An L. striatellus salivary carbonic anhydrase (LssaCA) directly binds to RSV NP in salivary glands

A. Immunofluorescence showing co-localization of RSV (red) and LssaCA (green) in L. striatellus salivary glands. Images represent three independent experiments with 30 SBPHs analyzed. Scale bar: 20 μm. psg, principal salivary gland; asg, accessory salivary gland.

B. Schematic of LssaCA protein sequence. SP, signal peptide; Carb_anhydrase domain, conserved eukaryotic-type carbonic anhydrase sequence. Triangles indicate seven predicted catalytically active residues.

C. Esterase activity of recombinant LssaCA protein expressed in sf9 insect cell and E. coli system. In LssaCA mutant protein, all seven predicted catalytically active residues were replaced. Mean and SD were calculated from 2 independent enzymatic assays. ****, P<0.0001.

D. GST pull-down assays demonstrating interaction between LssaCA (GST-LssaCA) and RSV NP (NP-His). GST served as negative control. Anti-GST and anti-His antibodies detected corresponding proteins.

E. MST assay showing binding between LssaCA (LssaCA-His) and RSV NP (GST-NP). GST served as negative control. Error bars represent SD.

F. Measurement of LssaCA tissure-specific expression by RT-qPCR and western blotting. Third-instar RSV-free nymphs were used to measure the gene expression in gut, salivary gland, fatbody, and remaining tissues (R-body) after dissection of the aforementioned tissues. Adult females and adult males were used to measure gene expression in ovaries and testes, respectively. Mean and SD were calculated from three independent experiments with 5 tissue samples each experiment. LssaCA protein was detected using anti-LssaCA antibodies.

G. Western blot analysis of LssaCA protein in RSV-free SBPH salivary glands. dsLssaCA treatment significantly reduces target protein levels compared to controls. LssaCA protein was detected using anti-LssaCA antibodies.

H. Western blot analysis showing LssaCA distribution in watery and gel saliva of RSV-free insects. LssaCA protein was detected using anti-LssaCA antibodies. Total saliva proteins were visualized by silver staining.

I. Western blot detection of LssaCA proteins in rice phloem following L. striatellus feeding. LssaCA protein was detected using anti-LssaCA antibodies.

J. Analyses of LssaCA levels regulated by RSV infection. Third-instar RSV-free and RSV-infected nymphs were used to measure gene expression (RT-qPCR) and protein accumulation (Western blotting). Mean and SD were calculated from three independent experiments with totally 14 tissue samples each experiment. ns, not significant.

All western blot images are representative of three independent experiments.

LssaCA enhances RSV infection in rice plants

A. Gene silencing efficiency determined by RT-qPCR.

B-C. Analysis of RSV infection levels in rice seedlings. Each plant seedling was fed upon by 5 infected insects for 2 days, followed by 14 days of cultivation. RSV levels were determined by RT-qPCR (B) and western blotting (C). RSV NP detected by anti-NP polyclonal antibodies indicated viral titers. Rubisco large subunit (RBCL) served as protein loading control.

D. Gene silencing efficiency determined by RT-qPCR.

E. RT-qPCR analysis of RSV titers in salivary glands. Each dot represents salivary glands from 5 RSV-infected third-instar nymphs.

F. RSV titers in L. striatellus saliva determined by RT-qPCR. Each dot represents one saliva sample from 10 insects fed with artificial diet.

G. RT-qPCR analysis of RSV titers in rice. Plants were fed upon by infected insects for 24 h (Left: schematic of L. striatellus feeding on specific rice stem sites), with titers assayed immediately post-feeding (Right). Each dot represents an individual rice seedling..

H. Western blot analysis of LssaCA in plants fed upon by 50 RSV-free insects for 2 days and continuously cultured for an additional 1 and 3 days. LssaCA protein was detected using anti-LssaCA antibodies. dpf, days post-feeding.

I-J. RSV infection in plants at 3 dpf. Five RSV-infected third-instar L. striatellus nymphs were fed upon healthy rice plants for 2 days, and viral titers in whole plant shoots were measured at 3 dpf by RT-qPCR (I) and western blotting (J). Each dot represents an individual plant. RSV NP detected by anti-NP polyclonal antibodies indicated viral titers. RBCL protein served as a protein loading control.

Mean and SD were calculated from 3 independent microinjection experiments. ns, not significant; *, P<0.05; **, P<0.01; ****, P<0.0001. All western blot images are representative of three independent experiments.

LssaCA interacts with rice thaumatin-like protein (OsTLP) to enhance its endo-β-1,3-glucanase activity

A. Schematic diagram of OsTLP protein sequence. SP, signal peptide; THM, conserved thaumatin family protein sequence. Glyco_hydro_64 domain, glycoside hydrolases of family 64.

B. Yeast two-hybrid assay showing interaction between OsTLP and LssaCA. SD, synthetically defined medium; Leu, Leucine; Trp, Tryptophan; His, Histidine; 3’AT, 3-amino-1,2,4-triazole; AD, transcription activation domain; BD, DNA-binding domain.

C. Pull-down assays showing interactions between LssaCA (LssaCA-His) and OsTLP (MBP-OsTLP). MBP served as a negative control. Anti-His and anti-MBP polyclonal antibodies were used for protein detection. Western blot images are representative of three independent experiments.

D. MST assay showing molecular interactions between LssaCA (LssaCA-His) and OsTLP (MBP-OsTLP). MBP served as a negative control. Error bars represent SD.

E. Endo-β-1,3-glucanase activity of purified recombinant OsTLP protein expressed as MBP-fusion protein. MBP was used for normalization.

F. Endo-β-1,3-glucanase activity in OsTLP-overexpressing transgenic plants (OsTLP OE) or wild-type plants (WT). Plant total soluble proteins were used for enzymatic activity assays. Units mg-1 means units per mg of plant total soluble proteins.

G. Regulation of OsTLP enzymatic activity by LssaCA binding.

H. OsTLP enzymatic activity measured at pH 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 to assess pH dependence.

I. RT-qPCR analysis of RSV infection in OsTLP OE and WT plants. RSV titers (NP copy number) were measured at 14 dpf. ns, not significant; *, P<0.05; ***, P<0.001; ****, P<0.0001.

RSV NP-LssaCA-OsTLP tripartite interaction further enhances OsTLP enzymatic activity

A. Pull-down assays showing interactions between OsTLP (MBP-OsTLP) and LssaCA-NP complex. LssaCA was expressed with a His tag, and RSV NP was expressed as a GST-fusion protein. LssaCA and NP were pre-incubated before co-incubation with OsTLP. Anti-His, anti-GST, and anti-MBP antibodies were used to detect corresponding proteins.

B. Endo-β-1,3-glucanase activity of OsTLP after co-incubation with LssaCA (LssaCA + GST) and LssaCA-NP complex (LssaCA + NP). LssaCA was pre-incubated with either GST or NP before addition to OsTLP. ****, P<0.0001.

LssaCA inhibits L. striatellus-transmitted RSV-induced callose deposition in rice plants

A. Callose concentration quantified by ELISA in leaves of rice plants fed on by RSV-free or RSV-infected L. striatellus. Non-fed plants served as the control (CK).

B. Bright blue fluorescence of cross-sections showing callose deposition at feeding sites. Samples were prepared from the leaf phloem of plants that were not fed or were fed on by RSV-free or RSV-infected L. striatellus. Thin sections were stained with 0.1% aniline blue at 24 h post-feeding and examined under a fluorescence microscope. xy, xylem; ph, phloem. Scale bars: 10 μm.

C. Average fluorescence intensity of callose deposition measured using ImageJ. Eight to ten random sites per sample were selected for intensity quantification.

D–H. Transcript levels of callose synthase genes (gls3, gls4, gls5, gls10) and OsTLP determined by RT-qPCR. Insects were allowed to feed on rice plants for 24 h prior to RNA extraction.

I. Callose concentration quantified by ELISA in leaves of rice plants fed on by dsGFP- or dsLssaCA-treated RSV-infected L. striatellus.

J. Bright blue fluorescence of cross-sections showing callose deposition at feeding sites. Samples prepared from the leaf phloem of plants fed on by dsGFP- or dsLssaCA-treated RSV-infected L. striatellus. Scale bars: 20 μm.

K. Average fluorescence intensity of callose deposition measured using ImageJ. Eight to ten random sites per sample were selected for intensity quantification.

Mean and SD were calculated from three independent experiments. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

LssaCA mediates in planta callose degradation by enhancing OsTLP enzymatic activity

A. Callose concentration quantified by ELISA in leaves of wild-type (WT), OsTLP overexpression (OsTLP OE), and OsTLP mutant (ostlp) plants.

B. Bright blue fluorescence of cross-sections showing callose deposition in leaf phloem. Plants were not fed on by insects. Scale bars: 10 μm. Average fluorescence intensity of callose deposition was measured using ImageJ. Eight to ten random sites per sample were selected for intensity quantification. xy, xylem; ph, phloem.

C. Callose concentration quantified by ELISA in leaves of ostlp plants fed on by dsGFP- or dsLssaCA-treated RSV-infected L. striatellus.

D. Bright blue fluorescence of cross-sections showing callose deposition at feeding sites. Samples were prepared from the leaf phloem of ostlp plants fed on by dsGFP- or dsLssaCA-treated RSV-infected L. striatellus. Scale bars: 10 μm. Average fluorescence intensity was quantified using ImageJ. Eight to ten random sites per sample were selected for intensity quantification.

Mean and SD were calculated from three independent experiments. ns, not significant; *, p < 0.05. ****, p < 0.0001.

Proposed model of RSV NP-LssaCA-OsTLP tripartite interaction facilitating callose degradation and promoting RSV infection.

In L. striatellus salivary glands (SG), RSV (red star) binds to LssaCA proteins (blue triangle) via the nucleocapsid protein (NP). Upon delivery into the rice phloem, RSV infection triggers callose synthesis, leading to callose deposition that restricts viral spread. However, LssaCA binds to OsTLP (yellow scissors), enhancing its β-1,3-glucanase activity and promoting callose degradation. The RSV NP-LssaCA complex formed in the insect’s SG can still interact with OsTLP in planta, resulting in significantly higher OsTLP β-1,3-glucanase activity. This enhanced enzymatic activity efficiently degrades callose, thereby facilitating RSV infection.