A new vector system for targeted integration and overexpression of genes in the crop pathogen .

BACKGROUND

Besides their ability to produce several interesting bioactive secondary metabolites, members of the species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation.

RESULTS

To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in through -mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)Cys transcriptional factor controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin.

CONCLUSION

By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (: ) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the species complex, and is obtainable from Addgene catalog #133094.