In this analysis of the imp and idpA genes of PoiBI, we confirmed the previously published assertions

that these two genes are not homologous, and that imp is well conserved over a wide range of phytoplasma strains (Kakizawa et al., 2009). Although the Imp of WX has been previously reported to be IdpA (Blomquist et al., 2001), Apoptosis inhibitor the identity of the Imp of PoiBI was not studied. In the present study, we were able to detect the expression of Imp in PoiBI-infected poinsettia plants using both immunohistochemical and Western blot analyses (Fig. 3a and b Fig. 4). In contrast, although we were able to detect expression of IdpA in PoiBI-infected plants immunohistochemically, we were not able to detect it by Western blotting, probably because immunohistochemical analysis is generally the more sensitive technique (Jiang et al., 1988; Friedlander et al., 1989; Gala et al., 1994). Our findings suggest that Imp is expressed at a higher level than IdpA in PoiBI. In comparing PoiBI strains from 26 different poinsettia cultivars, we found no variation

in their idpA, dnaD, or 16S rRNA genes. On the other hand, imp did exhibit some variation. All of the nucleotide substitutions in PoiBI imp resulted in amino acid changes; that is, no silent mutations were see more observed, suggesting that imp is prone to mutation. Although adaptive evolution of imp was not detected (Table 2), strong positive selection has been reported for the imp genes of AY-group phytoplasmas, indicating that Imp plays an important role in phytoplasma fitness (Kakizawa et al., 2006b, 2009). The imp gene of PoiBI might also play a crucial role correlated to the accumulation of amino acid substitutions. AY-group phytoplasmas express approximately ten times as much Amp as Imp, indicating that the Imp in this group is Amp (Kakizawa et al., 2009). Among phytoplasma

strains, amp gene sequences exhibit Thalidomide much more variation than imp gene sequences, and are subject to strong positive selection pressure (Kakizawa et al., 2009). In PoiBI, Imp was expressed at a higher level than IdpA (Fig. 3a and b), suggesting that the major membrane protein of PoiBI is Imp rather than IdpA, even though the Imp in closely related WX is IdpA (Blomquist et al., 2001). Therefore, the genes encoding Imps appear to differ among even closely related phytoplasma strains. Further analyses of imp and idpA sequences and gene expression among many strains of 16SrIII ribosomal group phytoplasmas, which include PoiBI and WX should yield more information about the diversity of Imps. The average nucleotide sequence identity between PoiBI imp genes and WX imp in our study was 97.2%, whereas that between PoiBI and WX idpA was 77.7%. Nonsynonymous substitutions outnumbered synonymous substitutions for both genes from the two strains, and dN/dS was > 1 for both comparisons (Table 2). The high value of dN/dS for idpA resulted solely from the differences between WX and PoiBI idpA, as there was no variation among the 26 PoiBI sequences.