'Candidatus Phytoplasma asteris', onion yellows strain (OY), a mildly pathogenic line (OY-M), is a phytopathogenic bacterium transmitted by Macrosteles striifrons leafhoppers. OY-M contains two types of plasmids (EcOYM and pOYM), each of which possesses a gene encoding the putative transmembrane protein, ORF3. A non-insect-transmissible line of this phytoplasma (OY-NIM) has the corresponding plasmids (EcOYNIM and pOYNIM), but pOYNIM lacks orf3. Here we show that in OY-M, orf3 is transcribed from two putative promoters and that on EcOYNIM, one of the promoter sequences is mutated and the other deleted. We also show by immunohistochemical analysis that ORF3 is not expressed in OY-NIM-infected plants. Moreover, ORF3 protein seems to be preferentially expressed in OY-M-infected insects rather than in plants. We speculate that ORF3 may play a role in the interactions of OY with its insect host.