Low levels were detected in the cultivars Burley 21, PD 4, VA 81, NC 567, Speight G-80, Kutsaga Mammoth 10, Kutsaga 110, and in the flue-cured breeding lines Cyst 913 and 9025-1. Differences in vegetative growth among genotypes grown in nematode-infested soil were detected only between those with the highest and lowest %SW and %RW. Genotype shoot and root weight were not correlated with nematode reproduction. Nevertheless, results from these experiments indicate that nematode reproductive parameters may be used to evaluate tobacco germ plasm for resistance to G. t. subsp. solanacearum in the greenhouse.}, number={1}, journal={PLANT DISEASE}, author={Herrero, S and Rufty, R and Barker, KR}, year={1996}, month={Jan}, pages={61–65} }