Invasion of midgut epithelial cells by a persistently transmitted virus is mediated by sugar transporter 6 in its insect vector
Fig 7
Knockdown of LsST6 expression interfered with virus entry into midgut epithelial cells of healthy planthoppers.
(A) RNA interference mediated by microinjection with dsLsST6. Third-instar and nonviruliferous nymphs were injected with dsLsST6 or dsGFP using an Auto-Nanoliter Injector, then fed on healthy rice seedling. LsST6 mRNA expression level was quantified by RT-qPCR at different times. (B) LsST6 RNA levels were reduced after injection with dsLsST6 as quantified by RT-qPCR. (C) RNA level of RSV quantified by RT-qPCR. (D) RNA levels of RSV and LsST6 were quantified by DIG-northern blot. Nonviruliferous SBPHs that had been injected with either dsLsST6 or dsGFP were fed on RSV-infected rice for a 2-day acquisition access period (AAP), then collected at 2, 4 and 8 days to quantify RNA. Level of the housekeeping gene actin was used as an internal control. (E) Percentage of RSV-positive insects and transmission efficency decreased significantly compared with the control that was injected with dsGFP. Total RNA was extracted from insects at 8 days after a 2-day AAP or from rice seedling at 21 days after a 10 h inoculation access period and used to detect RSV by RT-PCR using specific primers. (A–D) Mean ± SEM of three independent experiments (P < 0.01, one-way ANOVA, LSD test). (F) Localization of LsST6 and RSV in midgut epithelial cells after knockdown of LsST6 expression. Excised midguts were incubated with anti-LsST6 (green) and anti-RSV (red) antibodies and observed with LSCM. Scale bars, 50 μm.