Cloning, expression analysis, and sequence diversity of genes encoding two different immunodominant membrane proteins in poinsettia branch-inducing phytoplasma (PoiBI)

Abstract

Poinsettia branch-inducing phytoplasma (PoiBI) is a phytopathogenic bacterium that infects poinsettia, and is associated with the free-branching morphotype (characterized by many axillary shoots and flowers) of many commercially grown poinsettias. The major membrane proteins of phytoplasmas are classified into three general types, that is, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). These membrane proteins are often used as targets for the production of antibodies used in phytoplasma detection. Herein, we cloned and sequenced the imp and idpA genes of PoiBI strains from 26 commercial poinsettia cultivars. Although the amino acid sequences of the encoded IdpA proteins were invariant, those of the encoded Imp varied among the PoiBI isolates, with no synonymous nucleotide substitution. Western blotting and immunohistochemical analyses revealed that the amount of Imp expressed exceeded that of IdpA, in contrast to the case of a related phytoplasma-disease, western X-disease, for which the major membrane protein appears to be IdpA, not Imp. These results suggest that even phylogenetically close phytoplasmas express different types of major membrane proteins.

Immunodominant membrane proteins of phytoplasmas

“…If two IDP genes are present, one always encodes Imp. Furthermore, it appears that Imp can have a much more variable amino acid sequence with constant protein structure (Kakizawa et al, 2009) than Amp (Kakizawa et al, 2006a) or IdpA (Neriya et al, 2011). These observations suggest that Imp could represent an ancestral type of IDP from which Amp and IdpA have somehow emerged (Kakizawa et al, 2006b(Kakizawa et al, , 2009).…”

“…Thus, Amp, not Imp, appears to be the main IDP of OY. In contrast to OY phytoplasmas, in PoiBI phytoplasma, which carries imp and idpa, Imp is expressed at a higher level than IdpA, suggesting that Imp is the main IDP of PoiBI (Neriya et al, 2011). Taking this point into account and due to the high variability of the amino acid sequences of all three types of IDPs, conclusive evidence that Imp is the ancestor of Amp and/or IdpA is still lacking.…”

“…Using these methods, various studies strongly suggest that positive selection is acting on all three types of IDPs, but not on the genes adjacent to the IDPs (Kakizawa et al, 2006a(Kakizawa et al, , 2009Neriya et al, 2011;Siampour et al, 2013). For Imp and Amp proteins, most amino acid sites that are targets of positive selection are located within the hydrophilic regions of the proteins, suggesting that the selective pressure is acting mainly on the predicted extracellular region of each protein (Kakizawa et al, 2006a(Kakizawa et al, , 2009).…”

“…In PoiBI (Poinsettia branch inducing) phytoplasma, DNA fragments containing the idpA and imp genes were amplified using information from other phytoplasmas (genomic sequence of OY phytoplasma line M, OY-M) (Liefting & Kirkpatrick, 2003;Oshima et al, 2004) and sequenced (Neriya et al, 2011). In both phytoplasma strains, the following gene order was found ( Fig.…”

“…1.2) of WX and PoiBI phytoplasmas were predicted to contain two hydrophobic regions near the Cand N-termini of the protein and a large central hydrophilic region without cleavage sites leading to an extracellular domain, which is anchored to the membrane by two transmembrane regions (Blomquist et al, 2001;Neriya et al, 2011).…”