1.8.7S
MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE HAUSTORIAL H+-ATPase OF THE BIOTROPHIC RUST FUNGUS UROMYCES FABAE

C STRUCK, O ROMMEL, M HAHN and K MENDGEN

Universitšt Konstanz, D-78457 Konstanz, Germany

Background and objectives
The biotrophic way of life requires highly specialized adaptations of the parasites to the host organisms - particularly in nutrient-uptake mechanisms. Based on ultrastructural and cytochemical studies, it has been postulated that haustoria play a key role in nutrient uptake from infected plant cells driven by active transport processes across the host-parasite interface. The energy force for such transport is the activity of the plasma membrane H+-ATPase generating an electrochemical gradient across the plasma membrane. Recently, the activity of the vanadate-sensitive plasma membrane H+-ATPase was found to be several-fold higher in isolated rust haustoria than in fungal uredospores and germ tubes [1]. To study the molecular basis and mechanism of nutrient uptake by the plant parasitic rust fungi, we have isolated the gene (Uf-PMA1) encoding the plasma membrane H+-ATPase from Uromyces fabae, and functionally expressed the enzyme in yeast.

Results and conclusions
The open reading frame of a haustorial cDNA Uf-PMA1 clone encodes a protein of 962 amino acids, with a predicted molecular mass of 104,915 Da. Surprisingly, the PMA1-encoded rust protein is more similar to H+-ATPases from plants (55% identity) than that from ascomycetous fungi (36%). Our data indicate that the enzyme of the rust fungi is encoded by a single gene [2]. To study the regulation of the enzyme during rust development we analysed both the Uf-PMA1 cDNA and a mutant derivative (D76) deleted for the C-terminal 76 amino acids. When the rust PMA1 cDNA was expressed in Saccharomyces cerevisiae (the yeast strain used, YAK2, lacks both chromosomal H+-ATPase genes and was constructed in the laboratory of M. Boutry, Louvain-La-Neuve, Belgium), both the wild-type enzyme and the D76 mutant were able to support growth of the yeast strain lacking its own H+-ATPases. Western blot analysis using rust-specific antibodies against synthetic peptides of the H+-ATPase confirmed that the rust enzyme was expressed in the transformed yeast strain. Compared to the wild type, the D76 mutant enzyme displayed increased affinity to ATP, a higher vanadate sensitivity and a more alkaline pH optimum. These results indicate that the C-terminal region of the rust enzyme exhibits autoregulatory properties. Together with the identification of a haustorium-specific amino acid-proton co-transporter (see abstract of M. Hahn et al.), our results present for the first time molecular evidence for the mechanism of nutrient uptake by the rust haustoria.

References
1. Struck C, Hahn M, Mendgen K, 1996. Fungal Genetics and Biology 20, 30-35.
2. Struck C, Siebels C, Rommel O et al., 1998. Molecular Plant-Microbe Interactions (in press).