Isolation of the gene encoding an immunodominant membrane protein of the apple proliferation phytoplasma, and expression and characterization of the gene product.
An immunodominant membrane protein (IMP) of the apple proliferation (AP) phytoplasma was detected in preparations from AP-diseased periwinkle plants using monoclonal and polyclonal antibodies to the AP agent. Following isolation from Western blots and partial sequencing, degenerate oligonucleotides derived from the IMP sequence were used as probes to identify a DNA fragment containing the ORF encoding the IMP. Complete sequencing and subsequent analysis of the cloned DNA fragment revealed the presence of two ORFs, predicted to encode proteins with molecular masses of 25 kDa (P-318A) and 19 kDa (P-318B). Whilst database searches failed to assign a possible function to P-318A, analysis of P-318B predicted an amphiphilic membrane protein with a positively charged N-terminal portion, followed by a hydrophobic segment forming an alpha-helix, and a hydrophilic C-terminal part located outside of the cell. The amphiphilic nature of P-318B was confirmed by its solubility in Triton X-114. The gene encoding P-318B was expressed in Escherichia coli and the resulting protein was used to immunize rabbits. The antiserum obtained reacted specifically with P-318B. The same protein was also detected by an antiserum raised against antigen preparations from AP-diseased plants. The P-318B antiserum did not react with antigen preparations from plants infected with the closely related pear decline phytoplasma. However, in Southern hybridization studies, the gene encoding the IMP hybridized to genomic fragments of the pear decline and European stone fruit yellows phytoplasmas. It also showed significant sequence similarity to a gene encoding an antigenic membrane protein of the sweet potato witches' broom phytoplasma, but not to a gene encoding a major immunogenic membrane protein of an aster yellows group phytoplasma. Since it appears that most phytoplasmas possess a major immunogenic membrane protein which may have a function in pathogenesis, this work may be a basis for further studies on fundamental aspects of host-pathogen interactions. It also describes a new approach to obtain suitable immunogens to produce specific antibodies for detection and characterization of the non-culturable phytoplasmas.
Berg
Michael
M
Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, D-69221 Dossenheim, Germany.
Davies
David L
DL
Horticulture Research International, East Malling, Kent ME19 6BJ, UK.
Clark
Michael F
MF
Horticulture Research International, East Malling, Kent ME19 6BJ, UK.
Vetten
H Joseph
HJ
Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenvirologie, Mikrobiologie und biologische Sicherheit, D-38104 Braunschweiq, Germany.
Maie
Gernot
G
Deutsches Krebsforschungszentrum, Abteilung molekulare Biologie der Mitose, D-69120 Heidelberg, Germany.
Marcone
Carmine
C
Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, D-69221 Dossenheim, Germany.
Seemüller
Erich
E
Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, D-69221 Dossenheim, Germany.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D015153:Blotting, Western; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D016056:Immunodominant Epitopes / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D010944:Plants / Q000382:microbiology*; D011817:Rabbits; D019685:Rosales / Q000382:microbiology; D017422:Sequence Analysis, DNA; D008973:Tenericutes / Q000235:genetics* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D016056:Immunodominant Epitopes