FUNCTIONAL EXPRESSION OF SACCHAROMYCES-CEREVISIAE CYP51A1 ENCODING LANOSTEROL-14-DEMETHYLASE IN TOBACCO RESULTS IN BYPASS OF ENDOGENOUS STEROL BIOSYNTHETIC-PATHWAY AND RESISTANCE TO AN OBTUSIFOLIOL-14-DEMETHYLASE HERBICIDE INHIBITOR

Nicotiana tabacum protoplasts have been transformed by Agrobacterium t umefaciens containing a T-DNA in which the gene CYP51A1 encoding lanos terol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two tra nsformants strongly expressed the LAN14DM as shown by Northern and Wes tern experiments. These trans genic calli were killed by LAB 170250F ( LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-dem ethylase (OBT14DM) and LAN14DM) but were resistant to gamma-ketotriazo le (gamma-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. Howe ver, these transgenic calli were killed by mixtures of gamma-kt plus f ungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or f lusilazole which alone were not effective. Further analysis of the tra nsgenic calli grown in the presence of gamma-kt showed that their Delt a(5)-sterol content was close to that of untreated control calli obtai ned from protoplasts transformed with control plasmid; this is in agre ement with evidence that the LAN14DM expressed from the transgene coul d bypass the blocked OBT14DM by using the plant substrate obtusifoliol . In contrast, control calli when treated with gamma-kt, displayed a s terol content strongly enriched in 14 alpha-methyl sterols and depress ed in physiological Delta(5)-sterols. When the transgenic calli were c ultured in mixtures of gamma-kt and LAN14DM inhibitors sterol composit ions enriched in 14 alpha-methyl sterols were obtained, reflecting a s trong inhibition of both 'endogenous' OBT14DM and 'exogenous' LAN14DM. Taken together these results show that in tobacco calli transformed w ith CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues cr eates a bypass of the sterol biosynthetic pathway at the 14-demethylas e level when this latter is blocked by an OBT14DM herbicide inhibitor.