The low sequence identity score of IdpA is partially attributed to insertions and deletions in the PoiBI IdpA sequence relative to that of WX IdpA. In

a comparison of nucleotide sequences, the numbers of nonsynonymous and synonymous substitutions per site (dN and dS, respectively) and their ratio (dN/dS) are important indicators of selective pressure at the protein level. Calculated dN/dS values of <1, 1, and >1 imply stabilizing selection, neutral mutation, and diversifying positive selection, respectively. To investigate whether dnaD, imp, and idpA evolved under positive selection pressure, we determined the dN/dS value of the nucleotide sequences of these genes from PoiBI and WX. For dnaD, dN/dS was 0.444, which is < 1. For imp, dN/dS ranged from 1.278 to 1.556, but was this website not statistically significant (P = 0.3233–0.3716). In contrast,

the dN/dS value for idpA was 1.500 with P = 0.0639, which Cabozantinib purchase is significant at the 10% level (Table 2). To prepare materials for the production of anti-PoiBI-Imp and anti-PoiBI-IdpA antisera, we attempted to express His-tagged full-length and truncated forms of these proteins in E. coli. Attempts to express some proteins in E. coli were successful only for His-tagged full-length Imp and for His-tagged IdpA-N, which lacks the two transmembrane regions of IdpA, as well as half of the hydrophilic domain. These purified proteins were used to immunize rabbits. Use of the purified anti-Imp and anti-IdpA IgG in Western blots analysis of the Imp and IdpA hydrophilic domain proteins expressed in E. coli confirmed that the titers of the antibodies were similar (data not shown). To confirm that Imp and IdpA are expressed in PoiBI-infected poinsettia, crude protein extracts were prepared from the PoiBI-infected cultivar ‘Jester Red’ (‘infected’ extract) and from the healthy control cultivar ‘Flaming Sphere’ (‘control’ extract). Western blot analysis of these samples using anti-Imp antibody Carbohydrate revealed a distinct protein band in the infected extract, but not in the control extract (Fig. 3a). However, multiple attempts to detect IdpA using anti-IdpA antibody failed to detect IdpA in either the

infected or control extracts (Fig. 3b). The Western blot analysis estimated the molecular mass of Imp in the ‘infected’ as 19.6 kDa, which is the calculated mass of full-length Imp. This result suggests that the signal sequence of Imp is not cleaved, and that Imp exists as a membrane protein in infected poinsettia plants. In contrast, the strongest Imp band detected in E. coli extracts was smaller by approximately 3 kDa, suggesting that the Imp signal sequence is cleaved when the recombinant protein is expressed in E. coli. To investigate the localization of Imp and IdpA proteins in infected plants, we performed immunohistochemical analysis using anti-Imp and anti-IdpA antibodies. Both Imp and IdpA were specifically detected in the phloem of ‘Jester Red’ (‘infected’; Fig. 4b and d), but not in ‘Flaming Sphere’ (‘control’; Fig.