Interaction of viral pathogen with porin channels on the outer membrane of insect bacterial symbionts mediates their joint transovarial transmission

Abstract

Many hemipteran insects that can transmit plant viruses in a persistent and transovarial manner are generally associated with a common obligate bacterial symbiont Sulcia and its β-proteobacterial partner. Rice dwarf virus (RDV), a plant reovirus, can bind to the envelope of Sulcia through direct interaction of the viral minor outer capsid protein P2 with the bacterial outer membrane protein, allowing the virus to exploit the ancient oocyte entry path of Sulcia in rice leafhopper vectors. Here, we show that RDV can hitchhike with both Sulcia and its β-proteobacterial partner Nasuia to ensure their simultaneous transovarial transmission. Interestingly, RDV can move through the outer envelope of Nasuia and reside in the periplasmic space, which is mediated by the specific interaction of the viral major outer capsid protein P8 and the porin channel on the bacterial outer envelope. Nasuia porin-specific antibody efficiently interferes with the binding between RDV and the Nasuia envelope, thus strongly preventing viral transmission to insect offspring. Thus, RDV has evolved different strategies to exploit the ancient oocyte entry paths used by two obligate bacterial symbionts in rice leafhoppers. Our results thus reveal that RDV has formed complex, cooperative interactions with both Sulcia and Nasuia during their joint transovarial transmission. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

Keywords: Nasuia and Sulcia; obligate bacterial symbionts; porin channel; rice dwarf virus; rice leafhopper; transovarial transmission.

Figure 1.

RDV virions moved with the obligate bacterial symbionts Sulcia and Nasuia into the oocyte of female N. cincticeps. (a–c) Confocal micrographs show the colocalization of RDV with Sulcia or Nasuia in the epithelial plug of female insects at 6 days post-emergence. Panel b is an enlargement of boxed area in a. Panels in c are enlargements of boxed area in b. (d–f) Colocalization of RDV and Sulcia or Nasuia in the symbiont ball in the oocyte of female insects at 8 days post-emergence. Panel e is an enlargement of boxed area in d. Panels in f are enlargements of boxed area in e. Scale bars in a, b, d and e: 100 µm; c and f: 25 µm. (g–m) Transmission electron micrographs showing RDV particles distributed along the envelopes of Nasuia or Sulcia at the same follicular epithelial cells in the epithelial plug. Panel h is an enlargement of boxed area in g. Panels i and j are enlargements of boxed areas in h. Panels l and m are enlargements of boxed areas in k. Scale bars in g: 10 µm; h and k: 2 µm; i, j, l and m: 500 nm. (n) Model for the exploitation of bacterial symbionts Sulcia and Nasuia by RDV to enter the oocyte. RDV virions directly attach to the envelopes of Nasuia or Sulcia and then enter the oocyte from the epithelial plug along with Nasuia or Sulcia. Ep, epithelial plug; Fc, follicular cell; N, Nasuia, O, oocyte; Pd, pedicel; S, Sulcia; Sb, symbiont ball. Black arrows mark the entry route of virus-associated bacterial symbionts. Red arrows indicate RDV virions. All electron micrographs and immunofluorescence figures are representative of at least three replications.

Figure 2.

Electron micrographs showing distinct interaction strategies for RDV virions with the envelopes of Sulcia or Nasuia. (a–d) RDV virions in the invaginations formed by Sulcia envelopes. Panels b and d are enlargements of boxed areas in panels a and c, respectively. Scale bars in a and c: 2 µm; b and d: 500 nm. (e–h) RDV virions attached to the envelope of Nasuia close to the epithelial plug. Panels f and h are enlargements of boxed areas in panels e and g, respectively. Scale bar: 500 nm. (i–k) RDV virions embedded in the periplasmic space between outer and IMs of Nasuia in the epithelial plug. Panels j and k are enlargements of boxed areas of panels i and j, respectively. Scale bars: 500 nm. IM, inner membranes; N, Nasuia; OM, outer membranes; PS, periplasmic space; S, Sulcia. Red arrowheads indicate RDV virions. All micrographs are representative of at least three replications.

Figure 3.

RDV virion entry into Nasuia periplasmic space was mediated by specific interaction between RDV P8 and Nasuia porin. (a) Secondary structure prediction showed that Nasuia porin consisted of 18 β-strands and six transmembrane regions. (b) RDV virion consists of one minor outer capsid protein P2 and one major outer protein P8. Scale bar; 50 nm. (c) Yeast two-hybrid assay to detect interactions between P2N (15 nm domain, 1–688 aa) or RDV P8 and Sulcia OMP (BSA domain, 706–985 aa) or Nasuia porin. (d) GST pull-down assay to detect interactions between RDV P8 and Nasuia porin. Nasuia porin-GST acted as a bait protein with GST as a control. Pull-down samples were probed with GST antibody in a western blot. (e) Proposed model for the different binding strategies for RDV virions with the envelopes of Sulcia and Nasuia. IM, inner membranes; OM, outer membranes; OMP, outer membrane protein; PS, periplasmic space.

Figure 4.

Microinjection of Nasuia porin-specific antibody reduced RDV vertical transmission to the next insect generation. Newly emerged adult insects were injected with the purified RDV virions together with porin-specific antibody or pre-immune serum, and ovaries were dissected to detect RDV, Nasuia and Sulcia in 5 days. (a–d) At 5 days after microinjection, confocal micrographs showed the porin-specific antibody treatment inhibited the association of RDV with Nasuia, but not with Sulcia, in the epithelial plug. Scale bars: 100 µm. (e–h) At 7 days after microinjection, confocal microscopy showed that the Nasuia porin-specific antibody treatment strongly inhibited the association of RDV with Nasuia, but not with Sucia, in the ovaries. (i–k) Transcript levels of Nasuia 16S rRNA, Sulcia 16S rRNA and RDV P8 in ovaries or other parts of insects treated with porin-specific antibody or pre-immune serum, as determined by RT-qPCR assay in three independent experiments. (l) Accumulation levels of Nasuia, Sulcia and RDV P8 in ovaries or other parts of insects treated with porin-specific antibody or pre-immune serum, as determined by western blot using Nasuia porin-, Sulcia OMP- or RDV P8-specific IgGs, respectively. Insect actin was detected with actin-specific IgG as a control. (m) Vertical transmission rates of Nasuia, Sulcia and RDV after treatment with porin-specific antibody or pre-immune serum. Ep, epithelial plug; Fc, follicular cell; O, oocyte; Pd, pedicel. All images are representative of at least three replications. (i, j, k and m) Data are means ± s.e. from three independent experiments (independent-sample t-test at 0.05 level).