Abstract
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.
Highlights
The purpose of this work is to identify genes encoding hypothetical proteins that may play a role in resistance of Cercospora fungi to the photoactivated toxin cercosporin produced by these fungi for infection of host plants
Our work has focused on identifying cercosporin-autoresistance genes from Cercospora species, as cercosporin is almost universally toxic to cells due to its production of reactive oxygen species (ROS) [2]
We screened 13 expressed sequence tags (ESTs) from the library that are homologous to genes encoding hypothetical proteins
Summary
The purpose of this work is to identify genes encoding hypothetical proteins that may play a role in resistance of Cercospora fungi to the photoactivated toxin cercosporin produced by these fungi for infection of host plants. Mutants deficient in cercosporin production are significantly less virulent on their host plants, engineering cercosporin-resistant crop plants via expression of cercosporin resistance genes may be an ecologically friendly method for controlling these damaging diseases. Production of 1O2 and other ROS leads to peroxidation of cell membrane lipids in host plants and can damage nucleic acids, proteins and lipids in the target cells [1]. Cercospora fungi are immune to cercosporin toxicity, they may be a source of genes for engineering crop resistance [3], and serve as a model for understanding cellular resistance to 1O2
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