DOI: 10.5061/dryad.jh9w0vtqz
title: Rice stripe virus utilizes a <em>Laodelphax striatellus</em> salivary carbonic anhydrase to facilitate plant infection by direct molecular interaction

description: Plant viruses typically rely on insect vectors for transmission between
 plants, with insect salivary proteins playing critical roles in this
 process. In this study, we demonstrate how Laodelphax striatellus salivary
 carbonic anhydrase (LssaCA) promotes rice stripe virus (RSV) infection in
 plants. We discovered that LssaCA directly binds to RSV nucleocapsid
 protein (NP) in insect salivary glands. This LssaCA-NP complex interacts
 with a rice thaumatin-like protein (OsTLP) that possesses
 endo-β-1,3-glucanase activity, potentially degrading callose. Upon
 binding, the LssaCA-NP complex significantly enhances OsTLP enzymatic
 activity. We further clarify that both L. striatellus feeding and RSV
 infection induce callose deposition. The tripartite LssaCA-NP-OsTLP
 interaction enhances callose degradation, thereby facilitating RSV
 infection via its insect vector. This study provides new insights into
 complex virus-insect-plant tripartite interactions mediated by insect
 salivary proteins, with broad implications for numerous plant viruses
 transmitted by insect vectors.
description: Methods mentioned in the dataset
 <strong>RT-qPCR</strong> Gene
 transcript levels were determined by RT-qPCR. The RNA was extracted from
 <em>L. striatellus</em> tissues using a RNeasy Mini Kit
 (Qiagen, Hilden, Germany), and from plant leaves using TRIzol. Reverse
 transcriptional PCR (iScript cDNA Synthesis Kit, Bio-Rad, Hercules, CA,
 USA) and SYBR-Green-based qPCR (SYBR Green Supermix, Bio-Rad) was
 performed according to the manufacturer’s protocols. The primer pair used
 to amplify LssaCA was LssaCA-q-F/LssaCA-q-R. Viral RNA copies were
 measured using the primers NP-q-F/NP-q-R. <em>L.
 striatellus</em> translation elongation factor 2 (EF2) and rice
 actin were amplified as internal controls to ensure equal loading of cDNA
 isolated from different samples. The primer pairs EF2-q-F/EF2-q-R and
 actin-q-F/actin-q-R  were used to amplify <em>EF2</em> and
 <em>actin</em>, respectively. Water was used as a negative
 control. <strong>Microscale thermophoresis (MST)
 assays</strong> We conducted MST assays to detect
 the interactions between LssaCA and NP, and between LssaCA and OsTLP.
 First, 10 μM purified LssaCAsp- protein (His-fusion protein) was labelled
 with a Monolith NT Protein Labeling Kit RED-NHS (Nano Temper Technologies
 GMBH, München, Germany) using red fluorescent dye NT-647
 N-hydroxysuccinimide (amine-reactive), according to the manufacturer’s
 instructions. The binding assays were performed on a Monolith NT.115
 Microscale Thermophoresis instrument (Nano Temper Technologies GMBH) using
 capillaries treated using the standard method. The labeled protein
 LssaCAsp- was added to serially diluted NP (GST-NP, GST was used as a
 negative control) or OsTLPsp- (MBP-fusion protein, MBP was used as a
 negative control). The initial concentration of each protein was 40 µM
 with 0.1% (v/v) Tween 20. The KD Fit function of NanoTemper Analysis
 software version 1.2.214.1 was used for curve fitting and to calculate the
 dissociation constant (Kd).
 <em><strong>β-1,3-glucanase activity
 assays</strong></em> β-1,3-glucanase
 hydrolyzes laminarin by cleaving β-1,3-glucoside bond, thereby generating
 sugar termini. Enzyme activity was quantified by measuring the rate of
 reducing sugars production. One unit (U) of β-1,3-glucanase activity was
 defined as the amount of enzyme that hydrolyses laminarin to generate 1 μg
 reducing sugars per minute. The β-1,3-glucanase
 activity of OsTLP was measured using a beta-1,3-glucanase microplate assay
 kit  according to the manufacturer’s instructions. To determine the
 enzymatic activity of recombinant OsTLP proteins, MBP-fused OsTLPsp-
 protein and MBP control protein were adjusted to 0.5 mg/mL. The
 MBP-OsTLPsp- fusion protein was equilibrated in buffers of varying pH for
 the assay. To determine the enzymatic activity of OsTLP expressed in
 planta, 0.1 g of rice leaf tissue from either OsTLP over-expressing or
 wild-type plants was homogenized with 1 mL of assay buffer on ice. The
 homogenate was centrifuged at 12,000g at 4°C for 10 min, and the resulting
 supernatant was transferred into a new tube and kept on ice until
 analysis. For the assay, 50 μL of the protein solution
 was mixed with laminarin substrate and incubated at 37°C for 30 min. The
 reaction was terminated by incubating the reaction tube in a boiling water
 bath for 10 min. The reaction supernatant was transferred into a
 microplate and incubated at 90°C for 10 min before measuring the
 absorbance at 540 nm. 
 <strong>RT-qPCR</strong> Gene
 transcript levels were determined by RT-qPCR. The RNA was extracted from
 <em>L. striatellus</em> tissues using a RNeasy Mini Kit
 (Qiagen, Hilden, Germany), and from plant leaves using TRIzol. Reverse
 transcriptional PCR (iScript cDNA Synthesis Kit, Bio-Rad, Hercules, CA,
 USA) and SYBR-Green-based qPCR (SYBR Green Supermix, Bio-Rad) was
 performed according to the manufacturer’s protocols. The primer pair used
 to amplify LssaCA was LssaCA-q-F/LssaCA-q-R. Viral RNA copies were
 measured using the primers NP-q-F/NP-q-R. <em>L.
 striatellus</em> translation elongation factor 2 (EF2) and rice
 actin were amplified as internal controls to ensure equal loading of cDNA
 isolated from different samples. The primer pairs EF2-q-F/EF2-q-R and
 actin-q-F/actin-q-R  were used to amplify <em>EF2</em> and
 <em>actin</em>, respectively. Water was used as a negative
 control. <strong>Microscale thermophoresis (MST)
 assays</strong> We conducted MST assays to detect
 the interactions between LssaCA and NP, and between LssaCA and OsTLP.
 First, 10 μM purified LssaCAsp- protein (His-fusion protein) was labelled
 with a Monolith NT Protein Labeling Kit RED-NHS (Nano Temper Technologies
 GMBH, München, Germany) using red fluorescent dye NT-647
 N-hydroxysuccinimide (amine-reactive), according to the manufacturer’s
 instructions. The binding assays were performed on a Monolith NT.115
 Microscale Thermophoresis instrument (Nano Temper Technologies GMBH) using
 capillaries treated using the standard method. The labeled protein
 LssaCAsp- was added to serially diluted NP (GST-NP, GST was used as a
 negative control) or OsTLPsp- (MBP-fusion protein, MBP was used as a
 negative control). The initial concentration of each protein was 40 µM
 with 0.1% (v/v) Tween 20. The KD Fit function of NanoTemper Analysis
 software version 1.2.214.1 was used for curve fitting and to calculate the
 dissociation constant (Kd).
 <em><strong>β-1,3-glucanase activity
 assays</strong></em> β-1,3-glucanase
 hydrolyzes laminarin by cleaving β-1,3-glucoside bond, thereby generating
 sugar termini. Enzyme activity was quantified by measuring the rate of
 reducing sugars production. One unit (U) of β-1,3-glucanase activity was
 defined as the amount of enzyme that hydrolyses laminarin to generate 1 μg
 reducing sugars per minute. The β-1,3-glucanase
 activity of OsTLP was measured using a beta-1,3-glucanase microplate assay
 kit  according to the manufacturer’s instructions. To determine the
 enzymatic activity of recombinant OsTLP proteins, MBP-fused OsTLPsp-
 protein and MBP control protein were adjusted to 0.5 mg/mL. The
 MBP-OsTLPsp- fusion protein was equilibrated in buffers of varying pH for
 the assay. To determine the enzymatic activity of OsTLP expressed in
 planta, 0.1 g of rice leaf tissue from either OsTLP over-expressing or
 wild-type plants was homogenized with 1 mL of assay buffer on ice. The
 homogenate was centrifuged at 12,000g at 4°C for 10 min, and the resulting
 supernatant was transferred into a new tube and kept on ice until
 analysis. For the assay, 50 μL of the protein solution
 was mixed with laminarin substrate and incubated at 37°C for 30 min. The
 reaction was terminated by incubating the reaction tube in a boiling water
 bath for 10 min. The reaction supernatant was transferred into a
 microplate and incubated at 90°C for 10 min before measuring the
 absorbance at 540 nm.
description: # Rice stripe virus utilizes a *Laodelphax striatellus* salivary carbonic
 anhydrase to facilitate plant infection by direct molecular interaction
 Dataset DOI:
 [10.5061/dryad.jh9w0vtqz](https://doi.org/10.5061/dryad.jh9w0vtqz) ##
 **Description of the data and file** #### File: data.zip
 **Description:** In the dataset, the data for complete statistical data
 and image files. Each file is named with the related figure number
 descriptively. 
 
 **Fig1A Raw IF Images LssaCA-RSV salivary gland.** To test if RSV
 coloclize with LssaCA, we made the Immunofluorescence co-localization
 assay. This compressed archive provides the raw confocal microscopy images
 which shows the co-localization of LssaCA (green) and RSV NP (red) in
 salivary gland cells. The “.tif” files contain the original, unprocessed
 image data for each fluorescence channel (DAPI, Alexa 488, Alexa 594) from
 representative biological samples. This file is supported for Figure 1A
 of this dataset associated manuscript. **Fig1C Activity Enzymatic Assay
 LssaCA**. This file provides the source data for the specific activity of
 carbonic anhydrase. LssaCA and LssaCA mutant were purified from both E.
 coli and SF9 cells. and their enzymatic activity is measured. This file
 contains the calculated LssaCA enzymatic activity derived from raw kinetic
 data of the control and experimental groups—specifically, the absorbance
 change monitored at 405 nm over a 40-minute period.    These values were
 converted into a ratio relative to the total protein concentration of each
 sample to yield specific activity (Units/mg protein). Proteins from four
 purification batches are tested. Statistical analysis was performed on
 these specific activity values for Figure 1C of this dataset associated
 manuscript. **Fig1E MST GST-CP interaction binding affinity** and **Figure
 1E MST LssaCA-CP interaction binding affinity.** These files provides the
 data underlying the interaction between protein GST-NP and protein
 LssaCA-His , with GST serving as the negative control. It contains the
 normalized fluorescence change (ΔF_norm) of protein LssaCA-His across a
 titration series of GST-NP concentration. The data presented are the mean
 values from three independent experiments, which were subsequently used to
 fit a 1:1 binding model using MO.Affinity Analysis software. The resulting
 Kd value was used to generate the binding curve in Figure 1E of this
 dataset associated manuscript. **Fig1F LssaCA Expression organ tissue
 qPCR**. We compared LssaCA expression level in different organs by qRT PCR
 to find out where it is produced. This file contains the calculated mean
 normalized expression (2^-ΔCq^) of the target gene *LssaCA* relative to
 the reference gene *EF2* (*LssaCA/EF2*). This file provides the source
 data for Figure 1F of dataset associated manuscript. **Fig1J LssaCA
 Expression salivary gland qPCR.**  To detect if RSV infection influecne
 LssaCA expression in salivary gland, we compared LssaCA mRNA level in
 RSV-infected and RSV-free salivary gland by qRT PCR. This file contains
 the calculated mean normalized expression (2^-ΔCq^) of the target
 gene  *LssaCA*  relative to the reference gene *EF2* (
 *LssaCA/EF2*) .Statistical analysis was then performed for Figure 1J of
 the dataset associated manuscript. **Fig2A LssaCA Expression dsRNA
 Knockdown qPCR**.LssaCA was silence by dsRNA injection (dsLssaCA) and
 dsGFP was  microinjected as control. RSV titer inside sailvary gland was
 tested by qRT PCR.  "LssaCA/EF2" in this file is the  mean
 normalized expression (2^-ΔCq^) of the target gene *LssaCA* relative to
 the reference gene *EF2* . Statistical analysis was then performed for
 Figure 2A of the dataset associated manuscript.   **Fig 2B RSV NP
 Expresson rice qPCR**. LssaCA was silecned and then the influence on RSV
 transmission was tested. "*NP/ACTIN*" in this file is the
 calculated mean normalized expression (2^-ΔCq^) of the target gene *RSV
 NP* relative to the reference gene *actin* for the control group (dsGFP)
 and the experimental group (dsLssaCA). Statistical analysis was then
 performed for Figure 2B of the dataset associated manuscript. **Fig 2D
 LssaCA Expression dsRNA Knockdown qPCR**. SBPHs were micrinjected with
 dsRNA to silence the target genen. This file contains the calculated mean
 normalized expression (2^-ΔCq^) of the target gene *LssaCA* relative to
 the reference gene *EF2* for the control group (dsGFP) and the
 experimental group (dsLssaCA).  The data is name
 as"LssaCA/EF2". Statistical analysis was then performed for
 Figure 2D of the dataset associated manuscript. **Fig 2E RSV NP Expression
 salivary gland qPCR**.LssaCA was silenced by dsRNA microinjection, and
 then RSV in salivary gland was measured. " *NP/EF2*" in this
 file is  mean normalized expression (2^-ΔCq^) of the target gene *RSV NP*
 relative to the reference gene *EF2* for the control group (dsGFP) and the
 experimental group (dsLssaCA). Statistical analysis was then performed for
 Figure 2E of the dataset associated manuscript. **Fig2F RSV titer
 salivary**. Salivary secrated by dsRNA injected SBPHs were collected were
 collected to compare the RSV titer inside. Each sample was collected from
 the same quantity of SBPHs. RNA Level of RSV NP was measured by qRT PCR. 
 CqdsCA deletes CqdsGFP to obtain ΔCq . "RSV titer" in this file
 is  the calculated mean normalized expression (2^-ΔCq^) . Statistical
 analysis was then performed for Figure 2F of the dataset associated
 manuscript. **Fig2G RSV NP Expression rice qPCR**. Plants were treated
 with dsRNA microinjected SBPHs for one day, and the RSV level inside fed
 plant was measured immediately." *NP/Actin*" in this file is the
 calculated mean normalized expression (2^-ΔCq^) of *RSV NP* relative to
 the reference gene *actin* for the control group (dsGFP) and the
 experimental group (dsLssaCA). The statistical analysis was performed for
 Figure 2G of the dataset associated manuscript.^^ **Fig2H RSV NP
 Expression rice qPCR.** Plant were treated with dsRNA microinjected SBPHs
 for one day, and the RSV level inside fed plant was measured 3 days
 later." *NP/Actin*" in this file is the calculated mean
 normalized expression (2^-ΔCq^) of *RSV NP* relative to the reference
 gene *actin* for the control group (dsGFP) and the experimental group
 (dsLssaCA). The statistical analysis was performed for Figure 2H of the
 dataset associated manuscript. **Fig3D MST MBP-LssaCA interaction binding
 affinity** and **Fig3D MST OsTLP-LssaCA interaction binding affinity**.
 LssaCA and OsTLP interaction is measured by MST.  The MPB is the negtive
 control of MBO-OsTLP. Files contain the normalized fluorescence change
 (ΔF_norm) of protein LssaCA-His across a titration series of MBP-OsTLP (or
 MBP) concentration. The data presented are the mean values from three
 independent experiments, which were subsequently used to fit a 1:1 binding
 model using MO. Affinity Analysis software. The resulting Kd value was
 used to generate the binding curve in Figure 3D of this dataset associated
 manuscript. **Fig3E 3G 4B TLP enzymatic activity assay.** OsTLP was
 produced by E.coli, and  it was inoculated with LssaCA, BSA, LssaCA+NP,
 LssaCA+GST. The enzymatic activity was then measured to see if these added
 protein influecne OsTLP'S-1,3-glucanase activity . The measured
 optical density (OD) values were converted to the amount of reducing sugar
 in each sample using a standard curve. Enzyme activity was calculated
 based on the definition that one unit (U) equals the amount of enzyme
 required to produce 1 μmol of glucose per minute under the assay
 conditions, and the results were normalized to the total protein
 concentration of each sample. The resulting normalized specific activity
 values were used to plot the bar chart. Statistical analysis was performed
 for Figure 3E, Figure 3G and Figure 4B of this dataset associated
 manuscript. **Fig3F TLP Enzymatic activity assay (plant).** We compared
 the Endo-β-1,3-glucanase activity in OsTLP-overexpressing transgenic
 plants (OsTLP OE) and in 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. The measured optical density (OD)
 values were converted to the amount of reducing sugar in each sample using
 a standard curve. Enzyme activity was calculated based on the definition
 that one unit (U) of activity produces 1 μmol of glucose per minute, and
 the results were normalized to the total protein concentration of each
 sample. Statistical analysis was performed for Figure 3F of this
 dataset associated manuscript. **Fig3H** T**LP Enzymatic acticity assay
 (PH).** OsTLP was purified from *E. coli* and enzymatic activity measured
 at pH 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 to assess pH dependence. The
 measured optical density (OD) values were converted to the amount of
 reducing sugar in each sample using a standard curve. Enzyme activity was
 calculated based on the definition that one unit (U) of activity produces
 1 μmol of glucose per minute under the assay conditions, and the results
 were normalized to the total protein concentration of each
 sample. Statistical analysis was performed for Figure 3H of this
 dataset associated manuscript. **Fig3I RSV NP expression in rice qPCR.**
 OsTLP overexpressed and deleted plants were treated with RSV-infected
 SPBHs, and RSV infection level were tested by qRT PCR. *NP/Actin*" in
 this file is the calculated mean normalized expression (2^-ΔCq^) of *RSV
 NP* relative to the reference gene *actin* for the control group (WT) and
 the experimental groups (OsTLP-OE and *ostlp*). Statistical analysis was
 performed for Figure 3I of this dataset associated manuscript. **Fig5A 5I
 6A 6C Callose deposition rice ELISA.** WT, OsTLP OE and *ostlp* rice were
 fed by dsRNA microinjected SBPHs, to test the callose content in rice
 under different treatments. It contains the raw absorbance values measured
 at 450 nm for the samples, standards, and blank controls. The callose
 concentration for each sample was interpolated from a standard curve and
 then normalized to the fresh weight of the extracted tissue. Statistical
 analysis was performed for Figure 5A, Figure5I, Figure 6I and Figure 6C of
 this dataset associated manuscript. **Fig5C 5K 6B 6D Callose deposition
 quantification image J Aniline Blue.** This file contains data from the
 control group (wild-type) and the experimental groups (RSV-free,
 RSV-infected), featuring aniline blue-stained callose in rice leaf samples
 from representative biological replicates per treatment. The file includes
 the raw fluorescence intensity measurements (Integrated Density) for
 individual callose deposits, obtained using a fixed threshold in
 ImageJ. Statistical analysis was performed for Figure 5C, Figure5K, Figure
 6B and Figure 6D of this dataset associated manuscript. **Fig5D 5E 5F 5G
 GSLs Expression rice qPCR.** This file provides the raw qRT-PCR data. It
 contains the raw quantification cycle (Cq) values (reported as the mean of
 three technical replicates) for the target genes
 (*gsl3, gsl4, gsl5, gsl10*) and the reference gene *actin* across the
 control group (Mock) and the experimental groups (RSV-free, RSV-infected).
 Columns M to P calculate the mean normalized expression (2^-ΔCq^) for each
 respective target gene relative to actin. Statistical analysis was
 performed for Figure 5E-G of this dataset associated manuscript. **Fig5H
 TLP Expression rice qPCR.** This file provides the raw qRT-PCR data for
 Figure 5H. It contains the raw quantification cycle (Cq) values (reported
 as the mean of three technical replicates) for the target gene OsTLP and
 the reference gene actin across the control group (Mock) and the
 experimental groups (RSV-free, RSV-infected). The final column titled
 "*OsTLP/Actin* " calculates the mean normalized expression
 (2^-ΔCq^)relative to actin. Statistical analysis was performed for Figure
 5H of this dataset associated manuscript. ## Code/software Microsoft
 office