Fig. 2: Reduced RdCBP secretion into rice phloem during RGDV secretion from vector salivary glands. | Communications Biology

Fig. 2: Reduced RdCBP secretion into rice phloem during RGDV secretion from vector salivary glands.

From: A leafhopper saliva protein mediates horizontal transmission of viral pathogens from insect vectors into rice phloem

Fig. 2

a RT-qPCR assay of RdCBP transcript levels in the salivary glands from nonviruliferous (V−) and viruliferous (V+) insects. Quantitative data from three independent experiments are presented as mean ± SD (error bars) values. Significant differences were tested using a two-tailed Student’s t-test. ***P  <  0.0001. b Western blot assay of the RdCBP accumulation levels in the salivary glands of nonviruliferous (V−) and viruliferous (V+) insects. RdCBP and actin were detected by using RdCBP- and actin-specific IgGs, respectively. Data were representative of three biological replicates. c–h Immunofluorescence assay showing that the distribution areas of RdCBP were gradually reduced in type III secretory cells of PSGs after 3 (c, g). 7 (d, h), and 9 (e) days post-microinjection of purified viruses, respectively. Virus-infected salivary glands were immunostained with RdCBP-FITC (green), virus-rhodamine (red), and phalloidin-Alexa Fluor 647 (blue), respectively. The average percentages of distribution areas of RdCBP and RGDV from 20 type III cells were counted using ImageJ (f). Bars represent mean ± SD values. g, h are the large images clearly showing the distribution of viruses and RdCBP in the cytoplasm or salivary cavities of secretory cells. i Immunoelectron microscopy showing the distribution of RdCBP in the cavities but not in the cytoplasm in virus-infected regions. Blue arrows mark gold particles. Red arrows mark viruses. j Western blot assay of RdCBP in rice plants that had been fed on by viruliferous (V+) or nonviruliferous (V−) leafhoppers. Rubisco large subunit was used as a loading control, as detected by staining with Coomassie Brilliant Blue (CBB). Mock, rice plants not subjected to leafhopper feeding. Data were representative of three biological replicates. M protein marker. k–m Immunofluorescence detection of RdCBP and RGDV in cross-sections prepared from leaf phloem infested with nonviruliferous or viruliferous R. dorsalis. Nonviruliferous (V−, l) or viruliferous (V+, m) R. dorsalis adults were fed on healthy rice plants for 3 days, after which plants were then processed for immunofluorescence with RdCBP-FITC (green) and virus-rhodamine (red). Non-infested healthy rice leaves served as a mock treatment (k). n–p Immunofluorescence assay showing that during viral infection, RGDV Pns11 filaments competed with RdCBP for release into salivary cavities. Virus-infected salivary glands dissected from insects after 3 (n), 7 (o), and 9 (p) days post-microinjection of purified viruses. Salivary glands were immunostained with Pns11-rhodamine (red), RdCBP-FITC (green), and phalloidin-Alexa Fluor 647 (blue). q–s Immunoelectron microscopy showing the distribution of RdCBP during RGDV Pns11 filament-mediated viral secretion into salivary cavities. Blue arrows mark gold particles. Red arrows mark Pns11 filaments. Virus-infected salivary glands in (i) and (q–s) were immunolabeled with RdCBP-specific IgG as a primary antibody, followed by treatment with 15-nm gold particle-conjugated goat antibody against rabbit IgG as a secondary antibody. The relative intensities of western blot bands in the analyses of different proteins for (b) and (j) are shown below. APL apical plasmalemma, Cv cavity, SC salivary cytoplasm, Xy xylem, Ph phloem. All images are representative of at least three replicates. Bars: c–e 50 μm; k–m 25 μm; g, h, n–p 10 μm; i 1 μm; q–s 500 nm.

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