Similarly, there are only two possible configurations for the introduced Idasanutlin DNA – as a single copy or as multiple copies (Turgeon et al., 2010). In this study, PCR analysis clearly demonstrated the presence of nearly consistent hph and

amp genes in plasmid pSH75 and transformants but absence of the two genes in wild-type B. eleusines. It appears that PCR can confirm the genome integration rapidly, but may not detect multiple copies of insertion. Southern blot analysis may be used for further verification of single insertion or stability of the transformants. Biosynthesis of ophiobolin compounds as secondary metabolites can be a complex process and would require many enzymatic steps. Why fungi produce ophiobolin compounds remains unknown and the molecular pathway involved is not yet clear. Therefore, understanding the biosynthetic pathway in the filamentous fungus B. eleusines may help in improving ophiobolin yields via genetic engineering of the organism. REMI has been extensively used to tag pathogenicity genes or to study gene functions in numerous fungal pathogens (Bolker et al., 1995; Jin et al., 2005; Zhou et al., 2007). In addition, it can be used to clone the genes related to mutant characteristics by plasmid rescue in Eschericha coli (Kahmann & Basse, 1999) or by thermal asymmetric interlaced-(TAIL) PCR (Weld et al., Small molecule library 2006).

Therefore, REMI is an effective approach for isolating genes from fungal mutants, especially for those with little known genetic background. Screening and identifying ophiobolin A-deficient mutants of B. eleusines using REMI may lead to cloning the genes that influence or are potentially involved in the biosynthesis of ophiobolin compounds

using TAIL-PCR and/or plasmid rescue in E. coli. This information may be helpful in studying and unveiling the mechanism of ophiobolin production in filamentous fungi. In conclusion, a transformation system for B. eleusines has been developed using REMI. Screening and identification of ophiobolin A-deficient mutants were successively completed using bioassays coupled with HPLC and PCR techniques for confirmation. One stable ophiobolin A-deficient mutant was obtained. These techniques are relatively simple and provide a new approach for further studying the mechanism of microbial-based ophiobolin production. They may also help to improve Cytidine deaminase the yield of toxin production by transferring genes responsible for up-regulation of the biosynthetic pathways of B. eleusines. We thank Dr Gary Peng, Saskatoon Research Centre, Agriculture and Agri-Food Canada, for reviewing this manuscript and providing comments. We also thank Dr Sheng Qiang (Nanjing Agricultural University, China) and Dr Shiwen Huang (China National Rice Research Institute, China) for providing plasmid pSH75 and Rhizoctoni solani AG-1-IA, respectively. This work was financially supported by the National Natural Science Foundation of China (No.