Genes differentially expressed under naphthoquinone-producing conditions in the entomopathogenic fungus Ophiocordyceps unilateralis

The ant-pathogenic fungus Ophiocordyceps unilateralis BCC1869 produces six naphthoquinone (NQ) derivatives. These NQs can be found in fungal-infected ants or produced in culture. Also, the NQs have antibacterial, anticancer, and antimalarial activities and are red pigments with potential for use as natural colorants. Suppressive subtractive hybridization identified genes that were expressed under NQ–producing conditions but not under nonproducing conditions. On potato dextrose agar, the mycelia produced red pigments and secreted them into the medium and as droplets on top of the colony. High-performance liquid chromatography analysis indicated that the red pigment was predominantly erythrostominone with small amounts of its derivatives. For suppressive subtractive hybridization, the cDNA from O. unilateralis cultures on complete medium agar cultures (lacking NQs) were subtracted from those on potato dextrose agar (which produce and secrete NQs). Sixty-six unique expressed sequence tags (ESTs) were identified and include five transporter genes, two transcriptional regulator genes, and several genes in secondary metabolism and biodegradation. The transporter genes include an ATP-binding cassette transporter gene OuAtr1 and a major facilitator superfamily transporter gene OuMfs1. Expression of selected ESTs was further validated using quantitative reverse transcription PCR. Gene expression result indicates that OuAtr1 and OuMfs1 were dramatically upregulated (136- and 29-fold increase, respectively) during the NQ–producing stage compared with the NQ–nonproducing stage. Upregulation of other genes was also detected. This EST collection represents the first group of genes identified from this potential biocontrol agent and includes candidate genes for production and secretion of the red NQs. Roles of these genes could be further determined using a functional analysis.