Fig. 2

Infection route of ROLPs in the internal organs or tissues of its leafhopper vector R. dorsalis. Second-instar R. dorsalis nymphs were fed on ROLP-infected rice leaves for 4 days and then transferred to healthy rice seedlings. At different days post-first access to diseased plants (padp), organs or tissues dissected from insects were immunolabeled with Imp-rhodamine (red) and actin dye phalloidin-Alexa Fluor 647 (blue), then examined using immunofluorescence microscopy. a Alimentary canal of R. dorsalis. b Initial infection by ROLPs in the filter chamber (arrow) as early as 7 days padp. Panel ii is the enlarged image of the boxed area in panel i. c Extensive ROLP infection in the filter chamber at 7 days padp. d Extensive ROLP infection in the midgut epithelial cells at 14 days padp. Panels ii and iii are the enlarged images of boxed areas in panel i and ii, respectively. e, f Extensive ROLP infection in midgut epithelial cells (e) and visceral muscles (f) at 28 days padp. Panel ii is the enlarged image of boxed area in panel i. g Salivary gland of R. dorsalis. h Initial infection of ROLPs in type IV and V salivary cells at 14 days padp. Panel ii is the enlarged image of boxed area in panel i. i–k Extensive ROLP infection in type IV and V salivary cells at 21 or 28 days padp. l The distribution of ROLPs in the hemolymph from healthy or ROLP-infected insects. m Time course analysis of ROLP accumulation in infected insects by qPCR assay. At 7, 14, 21, and 28 days padp, a group of 15 R. dorsalis individuals were collected. Each insect was ground individually for DNA purification. Only ROLP-positive samples were used to measure phytoplasma titer. P values were estimated using Tukey’s honest significant difference (HSD) test. * P < 0.05, ** P < 0.01. n ROLP Imp protein accumulation in infected insects at different days padp, as detected by Western blotting assay with Imp-specific antibody (Imp2). Actin was used as a loading control. amg, anterior midgut; ec, epithelial cell; es, esophagus; fc, filter chamber; mmg, middle midgut; mt, malpighian tubule; pmg, posterior midgut; sg, salivary gland; vm, visceral muscle. Scale bars, 100 μm (a–k) and 5 μm (l)