Molecular characterisation of two plasmids from paulownia witches'-broom phytoplasma and detection of a plasmid-encoded protein in infected plants

Two plasmids were cloned from paulownia witches'-broom phytoplasma-Nanyang strain (PaWBNy). Southern blotting using pPaWBNy-1ORF1 probe confirmed the existence of the two plasmids. The 4485 bp plasmid, designated pPaWBNy-1, had a nucleotide content of 24 mol% G+C and contained six putative open reading frames (ORFs). The 3837 bp plasmid, designated pPaWBNy-2, had a nucleotide content of 25.9 mol% G+C and contained five putative ORFs which showed similarity with ORFs in pPaWBNy-1. The two plasmids contained a series of tandem repeats and encoded a replication-associated protein (RepA) and a single-stranded DNA-binding protein (SSB), which were necessary for the replication of plasmids. Seven putative proteins encoded by two plasmids were predicted to contain one or more hydrophobic transmembrane domains, respectively, and presumably to be localised to the membrane. ORF4 from pPaWBNy-1 was partially cloned and the recombinant protein with His-tag expressed in Escherichia coli. The fusion protein was used for immunisation and the polyclonal antiserum to ORF4 protein detected the native expression of ORF4 protein in Western blot analysis from infected but not healthy plants.