1. ¹Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany
2. ²Department of Plant Biology, University of Minnesota, Saint Paul, MN, USA
3. ³Institute for Microbiology, Friedrich-Schiller-University, Jena, Germany
4. ⁴Chair for Natural Product Chemistry, Friedrich-Schiller-University, Jena, Germany

Correspondence: C Hertweck, Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Beutenbergstr. 11a, Jena 07745, Germany. E-mail: christian.hertweck@hki-jena.de

Received 1 November 2007; Revised 31 January 2008; Accepted 31 January 2008; Published online 28 February 2008.

Abstract

The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of -tubulin sequences in the kingdom Fungi. A total of 49 new -tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism—mutualism shift in insensitive fungi.