Laodelphax striatellus feeding is necessary for Rice stripe virus (RSV)to infect rice.

A. The schematic diagram shows the two methods of rice inoculation with RSV: viruliferous insect feeding (VIF) and microinjection of purified virus (MPV). Three-leaf rice seedlings were used and viral infection was detected three weeks after inoculation.

B-D. Plant symptoms of rice stripe disease were observed in the VIF group (B-D), including heart leaf curling (C) and the striped spots on the leaves (D), but not in the MPV group (B and D). C shows an enlarged image of the VIF-group leaves.

A. E. Western blots to show the levels of RSV infection in plants inoculated with VIF or MPV. Anti-RSV antibodies were used to detect the RSV titers in the plants.

Characteristics of the L. striatellus saliva carbonic anhydrase (LssaCA).

A. RT-qPCR analyses of LssaCA transcript levels in different tissues. R-body, the remaining body after removal of the guts, salivary glands, fat bodies, and ovaries.

B. Western blots showing LssaCA protein distribution in L. striatellus salivary glands (SBPH SG) and rice plants (Rice). The weaker signal in dsLssaCA-treated SBPH indicates a specific knockdown of LssaCA by dsLssaCA. Rice plants fed by SBPH acquire LssaCA (+SBPH), whereas control plants do not contain LssaCA protein (-SBPH).

C. Western blots showing the distribution of LssaCA in watery and gel saliva. Total watery or gel saliva proteins were detected by silver staining.

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

E. Esterase activity of recombinantly expressed LssaCA protein.

The influence of LssaCA deficiency on RSV transmission and viral infection of plants.

A. The efficiency of gene silencing as determined by RT-qPCR. Each dot represents salivary glands from five RSV-infected third-instar nymphs.

B. RSV infection level in rice plants as determined by RT-qPCR. RSV infection levels are represented by the copy number of the NP gene. Each dot represents one rice plant fed on for 2 d and then grown for another 14 d.

C. RSV titers in L. striatellus salivary glands as determined by RT-qPCR. Each dot represents salivary glands from five RSV-infected third-instar nymphs.

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

E. RSV titers in rice plants as determined by RT-qPCR. Rice plants were fed for 24 h and RSV titers were assayed immediately post-feeding. Each dot represents one rice plant.

F. Schematic diagram to show the process of microinjecting an RSV particle solution into a plant. The RSV was pre-incubated with BSA or recombinantly expressed LssaCA protein before being microinjected into the plant phloem.

G. RT-qPCR to determine the level of RSV infection in microinjection-inoculated rice. The titers of RSV (copy number of NP) in rice plants were measured at 14 days post-inoculation (dpi). BSA was used as a negative control. *, p < 0.05.

DsLssaCA and dsGFP indicate LssaCA- and GFP-specific dsRNA, respectively. ****, p < 0.0001; **, p < 0.01; ns, not significant.

The interaction between OsTLP and LssaCA plays a role in regulating the enzymatic activity of OsTLP.

A. Schematic diagram of the 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 the 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 the interactions between LssaCA (LssaCA-His) and OsTLP (MBP-OsTLP). MBP was used as a negative control. Both anti-His and anti-MBP antibodies were used to detect corresponding proteins.

D. MST assay showing the interaction between LssaCA (LssaCA-His) and OsTLP (MBP-OsTLP). MBP was used as a negative control. Bars represent SE.

E. The endo-β-1,3-glucanase activity of the purified recombinant OsTLP protein (expressed as MBP-fusion protein). MBP was used to normalize results.

F. The endo-β-1,3-glucanase activity of OsTLP, which was overexpressed in transgenic plants (OsTLP OE) or wild-type plants (WT).

G. Regulation of OsTLP enzymatic activity by LssaCA. ****, p < 0.0001.

LssaCA inhibits RSV-induced callose deposition.

A. Bright blue fluorescence of cross-sections showing callose deposition at feeding sites. Samples were prepared from the leaf phloem of plants fed on by RSV-free or RSV-infected L. striatellus. Thin sections were stained with 0.1% aniline blue at 24 h after L. striatellus feeding and examined under a fluorescence microscope. Xy, xylem; ph, phloem. Scale bars: 20 μm.

B. Average fluorescence intensity of arbitrary area of callose deposition counted using ImageJ. Eight to ten random sites per sample were selected for the evaluation of fluorescence intensity. ****, p < 0.0001.

C. Callose concentration in leaves of rice plants fed on by RSV-free or RSV-infected L. striatellus. ****, p < 0.0001.

D–G. Transcript levels of callose synthase genes as determined by RT-qPCR. Insects were allowed to feed on rice plants for 24 h before total RNAs were extracted. *, p < 0.05; **, p < 0.01.

A. H. Bright blue fluorescence of cross-sections showing callose deposition at feeding sites. Samples were prepared from leaf phloem of plants fed on by dsGFP- or dsLssaCA-treated RSV-infected L. striatellus. Thin sections were stained with 0.1% aniline blue at 24 h after L. striatellus feeding and examined under a fluorescence microscope. Xy, xylem; ph, phloem. Scale bars: 20 μm.

I. Average fluorescence intensity of arbitrary area of callose deposition counted using ImageJ under a fluorescence microscope. Eight to ten random sites per sample were selected for the evaluation of fluorescence intensity. **, p < 0.01.

B. J. Callose concentration in leaves of rice plants fed on by dsGFP- or dsLssaCA-treated L. striatellus. **, p < 0.01.

OsTLP inhibits callose deposition to facilitate RSV infection.

A. Bright blue fluorescence of cross-sections showing callose deposition at feeding sites. Samples were prepared from the leaf phloem of plants fed on by RSV-infected L. striatellus. Scale bars: 20 μm. OsTLP OE, transgenic plants over-expressing OsTLP.

B. Average fluorescence intensity of arbitrary area of callose deposition counted using ImageJ under a fluorescence microscope. *, p < 0.05.

C. Promotion of RSV infection by OsTLP overexpression, as determined by RT-qPCR. Titers of RSV (copy number of NP) were measured at 14 days post-feeding. *, p < 0.05.

LssaCA-RSV NP interaction enhances the OsTLP enzymatic activity.

A. Immunofluorescence assay showing co-localization of RSV (shown in red) and LssaCA (shown in green) in L. striatellus salivary glands. Images are representative of three independent experiments with a total of 30 SBPHs analyzed. The scale bar represents 20 μm. psg, principal salivary gland; asg, accessory salivary gland.

B. GST pull-down assays show the interaction between LssaCA (GST-LssaCA) and RSV NP (NP-His). GST was used as a negative control. Anti-GST and anti-His antibodies were used to detect the corresponding proteins.

C. MST assay showing the interaction between LssaCA (LssaCA-His) and RSV NP (GST-NP). GST was used as a negative control. Bars represent SE.

D. Pull-down assays showing the interaction 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 preincubated before co-incubation with OsTLP. Anti-His and anti-GST antibodies were used to detect the corresponding proteins.

A. E. Regulation of OsTLP enzymatic activity by LssaCA and LssaCA-NP. (LssaCA + NP) indicates that the two proteins were pre-incubated before incubation with OsTLP. ****, p < 0.0001.

Proposed model of how RSV NP-LssaCA-OsTLP tripartite interaction leads to callose degradation to promote RSV infection of plants.

A. L. striatellus feeding on rice plants inoculates both RSV and LssaCA into the phloem sieve elements. Within the plants, RSV induces callose synthesis, causing callose deposition to inhibit RSV infection. However, LssaCA reverses this inhibition by binding to OsTLP to enhance its β-1,3-glucanase activity, which degrades the deposited callose. The interaction between RSV and LssaCA further enhances the OsTLP enzymatic activity and facilitates RSV infection.