Isolating intact phytoplasma particles from plants for sequencing applications and for testing host responses to phytoplasmas

‘Candidatus Phytoplasma’ species are obligate plant parasites limited to the phloem tissue. The difficulties to grow phytoplasma in culture seriously impedes the study of these prokaryotes. In this study, a protocol to extract intact phytoplasmas from infected periwinkle plants using filtration, differential and density-gradient centrifugations was optimized. Confocal and transmission electron microscopy was used to visualize intact phytoplasmas in the preparation. Quantitative-PCR and DNA-seq analyses revealed that phytoplasma DNA was significantly enriched following the extraction procedure. Challenging periwinkle plants with phytoplasma preparation induced the expression of one defence-related gene. This study provides an optimized procedure for phytoplasma DNA enrichment and for extracting intact phytoplasmas that can be used to examine host responses to them.