Rice stripe virus utilizes a Laodelphax striatellus salivary carbonic anhydrase to facilitate plant infection by direct molecular interaction
Abstract
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.
Dataset DOI: 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 TLP 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
Methods mentioned in the dataset
RT-qPCR
Gene transcript levels were determined by RT-qPCR. The RNA was extracted from L. striatellus 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. L. striatellus 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 EF2 and actin, respectively. Water was used as a negative control.
Microscale thermophoresis (MST) assays
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).
β-1,3-glucanase activity assays
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.
RT-qPCR
Gene transcript levels were determined by RT-qPCR. The RNA was extracted from L. striatellus 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. L. striatellus 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 EF2 and actin, respectively. Water was used as a negative control.
Microscale thermophoresis (MST) assays
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).
β-1,3-glucanase activity assays
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.