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. 2022 Apr 13;96(7):e0214021.
doi: 10.1128/jvi.02140-21. Epub 2022 Mar 7.

Flotillin 2 Facilitates the Infection of a Plant Virus in the Gut of Insect Vector

Wei Wang #  1 , Luqin Qiao #  2 , Hong Lu  1 , Xiaofang Chen  1   3 , Xue Wang  2 , Jinting Yu  1   3 , Jiaming Zhu  1   3 , Yan Xiao  1 , Yonghuan Ma  1   3 , Yao Wu  4 , Wan Zhao  1   3 , Feng Cui  1   3
Affiliations

Flotillin 2 Facilitates the Infection of a Plant Virus in the Gut of Insect Vector

Wei Wang et al. J Virol. .

Abstract

Most plant viruses require insect vectors for transmission. One of the key steps for the transmission of persistent-circulative plant viruses is overcoming the gut barrier to enter epithelial cells. To date, little has been known about viral cofactors in gut epithelial cells of insect vectors. Here, we identified flotillin 2 as a plasma membrane protein that facilitates the infection of rice stripe virus (RSV) in its vector, the small brown planthopper. Flotillin 2 displayed a prominent plasma membrane location in midgut epithelial cells. The nucleocapsid protein of RSV and flotillin 2 colocalized on gut microvilli, and a nanomolar affinity existed between the two proteins. Knockout of flotillin 2 impeded the entry of virions into epithelial cells, resulting in a 57% reduction of RSV levels in planthoppers. The knockout of flotillin 2 decreased disease incidence in rice plants fed by viruliferous planthoppers from 40% to 11.7%. Furthermore, flotillin 2 mediated the infection of southern rice black-streaked dwarf virus in its vector, the white-backed planthopper. This work implies the potential of flotillin 2 as a target for controlling the transmission of rice stripe disease. IMPORTANCE Plant viral diseases are a major threat to world agriculture. The transmission of 80% of plant viruses requires vector insects, and 54% of vector-borne plant viruses are persistent-circulative viruses, which must overcome the barriers of gut cells with the help of proteins on the cell surface. Here, we identified flotillin 2 as a membrane protein that mediates the cell entry of rice stripe virus in its vector insect, small brown planthopper. Flotillin 2 displays a prominent cellular membrane location in midgut cells and can specifically bind to virions. The loss of flotillin 2 impedes the entry of virions into the midgut cells of vector insects and substantially suppresses viral transmission to rice. Therefore, flotillin 2 may be a promising target gene for manipulation in vector insects to control the transmission of rice stripe disease and perhaps that of other rice virus diseases in the future.

Keywords: flotillin 2; insect vector; midgut; planthopper; plasma membrane protein; rice stripe virus; rice viral disease.

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Conflict of interest statement

The authors declare no conflict of interest.

We have no conflicts of interest to declare.

Figures

FIG 1
FIG 1
Characterization of small brown planthopper flotillins. (a) Diagram showing the PHB and flotillin (Flot) domains of flotillin 1 and flotillin 2. (b) Alignment of amino acid sequences of flotillin 1 and flotillin 2. Similar amino acid residues are shaded in gray. Identical amino acid residues are shaded in black. (c) Verification of anti-flotillin 2 polyclonal antibody in the recombinantly expressed flotillin 2-His and flotillin 1-Flag and the total protein of viruliferous planthoppers in Western blot assays. (d) Phylogenetic neighbor-joining tree of flotillins of the small brown planthopper and other insects and mammals. Bootstrap values higher than 60% are shown at the nodes. GenBank registration numbers of each protein are given in parentheses.
FIG 2
FIG 2
Flotillin 2 binds to RSV NP with specific affinity activity. (a) NP and flotillin 2 were coprecipitated from the total protein of viruliferous planthoppers using anti-NP monoclonal antibody and analyzed in Western blot. Mouse IgG was used as a negative control. (b) The recombinantly expressed N terminus of flotillin 2 with His tags (N-His) was coprecipitated with NP-Flag. The total protein from E. coli expressing the empty pET28a vector was applied as a negative control. (c) The C terminus of flotillin 2 with His-tags (C-His) was coprecipitated with NP-Flag. (d) The PHB domain of flotillin 2 with His-tags (PHB-His) was not coprecipitated with NP-Flag. (e) MST assay to reveal specific binding between NP-GST and flotillin 2-MBP. Solid curve shows fit of data points to a standard KD-fit function. Bars represent standard error (SE). (f) BLI binding profiles of NP-GST and flotillin 2-MBP. (g and h) Flotillin 1-Flag was not coprecipitated with NP-His (g) or NP from total protein of the viruliferous planthoppers (h). (i and j) Flotillin 1-Flag was not coprecipitated with flotillin 2-His (i) or flotillin 2 from the total protein of the viruliferous planthoppers (j).
FIG 3
FIG 3
Organ and plasma membrane localization of flotillin 2. (a) Relative transcript levels of flotillin 2 in various organs of nonviruliferous small brown planthopper after normalization according to the transcript level of EF2. Different letters indicate statistically significant differences. (b) Immunohistochemistry showing localization of flotillin 2 in planthopper midgut epithelial cells. Boxed regions are enlarged and shown in the two panels on the right. Flotillin 2 was labeled with anti-flotillin 2 polyclonal antibody (red). F-actin was stained with phalloidin (blue). The sample without antibody treatment is shown as negative control (NC). (c) Immunofluorescence labeling of recombinantly expressed flotillin 2-His, PHB-His domain, N-terminal-His (N-His), and C-terminal-His (C-His) of flotillin 2 in Drosophila S2 cells with anti-His monoclonal antibody (green). F-actin was labeled with phalloidin (red) and nuclei with Hoechst (blue). (d) Colloidal gold immunoelectron micrographs showing the colocalization of flotillin 2 and RSV NP on the microvilli (MV) of midgut epithelial cells. Images include enlarged images of the regions in blue boxes in the upper right corner panels. NP was labeled with the 10-nm colloidal gold-conjugated anti-NP monoclonal antibody, and flotillin 2 with the 5-nm colloidal gold-conjugated anti-flotillin 2 polyclonal antibody.
FIG 4
FIG 4
Flotillin 2 facilitates RSV infection in small brown planthoppers. (a) Comparison of relative flotillin gene transcription levels in the fourth-instar nymphs of viruliferous and nonviruliferous planthoppers, and in planthoppers fed on RSV-infected rice or uninfected rice for 8 days, as measured by qPCR. The flotillin gene transcription levels were normalized to that of EF2. Fold changes relative to those of nonvirulifous insects are shown. (b and c) Immunohistochemistry results showing the variation of RSV load in midgut epithelial cells after 12 h (b) or 10 days (c) of oral acquisition of RSV with the injection of dsRNA for flotillin 2 (dsFlotillin 2) compared to the injection of GFP dsRNA (dsGFP). Boxed regions are enlarged and shown in other three panels. NP was labeled with anti-NP monoclonal antibody (green), flotillin 2 with anti-flotillin 2 polyclonal antibody (red), and F-actin with phalloidin (blue). Fifteen midguts were analyzed for each group. NP fluorescence intensity decreased by 77% (P < 0.01) (b) and 86.2% (P < 0.001) (c). (d and e) Relative RNA and protein levels of RSV NP in planthoppers after 10 days of oral acquisition of RSV with the injection of dsFlotillin 2 (d) or dsFlotillin 1 (e) as measured by qPCR and Western blot. Control groups were injected with dsGFP. Relative transcription levels of flotillin 2 and flotillin 1 were also measured. Anti-NP monoclonal antibody and anti-human β-tubulin monoclonal antibody were used. The relative grayscale of NP to that of tubulin was compared between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 5
FIG 5
Flotillin 2 is critical for the transmission of RSV by small brown planthopper. (a) Germ-line insertion/deletion mutations in the flotillin 2 gene in G0 eggs and adults generated using CRISPR-Cas9. Target sequence is indicated in red. PAM sequence is indicated in green. Deleted nucleotides are indicated in gray, and inserted nucleotides are in blue. The length of the nucleotide alteration is indicated on the right (+, insertion; –, deletion). Black triangles indicate the genotypes of two successfully established flotillin 2-knockout lines (flot20+/20+, flot32+/32+). WT, wild type. (b) Protein levels of flotillin 2 in the two homozygous mutant lines and in WT planthoppers, assayed by Western blot using the anti-flotillin 2 polyclonal antibody. F, female. M, male. (c) Survival curves of flot20+/20+ and WT planthoppers throughout whole life spans. (d) Average numbers of eggs produced by single females of flot20+/20+ or WT planthoppers within 10 days. (e) Comparison of feeding behavior EPG waveforms. np, non-probing; N1+N2+N3, pathway phases; N4-a, watery salivation; N4-b, passive ingestion; N5, drinking from xylem. (f and g) Immunohistochemistry results showing RSV load and flotillin 2 expression in the midgut epithelial cells of flot20+/20+ and WT planthoppers after 12 h (f) or 15 days (g) of oral acquisition of RSV. NP was labeled with anti-NP monoclonal antibody (green), flotillin 2 with anti-flotillin 2 polyclonal antibody (red), and F-actin with phalloidin (blue). Eight midguts were analyzed for each group. The NP fluorescence intensity decreased by 79.4% (P < 0.01) (f) and 81.2% (P < 0.01) (g). (h) Relative RNA levels of RSV NP in single individuals of flot20+/20+ or WT planthoppers after 15 days of oral acquisition of RSV measured using qPCR. (i) The disease incidence and symptoms of the rice plants fed upon by flot20+/20+ or WT viruliferous planthoppers. *, P < 0.05; **, P < 0.01; ns, P > 0.05.
FIG 6
FIG 6
The role of flotillin 2 in the infection of insect vectors by other rice viruses. (a and b) The recombinantly expressed P10-His of SRBSDV was coprecipitated with SFflotillin 2-Flag (a) or SFflotillin 2 from the total protein of white-backed planthoppers (b) using the anti-His monoclonal antibody in Co-IP assays. Mouse IgG was used as negative control. The anti-His, anti-Flag, or anti-flotillin 2 antibodies were applied in Western blotting analyses. (c) and (d) The recombinantly expressed P8-His of RRSV was not coprecipitated with NLflotillin 2-Flag (c) or NLflotillin 2 from the total protein of brown planthoppers (d) in Co-IP assays. (e) Relative RNA levels of SRBSDV P10 in the white-backed planthopper after 10 days of oral acquisition of SRBSDV with the injection of dsRNA for SFflotillin 2 (dsSFflotillin 2) or dsRNA for GFP (dsGFP), as measured by qPCR. Relative transcription levels of SFflotillin 2 were measured at the same time. Transcription level of ribosomal L9 was quantified to normalize the cDNA template. (f) Relative RNA levels of RRSV P8 in the brown planthopper after 10 days of oral acquisition of RRSV with the injection of dsNLflotillin 2 or dsGFP, as measured by qPCR. Relative transcription levels of NLflotillin 2 were measured at the same time. Transcription level of 18S rRNA was quantified to normalize the cDNA template. *, P < 0.05; **, P < 0.01.
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