RESEARCH ARTICLE De Novo RNA Sequencing and Transcriptome Analysis of Colletotrichum gloeosporioides ES026 Reveal Genes Related to Biosynthesis of Huperzine A Guowei Zhang 1☯ , Wenjuan Wang 1☯ , Xiangmei Zhang 1 , Qianqian Xia 1 , Xinmei Zhao 2 , Youngjoon Ahn 3 , Nevin Ahmed 1,4 , Andreea Cosoveanu 5 , Mo Wang 1 , Jialu Wang 1 , Shaohua Shu 1 * 1 College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China, 2 College of Life Sciences, Xinyang Normal University, Xinyang, China, 3 College of Agriculture and Life Sciences, Seoul National University, Seoul 151–742, Republic of Korea, 4 Department of Plant Protection, Faculty of Agriculture, Benha University, Benha, Egypt, 5 Phytopathology Unit, Faculty of Biology, University of La Laguna, La Laguna, Tenerife, Spain ☯ These authors contributed equally to this work. * shushaohua@mail.hzau.edu.cn Abstract Huperzine A is important in the treatment of Alzheimer’s disease. There are major chal- lenges for the mass production of huperzine A from plants due to the limited number of huperzine-A-producing plants, as well as the low content of huperzine A in these plants. Various endophytic fungi produce huperzine A. Colletotrichum gloeosporioides ES026 was previously isolated from a huperzine-A-producing plant Huperzia serrata, and this fungus also produces huperzine A. In this study, de novo RNA sequencing of C. gloeosporioides ES026 was carried out with an Illumina HiSeq2000. A total of 4,324,299,051 bp from 50,442,617 high-quality sequence reads of ES026 were obtained. These raw data were as- sembled into 24,998 unigenes, 40,536,684 residues and 19,790 genes. The majority of the unique sequences were assigned to corresponding putative functions based on BLAST searches of public databases. The molecular functions, biological processes and biochemi- cal pathways of these unique sequences were determined using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) assignments. A gene encoding cop- per amine oxidase (CAO) (unigene 9322) was annotated for the conversion of cadaverine to 5-aminopentanal in the biosynthesis of huperzine A. This gene was also detected in the root, stem and leaf of H. serrata. Furthermore, a close relationship was observed between expression of the CAO gene (unigene 9322) and quantity of crude huperzine A extracted from ES026. Therefore, CAO might be involved in the biosynthesis of huperzine A and it most likely plays a key role in regulating the content of huperzine A in ES026. PLOS ONE | DOI:10.1371/journal.pone.0120809 March 23, 2015 1 / 16 a11111 OPEN ACCESS Citation: Zhang G, Wang W, Zhang X, Xia Q, Zhao X, Ahn Y, et al. (2015) De Novo RNA Sequencing and Transcriptome Analysis of Colletotrichum gloeosporioides ES026 Reveal Genes Related to Biosynthesis of Huperzine A. PLoS ONE 10(3): e0120809. doi:10.1371/journal.pone.0120809 Academic Editor: Brett Neilan, University of New South Wales, AUSTRALIA Received: May 12, 2014 Accepted: February 5, 2015 Published: March 23, 2015 Copyright: © 2015 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All sequencing files are available from the NCBI database (accession number SRP038110) http://www.ncbi.nlm.nih.gov/bioproject/? term=PRJNA238599. Funding: Funding for this work came from the National Natural Science Foundation of China (Grant No. 81202870, http://www.nsfc.gov.cn/) and the Ministry of Science and Technology of the People's Republic of China through the Project of International Scientific and Technological Cooperation between China and South Korea (Grant No. 2011DFA31290, http://www.most.gov.cn/). The funders had no role in