Plant Arbovirus Mobilizes a Vector Salivary Protein to Initiate Plant Infection

Plant arboviruses rely heavily on insects’ feeding activities for successful transmission. Insect salivary proteins have been suggested to be essential for successful viral infection, but their exact mechanisms are largely unknown. In this study, we reveal that salivary factors from Laodelphax striatellus are necessary for infection of Rice stripe virus (RSV) in plants. A salivary carbonic anhydrase (LssaCA) is identified as an essential factor in promoting RSV infection. LssaCA interacts with a rice thaumatin-like protein (OsTLP) that has endo-β-1,3-glucanase activity and can degrade callose in plants. RSV infection induces callose deposition, which can be reversed by LssaCA. Furthermore, LssaCA directly binds to the RSV nucleocapsid protein (NP) in salivary glands, and the LssaCA-RSV NP complex still binds OsTLP and further increases its glucanase activity. This study provides new insights into the tripartite virus-insect vector-plant interaction, which is relevant to many agriculturally important plant arboviruses whose transmission is facilitated by insect salivary proteins.

transmits RSV in a persistent-propagative manner. The virus initially infects the 94 midgut, then disperses from the hemolymph into the salivary glands, and is inoculated 95 into the plant host during L. striatellus feeding (Huo et al., 2022). L. striatellus 96 belongs to the order Hemiptera, whose members mainly feed from sieve tubes 97 through their mouthparts (stylets) that penetrate plant tissues and reach sieve tubes to 98 ingest the phloem sap (Tjallingii, 2006;van Bel & Will, 2016). RSV is secreted into 99 the rice phloem via the watery saliva (Huo et al., 2022;Wang & Blanc, 2021).

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In this study, we identified a molecular interaction among RSV, an L. striatellus 101 saliva protein, and a plant β-1,3-glucanase. The insect saliva protein directly binds to 102 the RSV nucleocapsid protein (NP) and then binds to a rice thaumatin-like protein to 103 activate its β-1,3-glucanase activity. The activation of β-1,3-glucanase helps facilitate 104 RSV infection of the host plant by inhibiting callose deposition in response to viral 105 infection. LssaCA-specific double-stranded RNA (dsLssaCA) was synthesized and delivered 128 into the hemocoel of RSV-infected 3 rd instar nymphs to interfere with the gene 129 expression. At 2 days post-microinjection (dpi), the LssaCA mRNA levels were 130 measured by RT-qPCR and compared with the dsGFP control. The results showed that 131 dsLssaCA treatment reduced the LssaCA mRNA level by 80% (Fig. 3A). The two 132 groups of insects were then allowed to feed on healthy rice seedlings (5 insects per 133 7 rice seedling) for 2 days. The RSV NP RNA levels (indicative of RSV titers) in the 134 plants were measured at 14 days post-feeding (dpf). Compared with the rice seedlings 135 fed by dsGFP-treated insects, those fed by dsLssaCA-treated insects had significantly 136 reduced RSV titers (Fig. 3B), indicating that LssaCA played an essential role in 137 mediating RSV infection of the rice plants.

LssaCA enhances RSV infection not before the viruses have been inoculated into
139 the plant phloem 140 To determine at which step LssaCA-deficiency affected RSV infection, we 141 analyzed the influence of LssaCA-deficiency on RSV titers during several steps of the 142 RSV transmission process, including the virus load in the salivary glands and saliva, 143 and the initial inoculation levels in planta. First, at 2 days after dsLssaCA treatment of 144 the viruliferous insects when LssaCA was significantly downregulated, RT-qPCR was 145 used to detect RSV titer in the insect salivary glands and the results showed that 146 LssaCA deficiency did not reduce RSV titers in this tissue (Fig. 3C). Then, the 147 LssaCA-deficient insects were allowed to feed on an artificial diet and their saliva 148 was collected for RSV level analysis, which revealed that LssaCA-deficiency did not 149 reduce the RSV titer in the secreted saliva (Fig. 3D). Finally, plants were fed with 10 150 viruliferous insects for 24 h respectively and the RSV titers were measured 151 immediately after feeding, which showed that LssaCA-deficiency did not affect the 152 RSV initial inoculation levels in planta (Fig. 3E). These results suggested that 153 LssaCA promoted RSV infection by a mechanism occurred not in insects or early 154 stage of viral entry in plants, but in planta after viral inoculation.

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As shown in Fig. 1 planta, we produced a recombinant LssaCA protein to pre-incubate with purified RSV 159 particle solution and used bovine serum albumin (BSA) as a control for comparison.

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The two groups were then microinjected into leaves on different plants, and at 14 dpi 161 8 their viral titers were measured using NP gene-specific RT-qPCR. The results showed 162 that compared to those inoculated with BSA plus RSVs particles, those inoculated 163 with LssaCA plus these same particles exhibited significantly higher RSV titer values 164 ( Fig. 3F-G). These findings clearly indicate that LssaCA plays an important role in 165 promoting RSV infection of plants after RSV has been inoculated into the plant.

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LssaCA interacts with rice thaumatin-like protein to increase its 167 endo-β-1,3-glucanase activity 168 Because LssaCA played its role in planta, we performed two experiments to   pull-down assays showed that OsTLP and LssaCA had a specific interaction (Fig. 4C).

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Previous studies have reported that TLP orthologs in apples, cherries, tomatoes,   212 Callose deposition on sieve plates is an important mechanism for slowing virus   246 We have confirmed that LssaCA binds to OsTLP to promote callose degradation 247 and facilitates RSV infection in planta, we then investigated whether there is a direct 248 interaction between LssaCA and RSV by which RSV directly participates in the 249 function played by LssaCA. By using RSV and LssaCA-specific antibodies to 250 conduct immunofluorescence assays (IFA), we found that LssaCA and RSV 251 co-localized in salivary glands (Fig. 7A), suggesting a potential molecular interaction 252 between them. A pull-down assay then revealed a specific interaction between 253 LssaCA and RSV NP (Fig. 7B). An MST assay determined that the K D of the 254 interaction between LssaCA and NP was 2.7 ± 2.2 μM, further confirming the 255 molecular interaction (Fig. 7C).

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As LssaCA interacts with both RSV NP and OsTLP, we measured the tripartite 257 interaction. Through pull-down assays, we discovered that when LssaCA was 258 pre-incubated with RSV to form an NP-LssaCA complex, the complex still had the 259 ability to bind to OsTLP ( Fig 7D). This result demonstrates a tripartite interaction 260 among these three proteins. 261 We then explored how the tripartite interaction affects OsTLP enzymatic activity.

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OsTLP was pre-incubated with LssaCA-NP complex or LssaCA control. After 263 incubation for 2 h, the enzyme activity was measured. The results showed that   Hao et al., 2008). In this study, we demonstrate a novel 304 mechanism by which an insect saliva protein binds to a plant β-1,3-glucanases protein, 305 thus activating the enzymatic activity and promoting callose degradation. 306 In this study, we found that plants fed on by viruliferous insects showed higher 307 callose deposition than did plants fed on by uninfected insects (Fig. 5A-C), 308 suggesting that arbovirus infection via insect saliva may represent a double stress to 309 the plant, inducing stronger callose deposition that must be overcome for successful               Table S1. Primers used in this study.  The RSV-specific antibody was produced using RSV ribonucleoproteins (RNPs) 499 as antigens. The LssaCA polyclonal antibody was produced using recombinantly 500 expressed LssaCA protein (with a His-tag at the C-terminus) as the antigen.

501
Tissue collection 502 Insect dissection and tissue collection were performed using previously 503 described procedures (Huo et al., 2018). In brief, insects were anesthetized at 4°C for 504 10 min, and the forelegs were severed at the coxa-trochanter joint using forceps. The    519 Saliva was collected as described previously (Huo et al., 2022). Watery saliva 520 was obtained by centrifuging the collected artificial diet at 10,000 ×g for 10 min to   529 Carbonic anhydrase activity was characterized by measuring its esterase activity.    The callose solution was prepared as follows: leaf material from the feeding site was 568 homogenized with PBS buffer (pH 7.4) and the mixture was centrifuged at 500-1000 569 g for 20 min. The supernatant was collected for the detection of callose concentration.

570
The absorbance at 450 nm was measured and callose concentrations were calculated 571 based on the standard curve.

RNA interference
573 24 We conducted RNAi analyses to determine the function of LssaCA in mediating 574 RSV infection and regulating callose deposition. The LssaCA-specific gene fragment 575 was PCR-amplified with the primer pair LssaCA-T7F/LssaCA-T7R (Table S1)  The negative control, GFP dsRNA, was synthesized and microinjected following the 582 same protocol. primer pair used to amplify LssaCA was LssaCA-q-F/LssaCA-q-R (Table S1). Viral 590 RNA copies were measured using the primers NP-q-F/NP-q-R (Table S1). L. 591 striatellus translation elongation factor 2 (EF2) and rice actin were amplified as 592 internal controls to ensure equal loading of cDNA isolated from different samples.

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The primer pairs EF2-q-F/EF2-q-R and actin-q-F/actin-q-R (Table S1) were used to 594 amplify EF2 and actin, respectively. Water was used as a negative control.

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Yeast two-hybrid assays 596 Yeast two-hybrid screening was performed to identify rice proteins interacting 597 with LssaCA. A high-complexity rice cDNA library was fused to Gal4 AD and    644 We conducted MST assays to detect the interactions between LssaCA and NP,

RSV inoculation through midrib microinjection
Midrib microinjection was performed as described previously (Zhao et al., 2017).  produce a reducing terminus. Therefore, the rate of reducing sugars production was 670 used to calculate enzyme activity. One unit of β-1,3-glucanase activity was defined as 671 the amount of enzyme that hydrolyses laminarin to generate 1 μg reducing sugars per 672 minute.

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The β-1,3-glucanase activity of OsTLP was measured using a beta-1,3-glucanase      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. E.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. F. 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. 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. 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. J. Callose concentration in leaves of rice plants fed on by dsGFP-or dsLssaCA-treated L. striatellus. **, p < 0.01.