Insect tissue-specific vitellogenin facilitates transmission of plant virus

Abstract

Insect vitellogenin (Vg) has been considered to be synthesized in the fat body. Here, we found that abundant Vg protein is synthesized in Laodelphax striatellus hemocytes as well. We also determined that only the hemocyte-produced Vg binds to Rice stripe virus (RSV) in vivo. Examination of the subunit composition of L. striatellus Vg (LsVg) revealed that LsVg was processed differently after its expression in different tissues. The LsVg subunit able to bind to RSV exist stably only in hemocytes, while fat body-produced LsVg lacks the RSV-interacting subunit. Nymph and male L. striatellus individuals also synthesize Vg but only in hemocytes, and the proteins co-localize with RSV. We observed that knockdown of LsVg transcripts by RNA interference decreased the RSV titer in the hemolymph, and thus interfered with systemic virus infection. Our results reveal the sex-independent expression and tissue-specific processing of LsVg and also unprecedentedly connect the function of this protein in mediating virus transmission to its particular molecular forms existing in tissues previously known as non-Vg producing.

Fig 1. Gene expression and protein distribution of Laodelphax striatellus vitellogenin (LsVg) in tissues of the female insects.

A. Distribution of LsVg mRNA in different tissues of female insects determined by qPCR. Both the mean and SD were calculated from three independent experiments, with four mRNA samples per experiment. Ef2, L. striatellus elongation factor 2 gene; HC, hemocyte; FB, fat body; SG, salivary glands; MG, midgut. B. Immunofluorescence staining to reveal the distribution of LsVg protein in L. striatellus tissues. LsVg was probed with mouse anti-LsVg monoclonal antibody Ab47Km and stained with Alexa Fluor 568 (shown in red). Nucleoli were stained with TO-PRO-3 (shown in blue). Images were examined using a Leica TCS SP8 confocal microscope. Images are representative of three independent experiments with a total of 15 SBPHs analyzed. The white arrow indicates the immune-reactive signal of the LsVg protein. The scale bar, 50 μm.

Fig 2. Localization of LsVg and RSV in female L. striatellus tissues.

RSV was probed with Alexa Fluor 488-labeled mouse anti-RSV monoclonal antibody (shown in green). LsVg was probed with mouse anti-LsVg monoclonal antibody Ab47Km and stained with Alexa Fluor 568 (shown in red). Nucleoli were stained with TO-PRO-3 (shown in blue). Images were examined using a Leica TCS SP8 confocal microscope. Images are representative of three independent experiments with a total of 15 SBPHs analyzed. The white arrow indicates the immune-reactive signal of the LsVg protein. The scale bar represents 20 μm.

Fig 3. Subunit composition of LsVn.

A. SDS-PAGE (10%) of purified LsVn. M is the molecular weight marker (kDa). The identified LsVn subunits are indicated by the arrows on the right. B. Mapping of vitellogenin-derived peptides identified by mass spectrometry onto the LsVg primary sequence. Peptides identified from SDS-PAGE bands are indicated by color: 178 kDa (shaded), 111 kDa (green), 67 kDa (blue) and 42 kDa (red). Pairs of arrows mark the span of LsVg or LsVn subunits. Shaded tetra-residues in bold font are the cleavage sites. Underlined sequences indicate synthetic peptides used for the production of subunit-specific antibodies. The predicted signal peptide sequence at the N-terminus is shown in bold. C. Verification of the composition of the LsVn subunit by western blot analysis. Purified LsVn was fractionated by SDS-PAGE (10%) and probed with the subunit-specific antibodies. Identified LsVn subunits are indicated by the arrows on the right. M, the molecular weight marker (kDa).

Fig 4. Tissue-specific processing of LsVg in female L. striatellus.

A. Confocal microscopy to reveal the distribution of different LsVg protein regions in the fat body or hemocytes. The LsVg N-terminus (recognized by antibody Ab42K) was present in both tissues, whereas the middle region (recognized by Ab67K2) and the C-terminus (recognized by Ab111K) existed only in hemocytes. LsVg probed with the LsVn subunit-specific antibody was stained with Alexa Fluor 568 (shown in red). RSV was stained with Alexa Fluor 488 (shown in green). Nucleoli were stained with TO-PRO-3 (shown in blue). Images were examined using a Leica TCS SP8 confocal microscope. The scale bar represents 20 μm. B. Western blots to determine the molecular weights and subunit distribution of proteins in the fat body (FB) or hemolymph (HL). Extracted hemolymph or fat-body proteins were fractionated by SDS-PAGE (10%) and probed with the subunit-specific antibodies Ab42K, Ab67K2 and Ab111K. M is the molecular weight marker (kDa). Identified LsVg subunits are indicated by the arrows on the right. C. Confocal microscopic images showing co-localization of the N-terminal small (Small) and C-terminal large (Large) subunits of LsVg. The large subunit was probed with antibody Ab111Km and stained with Alexa Fluor 488 (shown in green). The small subunit was probed with antibody Ab42K and stained with Alexa Fluor 568 (shown in red). Images were examined using a Leica TCS SP8 confocal microscope. The scale bar represents 20 μm. D. The mRNA abundance of LsVn subunits. The mRNA copy numbers were determined by SYBR Green-based qPCR. Each dot, square or triangle represents one fat-body sample collected from one female SBPH. NS, not significant. E. Western blots showing the influence of subunit-specific gene silencing on expression levels of multiple subunits. RNAi with dsRNA specific to either the N-terminal small (Small) or C-terminal large (Large) subunit dramatically decreased expression levels of both subunits. RNAi with dsGFP was used as a negative control and did not influence the expression of LsVg. Protein levels were detected with antibodies Ab67K2 or Ab42K. M is the molecular weight marker (kDa). Positions of the LsVg subunits (Small and Large) are indicated by the arrows on the right.

Fig 5. LsVg expression in SBPH nymphs and males.

A. LsVg mRNA distribution in different tissues of SBPH nymphs and males was revealed by qPCR. The mean and SD were calculated from three independent experiments, with four mRNA samples per experiment. Ef2, L. striatellus elongation factor 2 gene; HL, hemolymph; FB, fat body; SG, salivary glands; MG, midgut. TE, testis. B. Confocal microscopic image showing the existence of LsVg and its co-localization with RSV in hemocytes. LsVg was probed with LsVn-subunit specific antibodies Ab42K, Ab67K2 and Ab111K and stained with Alexa Fluor 568 (shown in red). RSV was stained with Alexa Fluor 488 (shown in green). Nucleoli were stained with TO-PRO-3 (shown in blue). C. Confocal microscopic image showing co-localization of the N-terminal small (Small) and C-terminal large (Large) subunits of LsVg. The large subunit was probed with antibody Ab111Km and stained with Alexa Fluor 488 (shown in green). The small subunit was probed with antibody Ab42K and stained with Alexa Fluor 568 (shown in red). D. Confocal microscopic image showing localization of LsVg and phagocytosed E. coli containing the gfp gene conferring green fluorescence. LsVg was probed with Ab42K and stained with Alexa Fluor 568 (shown in red). Images were examined using a Leica TCS SP8 confocal microscope. The scale bar represents 20 μm. E. LsVg is not cleaved in male L. striatellus. Extracted hemolymph proteins were fractionated by SDS-PAGE (10%) and probed with the subunit-specific antibodies Ab47Km, Ab67K2 and Ab111K. M, the molecular weight marker (kDa). Arrows on the right, identified LsVg proteins.

Fig 6. Influence of LsVg deficiency on RSV survival and transmission.

A and C. Treatment of RSV-infected (A) and RSV-free (C) three-instar nymphs with the dsRNA of LsVg (dsVg), which resulted in significantly lower LsVg expression levels compared with those after treatment with the dsRNA of GFP (dsGFP). LsVg was probed with antibody Ab111Km and stained with Alexa Fluor 488 (shown in green). Nucleoli were stained with TO-PRO-3 (shown in blue). Images were examined using a Leica TCS SP8 confocal microscope. The scale bar represents 20 μm. B. In RSV-infected SBPHs, LsVg-deficiency decreased the RSV titer in both the hemolymph and salivary glands but had no effect in the midgut or fat body. D. In RSV-free SBPHs, RSV delivered into dsVg-treated insects exhibited significantly decreased survival rates compared with those of dsGFP-treated insects following virus delivery. E. Influence of anti-Vg antibodies on RSV survival in L. striatellus hemolymph. NRS, normal rabbit serum. NS, not significant. **, p<0.01, *, p<0.05, ****, p<0.0001. The mean and SD were calculated from three independent experiments. CP, the RSV capsid protein; ef2, the L. striatellus elongation factor 2 gene.