An immunodominant membrane protein gene from the Western X-disease phytoplasma is distinct from those of other phytoplasmas.
Membrane proteins mediate several important processes, including attachment, in several Mollicute species. Phytoplasmas are non-culturable plant pathogenic mollicutes that are transmitted in a specific manner by certain phloem-feeding insect vectors. Because it is likely that phytoplasma membrane proteins are involved with some aspect of the transmission process, their identification, isolation and characterization are important first steps in understanding phytoplasma transmission. A 32 kDa immunodominant protein (IDP) from the Western X-disease (WX) phytoplasma was purified from infected plants by immunoprecipitation using monoclonal antibodies, and two peptides from a tryptic digest were sequenced. PCR primers designed from these sequences amplified a 145 bp product which hybridized with WX-related phytoplasmas in Southern blots. This PCR product was used to identify a 2.5 kbp ECO:RI-HIN:dIII fragment that was cloned and sequenced. A complete 864 bp ORF (idpA) was identified for which the putative translation product contained both of the tryptic digest peptide sequences that were used to design the PCR primers. Analysis of the predicted IdpA sequence indicated two transmembrane domains but no cleavage point. The amino acid sequence had no significant homology with other known phytoplasma IDP genes. The idpA ORF was cloned into an Escherichia coli expression vector and a fusion protein of the predicted size was identified in Western blots using a WX-specific antiserum. A rabbit polyclonal antiserum was prepared to the purified expression protein and this reacted with both the E. coli-expressed and native WX phytoplasma proteins. This newly identified WX IDP (IdpA) is distinct from other known mollicute membrane proteins.
Department of Plant Pathology, University of California at Davis, Davis, CA 95616, USA1.
Cheryl L
CL
Blomquist
Horticulture Research International, Wellesbourne, Warwickshire CV35 9EF, UK2.
Dez J
DJ
Barbara
Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, UK3.
David L
DL
Davies
Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, UK3.
Michael F
MF
Clark
Department of Plant Pathology, University of California at Davis, Davis, CA 95616, USA1.
Bruce C
BC
Kirkpatrick
D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D003001:Cloning, Molecular; D016056:Immunodominant Epitopes / Q000276:immunology*; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D008973:Tenericutes / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins; C435308:immunodominant membrane protein A, phytoplasma