Tools and techniquesA new transformant selection system for the gray mold fungus Botrytis cinerea based on the expression of fenhexamid-insensitive ERG27 variants

- The Botrytis cinerea strain B05.10 is sensitive to the fungicide fenhexamid.
- Expression of insensitive ERG27 variants confers resistance to fenhexamid.
- Expression of insensitive ERG27 variants does not result in secondary phenotypes.
- Expressing vectors conferring fenhexamid resistance (fenR) were generated.
- The new transformant selection system allows for the generation of triple mutants.

The gray mold fungus Botrytis cinerea features a wide host range and causes severe economic losses, making it an important object for molecular research. Thus far, genetic modification of the fungus mainly is relied on two selection systems (nourseothricin and hygromycin), while other selection systems hold significant disadvantages. To broaden the spectrum of available molecular tools, a new selection system based on the cheap and widely used fungicide fenhexamid (hydroxyanilide group) was established. Fenhexamid specifically targets the 3-ketoreductase ERG27 from the ergosterol biosynthesis pathway. We generated a set of expression vectors suitable for deletion or expression of genes of interest (GOIs) in B. cinerea based on fenhexamid-insensitive ERG27 variants. Expression of BcERG27F412I and Fusarium fujikuroi ERG27 in the sensitive B. cinerea strain B05.10 causes resistance towards fenhexamid (fenR) and allows for the selection of transformants and their genetic purification. A modified split-marker approach facilitates the site-specific integration and expression of GOIs at the bcerg27 locus. No undesired secondary phenotypes regarding virulence, stress responses, the formation of reproductive structures or conidial germination were observed in strains expressing fenhexamid-insensitive ERG27 variants. Thus, the fenR system represents a third reliable selection system for genetic modifications of fenhexamid-sensitive B. cinerea strains.

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