Rice stripe virus utilizes an Laodelphax striatellus salivary carbonic anhydrase to facilitate plant infection by direct molecular interaction

Reviewer #1 (Public review):

In this study, the authors identified an insect salivary protein LssaCA participating viral initial infection in plant host. LssaCA directly bond to RSV nucleocapsid protein and then interacted with a rice OsTLP that possessed endo-β-1,3-glucanase activity to enhance OsTLP enzymatic activity and degrade callose caused by insects feeding. The manuscript suffers from fundamental logical issues, making its central narrative highly unconvincing.

(1) These results suggested that LssaCA promoted RSV infection through a mechanism occurring not in insects or during early stages of viral entry in plants, but in planta after viral inoculation. As we all know that callose deposition affects the feeding of piercing-sucking insects and viral entry, this is contradictory to the results in Fig. S4 and Fig 2. It is difficult to understand callose functioned in virus reproduction in 3 days post virus inoculation. And authors also avoided to explain this mechanism.

(2) Missing significant data. For example, the phenotypes of the transgenic plants, the RSV titers in the transgenic plants (OsTLP OE, ostlp). The staining of callose deposition were also hard to convince. The evidence about RSV NP-LssaCA-OsTLP tripartite interaction to enhance OsTLP enzymatic activity is not enough.

(3) Figure 4a, there was the LssaCA signal in the fourth lane of pull-down data. Did MBP also bind LsssCA? The characterization of pull-down methods was rough a little bit. The method of GST pull-down and MBP pull-down should be characterized more in more detail.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

In this study, the authors identify an insect salivary protein participating viral initiate infection in plant host. They found a salivary LssaCA promoting RSV infection by interacting with OsTLP that could degrade callose in plants. Furthermore, RSV NP bond to LssaCA in salivary glands to form a complex, which then bond to OsTLP to promote degradation of callose.

The story focus on tripartite virus-insect vector-plant interaction and is interesting. However, the study is too simple and poor-conducted. The conclusion is also overstated due to unsolid findings.

We thank the reviewer for their constructive feedback. We have conducted additional experiments to strengthen our results and conclusions as detailed below:

(1) The comparison between vector inoculation and microinjection involves multiple confounding factors that could affect the experimental results, including salivary components, RSV inoculation titers, and the precision of viral deposition. The differential outcomes could be attributed to these various factors rather than definitively demonstrating the necessity of salivary factors. Therefore, we have removed this comparison from the revised manuscript and instead focused on elucidating the specific mechanisms by which LssaCA facilitates viral infection.

(2) We conducted new experiments to assess the function of LssaCA enzymatic activity in mediating RSV infection. Additional experiments revealed that OsTLP enzymatic activity is highly pH-dependent, with increased activity as pH decreases from 7.5 to 5.0 (Fig. 3H). However, the LssaCA-OsTLP interaction at pH 7.4 significantly enhanced OsTLP enzymatic activity without requiring pH changes. These results demonstrate that LssaCA-OsTLP protein interactions are crucial for mediating RSV infection. In contrast to pH-dependent mechanisms, our study demonstrated that LssaCA's biological function in mediating RSV infection is at least partially, if not completely, independent of its enzymatic activity. We have added these new resulted into the revised manuscript (Lines 220-227). We have also added a comprehensive discussion comparing the aphid CA mechanism described by Guo et al. (2023 doi.org/10.1073/pnas.2222040120) with our findings in the revised manuscript (Lines 350-371).

(3) We have repeated majority of callose deposition experiments, providing clearer images (Figures 5-6). In addition to aniline blue staining, we quantified callose concentrations using a plant callose ELISA kit to provide more precise measurements (Figure 5A, I, 6A, C and S8A). We utilized RT-qPCR to measure callose synthase expression in both feeding and non-feeding areas, confirming that callose synthesis was induced specifically in feeding regions, leading to localized callose deposition (Figures 5D-G and S8B-E). For sieve plate visualization, we examined longitudinal sections, which revealed callose deposition in sieve plates during SBPH feeding and RSV infection (Figure S7).

(4) We generated OsTLP mutant rice seedlings (ostlp) and use this mutant to directly demonstrate that LssaCA mediates callose degradation in planta through enhancement of OsTLP enzymatic activity (Lines 288-302 and Figure 6).

(5) We produced LssaCA recombinant proteins in sf9 cells to ensure full enzymatic activity and constructed a comprehensive CA mutant protein, in which all seven residues constituting the enzymatic active center mutated (LssaCA^H111D,LssaCA^N139H,LssaCA^H141D, LssaCA^H143D, LssaCA^E153H, LssaCA^H166D, LssaCA^T253E) (Fig. S1B). This LssaCA mutant protein demonstrated complete loss of enzymatic activity (Fig. 1C).

Major comments:

(1) The key problem is that how long the LssCA functioned for in rice plant. Author declared that LssCA had no effect on viral initial infection, but on infection after viral inoculation. It is unreasonable to conclude that LssCA promoted viral infection based on the data that insect inoculated plant just for 2 days, but viral titer could be increased at 14 days post-feeding. How could saliva proteins, which reached phloem 12-14 days before, induce enough TLP to degrade callose to promote virus infection? It was unbelievable.

We appreciate your insightful comment and acknowledge that our initial description may have been unclear. We agree that salivary proteins would not present in plant tissues for two weeks post-feeding or post-injection. Our intention was to clarify that when salivary proteins enhance RSV infection, this initial enhancement leads to sustained high viral loads. We measured viral burden at 14 days post-feeding or post-injection because this is the common measurement time point when viral titers are sufficiently high for reliable detection by qRT-PCR or western blotting. We have clarified this rationale in the revised manuscript (Lines 155-157).

To determine the actual persistence of LssaCA in plant tissues, we conducted additional experiments where insects were allowed to feed on a defined aera of rice seedlings for two days. We then monitored LssaCA protein levels at 1 and 3 days after removing the insects. Western blotting analysis revealed that LssaCA protein levels decreased post-feeding and remained detectable at 3 days post-feeding. These results are presented in Figure 2H and described in detail in Lines 184-193.

(2) Lines 110-116 and Fig. 1, the results of viruliferous insect feeding and microinjection with purified virus could not conclude the saliva factor necessary of RSV infection, because these two tests are not in parallel and comparable. Microinjection with salivary proteins combined with purified virus is comparable with microinjection with purified virus.

We thank the reviewer’s insightful comment. We agree that “the results of viruliferous insect feeding and microinjection with the purified virus could not conclude the saliva factor necessary of RSV infection”. However, due to the technical difficulty in collecting sufficient quantities of salivary proteins to conduct the microinjection experiment, we have removed these results from the revised manuscript.

(3) The second problem is how many days post viruliferous insect feeding and microinjection with purified virus did author detect viral titers? in Method section, authors declared that viral titers was detected at 7-14 days post microinjection. Please demonstrate the days exactly.

We thank the reviewer’s insightful comment. We typically measured RSV infection levels at both 7- and 14-days post-microinjection. However, since the midrib microinjection experiments have been removed from the revised manuscript, this methodology has also been removed accordingly.

(4) The last problem is that how author made sure that the viral titers in salivary glands of insects between two experiments was equal, causing different phenotype of rice plant. If not, different viral titers in salivary glands of insects between two experiments of course caused different phenotype of rice plant.

We thank the reviewer’s comment. When we compared the effects of LssaCA deficiency on RSV infection of rice plants, we have compared the viral titers in the insect saliva and salivary glands. The results indicated that the virus titers in both tissues have not changed by LssaCA deficiency, suggesting that the viruses inoculated into rice phloem by insects of different treatments were comparable. Please refer to the revised manuscript Figures 2D-G and Lines 161-173.

(5) The callose deposition in phloem can be induced by insect feeding. In Fig. 5H, why was the callose deposition increased in the whole vascular bundle, but not phloem? Could the transgenic rice plant directional express protein in the phloem? In Fig. 5, why was callose deposition detected at 24 h after insect feeding? In Fig. 6A, why was callose deposition decreased in the phloem, but not all the cells of the of TLP OE plant? Also in Fig.6A and B, expression of callose synthase genes was required.

We thank the reviewer for these insightful comments.

(1) Figure 5. The callose deposition increased in multiple cells within the vascular bundle, including sieve tubes, parenchymatic cells, and companion cells. While callose deposition was detected in other parts of the vascular bundle, no significant differences were observed between treatments in these regions, indicating that in response to RSV infection and other treatments, altered callose deposition mainly occurred in phloem cells. Please refer to the revised 5B, 5J, 6B, and 6D.

(2) Transgenic plant expression. The OsTLP-overexpressing transgenic rice plants express TLP proteins in various cells under the control of CaMV 35S promoter, rather than being directionally expressed in the phloem. However, since TLP proteins are secreted, they are potentially transported and concentrated in the phloem where they can degrade callose.

(3) Figure 5. The 24-hour time point for callose deposition detection was selected based on established protocols from previous studies. According to Hao et al. (Plant Physiology 2008), callose deposition increased during the first 3 days of planthopper infestation and decreased after 4 days. Additionally, Ellinger and Voigt (Ann Bot 2014) demonstrated that callose visualization typically begins 18-24 hours after treatment, making 24 hours an optimal detection time point.

(4) Figure 6, Phloem-specific changes. Similar to Figure 5, while callose deposition was detected in other parts of vascular bundle, significant differences between treatments were mainly observed in phloem cells, indicating that RSV infection specifically affects callose deposition in phloem tissue.

(5) Callose synthase gene expression. We performed RT-qPCR analysis to measure the expression levels of callose synthase genes. The results indicated that OsTLP overexpression did not significantly alter the mRNA levels of these genes, regardless of RSV infection status in SBPH.

Reviewer #2 (Public Review):

There is increasing evidence that viruses manipulate vectors and hosts to facilitate transmission. For arthropods, saliva plays an essential role for successful feeding on a host and consequently for arthropod-borne viruses that are transmitted during arthropod feeding on new hosts. This is so because saliva constitutes the interaction interface between arthropod and host and contains many enzymes and effectors that allow feeding on a compatible host by neutralizing host defenses. Therefore, it is not surprising that viruses change saliva composition or use saliva proteins to provoke altered vector-host interactions that are favorable for virus transmission. However, detailed mechanistic analyses are scarce. Here, Zhao and coworkers study transmission of rice stripe virus (RSV) by the planthopper Laodelphax striatellus. RSV infects plants as well as the vector, accumulates in salivary glands and is injected together with saliva into a new host during vector feeding.

The authors present evidence that a saliva-contained enzyme - carbonic anhydrase (CA) - might facilitate virus infection of rice by interfering with callose deposition, a plant defense response. In vitro pull-down experiments, yeast two hybrid assay and binding affinity assays show convincingly interaction between CA and a plant thaumatin-like protein (TLP) that degrades callose. Similar experiments show that CA and TLP interact with the RSV nuclear capsid protein NT to form a complex. Formation of the CA-TLP complex increases TLP activity by roughly 30% and integration of NT increases TLP activity further. This correlates with lower callose content in RSV-infected plants and higher virus titer. Further, silencing CA in vectors decreases virus titers in infected plants.

(1) Interestingly, aphid CA was found to play a role in plant infection with two non-persistent non-circulative viruses, turnip mosaic virus and cucumber mosaic virus (Guo et al. 2023 doi.org/10.1073/pnas.2222040120), but the proposed mode of action is entirely different.

We appreciate the reviewer’s insightful comment and have carefully examined the cited publication. The study by Guo et al. (2023) elucidates a distinct mechanism for aphid-mediated transmission of non-persistent, non-circulative viruses (turnip mosaic virus and cucumber mosaic virus). In their model, aphid-secreted CA-II in the plant cell apoplast leads to H⁺ accumulation and localized acidification. This trigger enhanced vesicle trafficking as a plant defense response, inadvertently facilitating virus translocation from the endomembrane system to the apoplast.

In contrast to these pH-dependent mechanisms, our study demonstrated that LssaCA’s biological function in mediating RSV infection is, if not completely, at least partially independent of its enzymatic activity. We performed additional experiments to reveal that OsTLP enzymatic activity is highly pH-dependent and exhibits increased enzymatic activity as pH decreases from 7.5 to 5.0 (Fig. 3H); however, the LssaCA-OsTLP interaction occurring at pH 7.4 significantly enhanced OsTLP enzymatic activity without any change in buffer pH (Fig. 3G). These results demonstrate the crucial importance of LssaCA-OsTLP protein interactions, rather than enzymatic activity alone, in mediating RSV infection.

We have incorporated these new experimental results and added a comprehensive discussion comparing the aphid CA mechanism described by Guo et al. (2023) with our findings in the revised manuscript. Please refer to Figures 3G-H, Lines 220-227 and 350-371 for detailed information.

(2) While this is an interesting work, there are, in my opinion, some weak points. The microinjection experiments result in much lower virus accumulation in rice than infection by vector inoculation, so their interpretation is difficult.

We acknowledge the reviewer's concern regarding the lower virus accumulation observed in microinjection experiments compared to vector-mediated inoculation. We have removed these experiments from the revised manuscript. To address the core question raised by these experiments, we have conducted new experiments that directly demonstrate the importance of LssaCA-OsTLP protein-protein interactions in mediating RSV infection. These results demonstrate the crucial importance of LssaCA-OsTLP protein interactions, rather than enzymatic activity alone, in mediating RSV infection. Additionally, we have incorporated a comprehensive discussion examining carbonic anhydrase activity, pH homeostasis, and viral infection. Please refer to the detailed experimental results and discussion in the sections mentioned in our previous response (Figures 3G-H, Lines 220-227 and 350-371).

(3) Also, the effect of injected recombinant CA protein might fade over time because of degradation or dilution.

We appreciate the reviewer’s insightful comment. This is indeed a valid concern that could affect the interpretation of microinjection results. To address the temporal dynamics of CA protein presence in planta, we conducted time-course experiments to monitor the retention of naturally SBPH-secreted CA proteins in rice plants. Our analysis at 1- and 3- days post-feeding (dpf) revealed that CA protein levels decreased progressively following SBPH feeding, but could also been detected at 3dpf (Fig. 2H). Please refer to Figures 2H and lines 184-193 for detailed information.

(4) The authors claim that enzymatic activity of CA is not required for its proviral activity. However, this is difficult to assess because all CA mutants used for the corresponding experiments possess residual activity.

We appreciate the reviewer’s insightful comment. We constructed a comprehensive CA mutant protein in which all seven residues constituting the enzymatic active center mutated (LssaCA^H111D, LssaCA^N139H, LssaCA^H141D, LssaCA^H143D, LssaCA^E153H, LssaCA^H166D, LssaCA^T253E) (Fig. S1B). This LssaCA mutant protein demonstrated complete loss of enzymatic activity (Fig. 1C). However, since we have removed the recombinant CA protein microinjection experiments from the revised manuscript, we lack sufficient direct evidence to definitively demonstrate that CA enzymatic activity is dispensable for its proviral function. To address the core question raised by these experiments, we have conducted new experiments that provide direct evidence for the importance of LssaCA-OsTLP protein-protein interactions in mediating RSV infection. Additionally, we have incorporated a comprehensive discussion examining carbonic anhydrase activity, pH homeostasis, and viral infection. Please refer to the detailed experimental results and discussion in the sections mentioned in our previous response (Figures 3G-H, Lines 220-227 and 350-371).

(5) It remains also unclear whether viral infection deregulates CA expression in planthoppers and TLP expression in plants. However, increased CA and TLP levels could alone contribute to reduced callose deposition.

We have compared LssaCA mRNA levels in RSV-free and RSV-infected L.striatellus salivary glands, which indicated that RSV infection does not significantly affect LssaCA expression (Figure 1J). By using RSV-free and RSV-infected L.striatellus to feed on rice seedlings, we clarified that RSV infection does not affect TLP expression in plants (Figure 5H).

Reviewer #1: (Recommendations For The Authors):

Other comments:

(1) Most data proving viral infection and LssaCA expression were derived from qPCR assays. Western blot data are strongly required to prove the change at the protein level.

We agree that western blot data are required to prove the change at the protein level. In the revised manuscript, we have added western-blotting results (Figures 1F, 1I, 2C, 2J, and S6).

(2) Line 145, data that LssaCA was significantly downregulated should be shown.

Thank you and the data has been added to the revised manuscript. Please refer to Line 165 and Figure 2D.

(3) Lines 159-161, how did authors assure that the dose of recombinant LssCA was closed to the release level of insect feeding, but not was excessive? How did author exclude the possibility of upregulated RSV titer caused by excessive recombinant LssCA?

We appreciate this important concern regarding dosage controls. While microinjection of recombinant proteins typically yields viral infection levels significantly lower than those achieved through natural insect feeding, higher protein concentrations are often required to achieve high viral infection levels. In this experiment, we compared RSV infection levels following microinjection of BSA+RSV versus LssaCA+RSV, with the expectation that any observed upregulation in RSV titer would be specifically attributable to recombinant LssaCA rather than excessive protein dosing. However, given the low RSV infection levels observed with viral microinjection, we have removed their corresponding results from the revised manuscript.

(4) Lines 124-125, recombinantly expressed LssaCA protein should be underlined, but not the LssaCA protein itself.

We have clearly distinguished recombinantly expressed LssaCA from endogenous LssaCA protein throughout the manuscript, ensuring that all references to recombinant proteins are properly labeled as such.

(5) LssaCA expression in salivary glands of viruliferous and nonviruliferous insects is required. LssaCA accumulation in rice plant exposed to viruliferous and nonviruliferous insects is also required.

We have measured LssaCA mRNA levels in salivary glands of viruliferous and nonviruliferous insects (Figure 1J), and protein levels in rice plant exposed to viruliferous and nonviruliferous insects (Figure 1I).

(6) Fig. 4G, the enzymatic activities of OsTLP were too low compared with that in Fig. 4E and Fig. 7E. Why did the enzymatic activities of the same protein show so obvious difference?

We apologize for the error in Fig. 4G. The original data presented relative fold changes between OsTLP+BSA and OsTLP+LssaCA treatment, with OsTLP+BSA normalized to 1.0 and OsTLP+LssaCA values expressed as fold changes relative to this baseline. However, the Y-axis was incorrectly labeled as “β-1,3-glucanase (units mg^-1)”, which suggested absolute enzymatic activity values. We have now corrected the figure (revised Figure 3G) to display the actual absolute enzymatic activity values with the appropriate Y-axis label “β-1,3-glucanase (units mg^-1)”.

(7) Fig. 7E, was the LssaCA + NP and LssaCA + GST quantified?

Yes, all proteins were quantified, and enzymatic activity values were calculated and expressed as units per milligram of proteins (units mg^-1).

Minor comments:

(1) The keywords: In fact, the LssaCA functioned during initial viral infection in plant, but not viral horizontal transmission.

We appreciate the reviewer’s insightful comment. We have revised the manuscript title to “Rice stripe virus utilizes an Laodelphax striatellus salivary carbonic anhydrase to facilitate plant infection by direct molecular interaction” and changed the keyword from “viral horizontal transmission” to “viral infection of plant”.

(2) Fig. 2A, how about testes? Was this data derived from female insects? Fig. 2C, is the saliva collected from nonviruliferous insects? Fig. 2E, what is the control?

We appreciate the reviewer’s insightful comments.

(1) Fig. 2A: The data present mean and SD calculated from three independent experiments, with 5 tissue samples per experiment. Since 3^rd instar nymphs were used for feeding experiments in this study, we also used 3^rd instar RSV-free nymphs to measure gene expression in guts, salivary glands and fat bodies. R-body represents the remaining body after removing these tissues. Female insects were used to measure gene expression in ovaries, and gene expression in testes was also added. We have added this necessary information to the revised manuscript (please refer to new Figure 1F and Lines 402-403).

(2) Fig. 2C: Yes, saliva was collected from nonviruliferous insects.

(3) Fig. 2E: The control consisted of 100 mM PBS, as described in the experimental section (Lines 643-644): “A blank control consisted of 2 mL of 100 mM PBS (pH 7.0) mixed with 1 mL of 3 mM p-NPA.” In the revised manuscript, we recombinantly expressed LssaCA and its mutant proteins in both sf9 cells and E.coli. Therefore, we have used the mutant proteins as controls to demonstrate specific enzymatic activity. Please refer to Figure 1C, Lines 115-122 and 621-635 for detailed information.

(3) Some figure labeling appeared unprofessional. For example, "a-RSV", "loading" in Fig. 1, "W-saliva", "G-saliva" in Fig. 2, and so on, the related explanations were absent.

We appreciate the reviewer’s insightful comments. We have thoroughly reviewed all figures to ensure professional labels. Specifically, we have:

(1) Used proper protein names to label western blots and clearly explained the antibodies used for protein detection.

(2) Provided comprehensive explanations for all abbreviations used in figures within the corresponding figure legends.

(3) Ensured consistent and clear labeling throughout all figures.

Please refer to the revised Figures 1-3 for these corrections.

(4) Lines 83-84, please cite references on callose preventing viral movement. I do not think the present references were relevant.

We have added a more relevant reference (Yue et al., 2022, Line 82), which revealed that palmitoylated γb promotes virus cell-to-cell movement by interacting with NbREM1 to inhibit callose deposition at plasmodesmata.

(5) The background of transgenic plants of OsTLP OE should be characterized. And the overexpression of OsTLP should be shown. Which generation of OsTLP OE did authors use?

The background of transgenic plants of OsTLP OE and its generation used have been shown in the “Materials and methods” section (Line 782-786) and has been mentioned in the main text (Line 214). T² lines have been selected for further analysis (Line 789).

(6) Fig. 5A, the blank, which derived from plants without exposure to insect, was absent.

We appreciate the reviewer’s insightful comments. We have added the non- fed control in the revised Figure 5A-C.

(7) Fig. 7A, the nonviruruliferous insects were required to serve as a control.

Immunofluorescence localization of RSV and LssaCA in uninfected L. striatellus salivary glands have been added to the revised manuscript (Figure S2).

(8) The manuscript needs English language edit.

The manuscript has undergone comprehensive English language editing to improve clarity, grammar, and overall readability.

Reviewer #2 (Recommendations For The Authors):

(1) The first experiment compares vector inoculation vs microinjection of RSV in tissue. I am not sure that your claim (saliva factors are necessary for inoculation) holds, because the vector injects RSV directly into the phloem, whereas microinjection is less precise and you cannot control where exactly the virus is deposed. However, virus deposited in other tissues than the phloem might not replicate, and indeed you observe, compared to natural vector inoculation, highly reduced virus titers.

We appreciate the reviewer’s insightful comments. We agree that the comparison between vector inoculation and microinjection involves multiple confounding factors that could affect the experimental results, including salivary components, RSV inoculation titers, and the precision of viral deposition. As the reviewer correctly points out, the differential outcomes could be attributed to these various factors rather than definitively demonstrating the necessity of salivary factors. Therefore, we have removed this comparison from the revised manuscript and instead focused on elucidating the specific mechanisms by which LssaCA facilitates viral infection.

(2) Next the authors show that a carbonic anhydrase (CA) that they previously detected in saliva is functional and secreted into rice. I assume this is done with non-infected insects, but I did not find the information. Silencing the CA reduces virus titers in inoculated plants at 14 dpi, but not in infected planthoppers. At 1 dpi, there is no difference in RSV titer in plants inoculated with CA silenced planthoppers or control hoppers. To see a direct effect of CA in virus infection, purified virus is injected together with a control protein or recombinant CA into plants. At 14 dpi, there is about double as much virus in the CA-injected plants, but compared to authentic SBPH inoculation, titers are 20,000 times lower. Actually, I believe it is not very likely that the recombinant CA is active or present so long after initial injection.

We appreciate the reviewer’s insightful comments.

(1) Our previous study identified the CA proteins from RSV-free insects. We have added this information to the revised manuscript (Line 110).

(2) We acknowledge the reviewer's concern regarding the lower virus accumulation observed in microinjection experiments compared to vector-mediated inoculation. We have removed these experiments from the revised manuscript and instead focused on elucidating the specific mechanisms by which LssaCA facilitates viral infection.

(3) We didn’t intend to suggest that LssaCA proteins presented for 14 days post-injection. We measured viral titers at 14 days post-feeding or post-injection because this is the common measurement time point when viral titers are sufficiently high for reliable detection by RT-qPCR or western blotting. We have clarified this rationale in the revised manuscript (Lines 155-157). To determine the actual persistence of LssaCA in plant tissues, we monitored LssaCA protein levels at 1 and 3 dpf. Western blotting analysis revealed that LssaCA protein levels decreased post-feeding and remained detectable at 3 dpf. These results are presented in Figure 2H and described in detail in Lines 184-193.

(3) Then the authors want to know whether CA activity is required for its proviral action and single amino acid mutants covering the putative active CA site are created. The recombinant mutant proteins have 30-70 % reduced activity, but none of them has zero activity. When microinjected together with RSV into plants, RSV replication is similar as injection with wild type CA. Since no knock-out mutant with zero activity is used, it is difficult to judge whether CA activity is unimportant for viral replication, as claim the authors.

We appreciate the reviewer’s insightful comment. We constructed a comprehensive CA mutant protein in which all seven residues constituting the enzymatic active center mutated (LssaCA^H111D, LssaCA^N139H, LssaCA^H141D, LssaCA^H143D, LssaCA^E153H, LssaCA^H166D, LssaCA^T253E) (Fig. S1B). This LssaCA mutant protein demonstrated complete loss of enzymatic activity (Fig. 1C). However, since we have removed the recombinant CA proteins microinjection experiments from the revised manuscript, we lack sufficient direct evidence to definitively demonstrate that CA enzymatic activity is dispensable for its proviral function. To address the core question raised by these experiments, we have conducted new experiments that provide direct evidence for the importance of LssaCA-OsTLP protein-protein interactions in mediating RSV infection. Additionally, we have incorporated a comprehensive discussion examining carbonic anhydrase activity, pH homeostasis, and viral infection. Please refer to the detailed experimental results and discussion in the sections mentioned in our previous response (Figures 3G-H, Lines 220-227 and 350-371).

(4) Next a yeast two hybrid assay reveals interaction with a thaumatin-like rice protein (TLP). It would be nice to know whether you detected other interacting proteins as well. The interaction is confirmed by pulldown and binding affinity assay using recombinant proteins. The kD is in favor of a rather weak interaction between the two proteins.

We have added a list of rice proteins that potentially interact with LssaCA (Table S1) and have measured interactions with additional proteins (unpublished data). Despite the relatively weak binding affinity, the functional significance of the LssaCA-OsTLP interaction in enhancing TLP enzymatic activity is substantial.

(5) Then the glucanase activity of TLP is measured using recombinant TLP-MBP or in vivo expressed TLP. It is not clear to me which TLP is used in Fig. 4G (plant-expressed or bacteria-expressed). If it is plant-expressed TLP, why is its basic activity 10 times lower than in Fig. 4F?

Fig. 4G is the Fig. 3G in the revised manuscript. A E. coli-expressed TLP protein has been used. We apologize for the error in our original Fig. 4G. The original data presented relative fold changes between OsTLP+BSA and OsTLP+LssaCA treatment, with OsTLP+BSA normalized to 1.0 and OsTLP+LssaCA values expressed as fold changes relative to this baseline. However, the Y-axis was incorrectly labeled as “β-1,3-glucanase (units mg^-1)”, which suggested absolute enzymatic activity values. We have now corrected the figure to display the actual absolute enzymatic activity values with the appropriate Y-axis label “β-1,3-glucanase (units mg^-1)”.

(6) There is also a discrepancy in the construction of the transgenic rice plants: did you use TLP without signal peptide or full length TLP? If you used TLP without signal peptide, you should explain why, because the wild type TLP contains a signal peptide.

We cloned the full-length OsTLP gene including the signal peptide sequence (Line 782 in the revised manuscript).

(7) The authors find that CA increases glucanase activity of TLP. Next the authors test callose deposition by aniline blue staining. Feeding activity of RSV-infected planthoppers induces more callose deposition than does feeding by uninfected insects. In the image (Fig. 5A) I see blue stain all over the cell walls of xylem and phloem cells. Is this what the authors expect? I would have expected rather a patchy pattern of callose deposition on cell walls. Concerning sieve plates, I cannot discern any in the image; they are easier to visualize in longitudinal sections than in transversal section as presented here.

We appreciate the reviewer’s insightful comment.

(1) Callose deposition pattern: While callose deposition was detected in other parts of the vascular bundle, significant differences between treatments were mainly observed in phloem cells, indicating that phloem-specific callose deposition is the primary response to RSV infection and SBPH feeding (Figures 5B and 5J).

(2) Sieve plate visualization: We have examined longitudinal sections to visualize sieve plates, which revealed callose deposition in sieve plates during SBPH feeding and RSV infection (Figure S7).

(3) Quantitative analysis: In addition to aniline blue staining, we quantified callose concentrations using a plant callose ELISA kit to provide more precise measurements (Figure 5A, 5I and S8A).

(4) Gene expression analysis: We utilized RT-qPCR to measure callose synthase expression in both feeding and non-feeding areas, confirming that callose synthesis was induced specifically in feeding regions, leading to localized callose deposition (Figures 5D-H).

These experimental results collectively demonstrate that RSV infection induces enhanced callose synthesis and deposition, with this response occurring primarily in phloem cells, including sieve plates, within feeding sites and their immediate vicinity.

(8) I do not quite understand how you quantified callose deposition (arbitrary areas?) with ImageJ. Please indicate in detail the analysis method.

We have added more detailed information for the methods to quantify callose deposition (Lines 673-678).

(9) More callose content is also observed by a callose ELISA assay of tissue extracts and supported by increased expression of glucanase synthase genes. Did you look whether expression of TLP is changed by feeding activity and RSV infection? Silencing CA in planthoppers increases callose deposition, which is inline with the observation that CA increases TLP activity.

We measured OsTLP expression following feeding by RSV-free or RSV-infected SBPH and found that gene expression was not significantly affected by either insect feeding or RSV infection. These results have been added to the revised manuscript (Lines 275-277 and Figure 5H).

(10) Next, callose is measured after feeding of RSV-infected insects on wild type or TLP-overexpressing rice. Less callose deposition (after 2 days) and more virus (after 14 days) is observed in TLP overexpressors. I am missing a control in this experiment, that is feeding of uninfected insects on wild type or TLP overexpressing rice, where I would expect intermediate callose levels.

We appreciate the reviewer’s insightful comment and fully agree with the prediction. In the revised manuscript, we have constructed ostlp mutant plants and conducted additional experiments to further clarify how callose deposition is regulated by insect feeding, RSV infection, LssaCA levels, and OsTLP expression. Specifically:

(1) Both SBPH feeding and RSV infection induce callose deposition, with RSV-infected insect feeding resulting in significantly higher callose levels compared to RSV-free insect feeding (Fig. 5A-C).

(2) LssaCA enhances OsTLP enzymatic activity, thereby promoting callose degradation (Fig. 5I-K).

(3) OsTLP-overexpressing (OE) plants exhibit lower callose levels than wild-type (WT) plants, while ostlp mutant plants show higher callose levels than WT (Fig. 6A-B).

(4) In ostlp knockout plants, LssaCA no longer affects callose levels, indicating that OsTLP is required for LssaCA-mediated regulation of callose (Fig. 6C-D).

These additional data address the reviewer’s concern and support the conclusion that OsTLP plays a central role in modulating callose levels in response to RSV infection and insect feeding.

(11) Next the authors test for interaction between virions and CA. Immunofluorescence shows that RSV and CA colocalize in salivary glands; in my opinion, there is partial and not complete colocalization (Fig. 7A).

We agree with the reviewer’s observation. CA is primarily produced in the small lobules of the principal salivary glands, while RSV infects nearly all parts of the salivary glands. In regions where RSV and CA colocalize within the principal glands, the CA signal appears sharper than that of RSV, likely due to the relatively higher abundance of CA compared to RSV in these areas. This may explain the partial, rather than complete, colocalization observed in our original Figure 7A. In the revised manuscript, please refer to Figure 1A.

(12) Pulldown experiments with recombinant RSV NP capsid protein and CA confirm interaction, binding affinity assays indicate rather weak interaction between CA and NP. Likewise in pull-down experiments, interaction between NP, CA and TLP is shown. Finally, in vitro activity assays show that activity of preformed TLP-CA complexes can be increased by adding NP; activity of TLP alone is not shown.

We performed two independent experiments to confirm the influence on TLP enzymatic activity by LssaCA or by the LssaCA-RSV NP complex. In the first experiment, we compared the enhancement of TLP activity by LssaCA using TLP alone as a control (Figure 3G). In the second experiment examining the LssaCA-RSV NP complex effect on TLP activity, we used the LssaCA-TLP combination as the baseline control rather than TLP alone (Figure 4B), since we had already established the LssaCA enhancement effect in the previous experiment.

(13) For all microscopic acquisitions, you should indicate the exact acquisition conditions, especially excitation and emission filter settings, kind of camera used and objectives. Use of inadequate filters or of a black & white camera could for example be the reason why you observe a homogeneous cell wall label in the aniline blue staining assays. Counterstaining cell walls with propidium iodide might help distinguish between cell wall and callose label.

Thank you for your insightful suggestions. We have added the detailed information to the revised manuscript (Lines 656-659 and 673-678).

(14) You should provide information whether CA is deregulated in infected planthoppers, as this could also modify its mode of action.\

We have compared LssaCA mRNA levels in RSV-free and RSV-infected L.striatellus salivary glands. The results indicated that RSV infection does not significantly affect LssaCA expression (Figure 1J).

(15) You should show purity of the proteins used for affinity binding measurements.

We have included SDS-PAGE results of purified proteins in the revised manuscript (Figure S3).

(16) L 39: Not all arboviruses are inoculated into the phloem.

Thank you. We have revised this description (Lines 40, 73, 95 and 97).

(17) L 76: Watery saliva is also injected in epidermis and mesophyll cells.

Thank you. We have revised this description (Line 73).

(18) L 79: What do you mean by "avirulent gene"?

Thank you for your valuable comments. We have revised this description as “certain salivary effectors may be recognized by plant resistance proteins to induce effector-triggered immunity”. Please refer to Lines 76-77 for detail.

(19) L 128: Please add delivery method.

Thank you. We have added the delivery methods (Line 134).

(20) L 195: Please explain "MST".

Explained (Line 124). Thank you.

(21) L 203: Please add the plant species overexpressing TLP.

Added (Line 214). Thank you.

(22) L 213: Callose deposition has also a role against phloem-feeding insects.

We appreciate the reviewer’s insight comment. We have added this information to the revised manuscript (Line 252).

(23) L 626: What is a "mutein"?

"mutein" is an abbreviation for mutant proteins. Since the recombinant protein microinjection experiments have been removed from the revised manuscript, the term “mutein” has also been removed. For all other instances, we now use the full term “mutant proteins”.

(24) Fig. 1E: what is "loading"? You should rather show here and elsewhere (or add to supplement) complete protein gels and Western blot membranes and not only bands of interest.

Thank you for your valuable suggestion. Although Figure 1E has been removed from the revised manuscript, we have carefully reviewed all figures to ensure that the term “loading” has been replaced with the specific protein names where appropriate.

(25) Fig. 2C: Please indicate which is the blot and which is the silver stained gel and add mass markers in kDa to the silver stained gel.

Thank you for your suggestion. We have revised figure to include labeled silver-stained gels with indicated molecular weight markers (Figure 1H in the revised manuscript).