A new technique for manipulating host–parasite interactions by low speed centrifugation

Abstract

An apparatus and a method for centrifuging living, inoculated plant tissues were developed for studies of a cytoplasm-related host response of barley coleoptiles to penetration by the powdery mildew fungus Erysiphe graminis hordei. When the coleoptiles were attached to the apparatus with the long axes of the cells parallel to the centrifugal force, cytoplasm-rich and cytoplasm-poor zones were produced, permitting simultaneous comparisons of interactions in the presence or absence of host cytoplasm. The inner epidermis was inoculated and centrifuged for 12–14 h at 4750 × g. Desiccation was prevented by addition of liquids at the cut end of each coleoptile; the rest of the coleoptile was dry and suitable for inoculation with E. graminis conidia. Of the host cells, 50–80% had well-compacted cytoplasm, conidia developed penetration and infection structures, and the host response was restricted to cytoplasm-rich zones and was shown to be cytoplasm-dependent. Cytoplasmic streaming resumed within 10 min after centrifugation stopped, and 90–100% of the epidermal cells were actively streaming 3 h later. This centrifugation method could be adapted and applied to studies of the other host–parasite interactions.