Isolation of the gene encoding an immunodominant membrane protein of the apple proliferation phytoplasma, and expression and characterization of the gene product

Abstract

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 a-helix, and a hydrophilic C-terminal part located outside of the cell. The amphiphilic nature of P-318B was confirmed by its solubility in T r i t o n 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 APdiseased 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.

Comparative Genome Analysis of “CandidatusPhytoplasma australiense” (Subgrouptuf-Australia I;rp-A) and “Ca. Phytoplasma asteris” Strains OY-M and AY-WB

“…One indication of a functional sec-dependent pathway is the presence of proteins with N-terminal signal peptides that can be cleaved (9,10,36). This signal peptide has been found to precede major membrane proteins of phytoplasmas from other groups (10,12). Such proteins can be secreted via the Sec protein translocation system and might act as part of the virulence machinery, as reported previously for Streptococcus pyogenes (9,69).…”

Preparation of Antibody Against Immunodominant Membrane Protein (IMP) of Candidatus Phytoplasma aurantifolia

“…Both polyclonal and monoclonal antibodies have been generated against phytoplasmas for their detection and differentiation. The relative sensitivities of polyclonal antibodies produced against several phytoplasmas such as: aster yellows, peanut witches' broom, phytoplasma associated with faba bean, sesamum phyllody, apple proliferation and sandal spike phytoplasma were determined using indirect ELISA procedure (20,22,38,39). Polyclonal antibodies can be provided rapidly, at less expense, with less technical skill than required to produce monoclonal antibodies but it has mainly the disadvantage of cross reactivity with related antigens and limited products obtained in this way.…”

“…These antibodies have some disadvantages such as low titers, cross reactivity with plant components and weak specificity to the target phytoplasma. To overcome these difficulties, recombinant IMP has been successfully used to produce antibody against several phytoplasmas causing apple proliferation (AP) (22), Western X-disease (WX) (23) and onion yellows (OY) (13). Moreover, traditional approach to prepare antibody against phytoplasmas is based on immunizing animal with antigen preparations from infected plants.…”

Identification and Characterization of Phytoplasmal Genes, Employing a Novel Method of Isolating Phytoplasmal Genomic DNA

“…Therefore, phytoplasmal DNA is obtained with a high host (plant or insect) background, hampering attempts to study structural features and genome organization. In the last 15 years, some progress has been made towards enabling the enrichment of phytoplasmal fractions from extracts of infected plants and insects (4,20,21,42,47,48). In some cases, it became possible to produce phytoplasma-specific polyclonal and monoclonal antibodies, greatly facilitating diagnosis (6,9).…”