Functional characterization of the principal sigma factor RpoD of phytoplasmas via an in vitro transcription assay

Phytoplasmas (class, Mollicutes) are insect-transmissible and plant-pathogenic bacteria that multiply intracellularly in both plants and insects through host switching. Our previous study revealed that phytoplasmal sigma factor rpoD of OY-M strain (rpoD(OY)) could be a key regulator of host switching, because the expression level of rpoD(OY) was higher in insect hosts than in plant hosts. In this study, we developed an in vitro transcription assay system to identify RpoD(OY)-dependent genes and the consensus promoter elements. The assay revealed that RpoD(OY) regulated some housekeeping, virulence, and host–phytoplasma interaction genes of OY-M strain. The upstream region of the transcription start sites of these genes contained conserved –35 and –10 promoter sequences, which were similar to the typical bacterial RpoD-dependent promoter elements, while the –35 promoter elements were variable. In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools. The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns. These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.