Cloning of b-tubulin and succinate dehydrogenase genes from Uromyces fabae and establishing selection conditions for their use in transformation

The obligate biotrophic nature of rust fungi calls for an in planta selection scheme as a means of developing a rust transformation technology. We show that the fungicides benomyl (used as its formulated product benlate) and carboxin suppress morphogenesis of the rust fungus Uromyces fabae in vitro and disease in planta, the latter without affecting the health of the host. The limits of their applicability were determined regarding concentration, method of application and optimal time intervals of treatment. Besides procedures for selection, a stable transformation system will also need to include genetic markers allowing to enrich for transformed cells within a large background of untransformed cells. Since the molecular targets of benlate and carboxin had been identified as b-tubulin and succinate dehydrogenase, respectively, the corresponding genes (Uf-TBBI and Uf-SUCDHI) were cloned and characterized. Molecular phylogenies demonstrate that both are typical homologs to those of other Basidiomycota. RT-PCR analysis confirmed that both genes are constitutively expressed in all developmental stages of the mitotic uredospore multiplication cycle. Since homologs of Uf-TBB1 and Uf-SUCDH1 have been successfully used as selection markers in other fungal systems, they provide valuable tools to develop additional corner stones of a stable transformation system for rust fungi.