1.2.23
STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE Mlo GENE

A DEVOTO, P PIFFANELLI, R PANSTRUGA and P SCHULZE-LEFERT

The Sainsbury Laboratory, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Background and objectives
Recessive alleles of the Mlo locus in barley mediate a broad, non-race-specific resistance reaction to the powdery mildew fungus. Moreover, under pathogen-free conditions, mlo plants exhibit a spontaneous leaf cell death phenotype. The HvMlo gene has been isolated and sequenced [1] and a large number of Mlo homologues sharing common structural features with the barley gene have been identified in various other monocots and in Arabidopsis thaliana. The Mlo gene was predicted to encode an integral membrane protein containing seven transmembrane-spanning domains.These features are structurally reminiscent of the large family of mammalian G-protein coupled receptors. As the number and orientation of membrane-spanning helices is an important indicator for the function of an integral membrane protein, a rigorous experimental examination of the deduced Mlo membrane topology is carried out. We tested the topology predictions for Mlo by scanning N-glycosylation mutagenesis [2].

If Mlo represents a founder of plant GPCRS, it would be expected to reside in the plant plasma membrane since this class of molecules is known to relay extracellular signals into amplified intracellular responses. In order to localize Mlo within the cell, both a biochemical and a GFP-based in vivo approach have been undertaken.

Results and conclusions
The barley Mlo cDNA has been cloned in an expression vector. Modification of sequences adjacent to the initiator ATG according to the Kozak consensus enabled high-level expression of an in vitro transcribed/translated Mlo protein of the expected molecular weight. A series of mutants in which single N-acceptor sites are placed in each of the predicted lumenal and cytoplasmic loops is currently being generated by overlap extension using a PCR approach (SOE). Accessibility of the acceptor site for the glycosyl-transferase will be tested in the presence of dog pancreas microsomes. Expected shifts in the molecular weight will be visualized by SDS-PAA size separation.

Various methods of subcellular fractionation of barley leaf cells have been tested and optimized. Highly pure plasma membrane vesicles have been purified by a two-phase partitioning protocol. Polyclonal antibodies directed against different domains of the Mlo protein have been raised and are currently tested on immunoblots containing various subcellular protein fractions. We have obtained the first evidence for specific Mlo immunoblot signals in microsomal fractions. In vivo localization of Mlo-GFP fusion proteins by confocal laser microscopy in barley leaf epidermal cells has also been undertaken.

References
1. Boschges R, Holiricher K, Panstruga R et al., 1997. Cell 88, 695-709.
2. Popov M, Tam LY, Li J, Reithmeier RAF, 1997. Journal of Biological Chemistry 272, 18325-18332.