1.5.9
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
Despite many attempts in laboratories around the world, no phytoplasma has yet been grown in in vitro culture, suggesting that their hosts, both insect and plant, may act as more than just convivial places to grow. We postulate that phytoplasmas require for growth an intimate association between the cellular membranes of the host and of the pathogen, and our studies are directed towards investigating the molecular basis of any such association.

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 [1] 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
Using a chlorante aster yellows isolate, micro-sequencing of the N-terminals of intact immunopurified protein and of enzymatic or chemical cleavage products from the protein allowed us to design degenerate primers (used in both inverse and standard PCR) which eventually led to our cloning two segments of phytoplasma genomic DNA. The major ORF of one of these showed very high homology with a bacterial acetate kinase; expressed protein from this ORF did not react with antibodies against the major membrane protein (MMP) and this ORF was not studied further. The second 2.2-kbp DNA segment contained one complete and two partial ORFs. Database searches found no homologies with the smaller partial ORF, but the larger one was very similar to the C-terminal 50% of a bacterial chaperonin (GroEL).

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.

References
1. Milne RG, Ramasso E, Lenzi R et al., 1995. Netherlands Journal of Plant Pathology 101, 57-67.