CLONING AND SEQUENCING OF A MAJOR MEMBRANE PROTEIN FROM CHLORANTE (ASTER YELLOWS) PHYTOPLASMA
MF CLARK1, DL DAVIES1 and DJ BARBARA2
1Entomology and Plant Pathology Department, HRI, East Malling, Kent ME19 6BI, UK; 2Plant Pathology and Microbiology Department, Horticulture Research International, Wellesbourne, Warwickshire CV35 9EF, UK
Background and objectives
Purification and aetiological studies at HRI-East Malling and elsewhere have shown that for most phytoplasmas studied, a single immunodominant protein comprises the major proportion of the proteins in semi-purified cell membrane preparations. Furthermore, ISEM studies  suggest that at least part of this protein is exposed on the outside of the phytoplasma cell membrane and is, therefore, a strong candidate for being involved in the interaction of the pathogen with its host. We report on progress in studies to clone and characterize this membrane protein.
Results and conclusions
No significant homologies were found with the complete ORF (or its putative translation product), but this was identified as the gene encoding the MMP because (i) there was complete agreement between the predicted amino-acid sequence and the several short segments derived through the original microsequencing of the cleavage products; and (ii) expressed protein reacted with antibodies against the MMP.
The predicted properties of the translation product were strongly suggestive of a membrane-bound protein of the correct size situated on the exterior of the phytoplasma cell. An N-terminal hydrophobic bacterial export domain with associated cleavage point would leave a 201-aa, largely hydrophilic, mature protein anchored on the exterior of the cell by a transmembrane domain close to the C terminal, with only a short terminal sequence within the cell.
In conventional bacterial expression systems the protein appears to be toxic, and we are now working to overcome this prior to studying the protein's functions and its possible involvement in host-pathogen cellular interactions.