@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_noar_daub_2015, title={Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance}, volume={10}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0140676}, abstractNote={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.}, number={10}, journal={PLOS ONE}, author={Beseli, Aydin and Noar, Roslyn and Daub, Margaret E.}, year={2015}, month={Oct} }
 @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} }