New ex vivo reporter assay system reveals that σ factors of an unculturable pathogen control gene regulation involved in the host switching between insects and plants

Analysis of the environmental regulation of bacterial gene expression is important for understanding the nature, pathogenicity, and infection route of many pathogens. "Candidatus Phytoplasma asteris", onion yellows strain M (OY-M), is a phytopathogenic bacterium that is able to adapt to quite different host environments, including plants and insects, with a relatively small ~850 kb genome. The OY-M genome encodes two sigma (σ) factors, RpoD and FliA, that are homologous to Escherichia coli σ(70) and σ(28) , respectively. Previous studies show that gene expression of OY-M dramatically changes upon the response to insect and plant hosts. However, very little is known about the relationship between the two σ factors and gene regulatory systems in OY-M, because phytoplasma cannot currently be cultured in vitro. Here, we developed an Escherichia coli-based ex vivo reporter assay (EcERA) system to evaluate the transcriptional induction of phytoplasmal genes by the OY-M-derived σ factors. EcERA revealed that highly expressed genes in insect and plant hosts were regulated by RpoD and FliA, respectively. We also demonstrated that rpoD expression was significantly higher in insect than in plant hosts and fliA expression was similar between the hosts. These data indicate that phytoplasma-derived RpoD and FliA play key roles in the transcriptional switching mechanism during host switching between insects and plants. Our study will be invaluable to understand phytoplasmal transmission, virulence expression in plants, and the effect of infection on insect fitness. In addition, the novel EcERA system could be broadly applied to reveal transcriptional regulation mechanisms in other unculturable bacteria.