Plant materials, virus inoculation and plant growth conditions

The susceptible rice cultivar Huai 5 (Oryza sativa japonica) was used for virus inoculation. Rice seeds were submerged in water inside a container for two days, and then sown in a 1 L beaker and kept in a greenhouse at 25°C with a light/dark cycle of 16/8 h. Nine-day-old rice seedlings were exposed to viruliferous SBPHs (approximately three viruliferous insects per seedling) in each of the inoculation chamber for 3 days. The seedlings that were mock-inoculated with the virus-free SBPH were used as controls. After the insects were removed from the plants by gentle brushing, the inoculated seedlings were transplanted to an insect-free glasshouse at 25 ± 3 °C under natural sunlight. The aerial parts of the whole plants were collected 50 days post-inoculation (dpi), a time when the dwarf symptom could be significantly observed in virus-inoculated seedlings.

To verify that the dwarf symptoms were caused by RBSDV infection, RBSDV genome segments S9-1 and S10 was examined by RT-PCR analysis [26,27]. PCR was performed with the virus specific primers listed in Table S1. The expected sizes of S9-1 and S10 were 1044 bp and 1677 bp, respectively. The amplified fragments of the expected size were confirmed by electrophoresis in 1% (w/v) agarose gels.

Protein samples preparation

Proteome analysis was performed in three biological repeats. The aerial part of the rice seedlings were used for protein extraction. Proteins were extracted using the TCA/acetone precipitation method according to Damerval et al. with several modifications [28]. One gram of rice was ground to a fine powder in liquid nitrogen, and the powder was later precipitated by 30 ml of cold acetone (-20°C) with 10% w/v TCA and 0.07% β-mercaptoethanol at -20 °C overnight. After centrifugation at 13,000 g at 4 °C for 30 min, the pellets were washed three times with 30 ml of ice-cold acetone containing 0.07% β-mercaptoethanol and lyophilized. The resulting powder was dissolved in lysis buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 65 mM DTT and 2% IPG buffer (pH 3-10)). After incubation at 30 °C for 1 h, the suspension was centrifuged at 40,000 g at 4 °C for 30 min to remove the insoluble debris. The protein concentration was quantified using a 2-D Quant kit (GE Healthcare).

2-DE, gel staining and image analysis

The 2-DE was performed as described by Xu et al. [29] with some modification. The IPG strips (24 cm, pH 4-7 nonlinear, GE Healthcare) were passively rehydrated at 25 °C for 12 h with 450 μl of rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 30 mM DTT, 0.5% IPG buffer (pH 4-7) and 0.004% bromophenol blue) containing 800 μg of protein. Isoelectric focusing was performed on an Ettan IPGphor II (GE Healthcare) at 20 °C, under the following conditions: 100V for 1 h, 250 V for 1 h, 500V for 1 h, 1000V for 1 h, 8000 V for 3 h, and 8000 V with a total of 68000 vhs. After focusing, the strips were incubated in 10 ml of equilibration buffer (6 M urea, 2% w/v SDS, 75 mM Tris-HCl at pH 8.8, 30% v/v glycerol, 0.002% w/v bromophenol blue, and 1% w/v DTT) for 15 min and subsequently in equilibration buffer containing 2.5% w/v iodoacetamide instead of 1% DTT for another 15 min. For the second dimension electrophoresis, the proteins were separated on 12.5% vertical polyacrylamide gels using an Ettan Dalt six electrophoresis system (GE Healthcare). The electrophoresis was performed at 2 watts/gel for 45 min followed by 17 watts/gel until the dye front reached approximately 1cm from the bottom of the gels.

2-DE gels were stained by a modified colloidal Coomassie G-250 staining [30] and scanned by UMAX PowerLook 2100XL scanner (UMAX Systerms, Germany). A total of six gels (three of the best-matched replicate gels from three independent experiments for each treatment) were analyzed using ImageMaster 2D Platinum software version 7.0 (GE Healthcare). The images were analyzed according to the manufacturer’s instructions. Spot detection and gel matching were performed automatically. Protein expression was quantified using the normalized percentage volumes (vol. %), a ratio of the volume of a particular spot to the total volume of all of the spots present on a gel. The match analysis was manually edited to correct the mismatched spots. To calculate the significant differences, the expression levels of the mock- and RBSDV- inoculation groups were analyzed using Student’s t-test. The protein spots with vol. % ≥1.5 and P ≤ 0.05 were defined as differentially expressed proteins (DEPs).

In-gel digestion, MALDI-TOF/TOF-MS and data analysis

The DEPs were manually excised from their gels for proteolytic digestion [29]. The excised gel pieces were destained with 200 μl of 100 mM ammonium bicarbonate (NH₄HCO₃) in 30% acetonitrile. After removing the destaining buffer, the gel pieces were dried in a SpeedVac and rehydrated with 30 μl of 50 mM NH₄HCO₃ containing 50 ng trypsin (sequencing grade; Promega, Madison, WI, USA). The peptides were extracted three times with 0.1% trifluoroacetic acid (TFA) in 60% acetonitrile after an overnight digestion at 37 °C. The supernatants were pooled together and lyophilized, and the resulting peptides were dissolved in 50% acetonitrile containing 0.5% TFA and kept at -80 °C until use in mass spectrometry. A protein-free gel piece was used as a control to identify the autoproteolysis products derived from trypsin.

MALDI-TOF/TOF-MS was conducted with a 4800 MALDI TOF/TOF™ Analyzer (Applied Biosystems, Foster City, CA, USA) according to Meisrimler et al. [31]. The spectra of the proteins were searched against the NCBI nonredundant protein (NCBInr) database with the taxonomy Oryza sativa 2010.12.10 using MASCOT 2.1 (MatrixScience, London, UK). The spectra that were not identifiable were researched in the NCBInr database with the taxonomy of all entries. The search parameters were set as follows: enzyme: trypsin; fix modifications: carbamidomethyl (C); mass values: monoisotopic; peptide mass tolerance: ± 100 ppm; fragment mass tolerance: ± 0.8 Da; peptide charge state: 1+; max missed cleavages: 1. Proteins identified by MALDI-TOF/TOF-MS with C.I. % scores above 95% were deemed significant.

Measurements of photosynthesis and H₂O₂ level

The net photosynthetic rates, stomatal conductance, intercellular CO₂ concentrations and transpiration speeds of the third and fourth rice leaves of each sample were measured using a LI-6400 XT portable photosynthesis system with a Red/Blue LED light source (LI-COR Inc., Lincoln, NE, USA). At least ten leaves were measured for each sample.

The H₂O₂ accumulation in mock- or virus- inoculated leaves was measured according to Wan et al. [25]. One gram of rice seedling leaves were ground with a pestle and mortar in liquid nitrogen to a fine powder, and suspended in 10 ml of 5 mM titanium sulfate. After incubation for 1 h at room temperature, debris was discarded by centrifugation at 12,000 g for 10 min at 25°C. The oxidation of titanium sulfate was recorded by reading the A410 using a PerkinElmer Lambda 25 UV/VIS spectrometer. The H₂O₂ concentrations were calculated according to a standard calibration plot, and expressed as μmol H₂O₂ g^-1 FW.

Quantitative real-time PCR

Quantitative real-time PCR was performed to quantify the transcriptional levels of genes corresponding to the DEPs. The sequences of the identified proteins were searched against the NCBI database using tBLASTn. The corresponding gene sequences were selected for primer design, and the primers designed using primer express 3.0 software were shown in Table S1. Total RNA was extracted with Trizol (Invitrogen) and treated with DNase I (Promega, WI, USA) to eliminate the DNA. The integrity of the RNA samples was assessed by agarose gel electrophoresis, and RNA concentrations were measured using a spectrophotometer. Reverse transcription was performed using 1 μg of RNA. The cDNA was synthesized using an M-MLV RTase cDNA synthesis kit (TaKaRa). The iQ5 Real-Time PCR System (Bio-Rad) was used to perform RT-qPCR. The rice UBQ5 gene was used as internal standard, and experiments were performed in triplicate. Amplification was performed in a 20 μl reaction mixture containing 1μl of cDNA, 1 μl of forward and reverse primers, 8 μl of ddH₂O and 10 μl of SsoFast^TM EvaGreen supermix (Bio-Rad). After an initial denaturation at 95 °C for 20 s, 40 cycles of amplification were carried out: denaturation at 95 °C for 10 s, annealing at 58 °C for 15 s and extension at 72 °C for 15 s. Melting curves were obtained and quantitation of the data was performed using the iQ5 software (Bio-Rad) with the relative quantification (ddCt) model.

RBSDV infection elevated the H₂O₂ level in rice

The rice seedlings infected with RBSDV exhibited notable stunting symptoms (Figure 1A). The height and width of the infected plants were only 46.2% and 56.7% of that of the control. The virus genome segments S9-1 and S10 could be detected by RT-PCR in the virus-infected rice leaves but not in the mock-infected plants (Figure 1B), indicating that the virus successfully infected the rice plant and consequently led to the dwarf. To verify whether the level of H₂O₂ was changed by RBSDV-infection in rice, the content of H₂O₂ was measured. An increasing H₂O₂ was found in RBSDV infected rice plant. The H₂O₂ concentration in virus infected plants was 2.64 μmol g^-1 FW whereas it was 1.75 μmol g^-1 FW in the control. As shown in Figure 1C, the H₂O₂ content in RBSDV infected plants was 1.5 times as much as the control.

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Figure 1. Long-term RBSDV infection resulted in an increase of H₂O₂ in rice.

(A) An RBSDV - infected rice plant (right) is compared to the mock-inoculated plant (left) at 50 dpi. (B) RT-PCR analysis of the RBSDV genome segments S9-1 and S10 in mock-inoculated rice (lanes 1 and 2) and in RBSDV-inoculated rice (lanes 3 and 4). (C) The accumulation of endogenous H₂O₂ in rice.

https://doi.org/10.1371/journal.pone.0081640.g001

Proteomic analysis of rice plants in response to RBSDV infection

To reveal the changes in protein expression under RBSDV stress in rice, the proteome profiles of mock- and virus-infected plants were analyzed by 2-DE. Approximately 1800 detectable spots were visualized on each gel by Coomassie Brilliant Blue staining (Figure 2). Three independent experiments were conducted to ensure that the protein abundance changes were reproducible and significant. 72 spots that showed at least a 1.5-fold increase or decrease in abundance (P≤0.05) were considered to change significantly between the mock- and virus-infected plants. Of these spots, 25 spots were down-regulated (marked in Figure 2A) and 47 spots were up-regulated (marked in Figure 2B).

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Figure 2. The mock- and RBSDV-infected rice proteomes display 72 differentially expressed protein spots.

The Proteins (800 μg) were separated on 24 cm pI 4-7 non-linear gradient IPG strips and with 12.5% SDS-PAGE. The gels were stained with CBB G-250 according to the blue silver method. A total of 72 proteins were differentially expressed in response to RBSDV infection. The down-regulated proteins were labeled in the 2-DE gel image of the mock-infected rice (A), and the up-regulated proteins were marked in the gel of the RBSDV-infected rice (B).

https://doi.org/10.1371/journal.pone.0081640.g002

To identify the proteins that were differentially expressed due to the RBSDV infection, all 72 spots with a threshold greater than 1.5-fold were excised and analyzed by MALDI-TOF/TOF-MS. 69 spots were successfully identified. 43 of these spots have been deposited in the current database as putative functional proteins. The remainder of the spots was without specific function in the database and was annotated using the Uniprot Knowledgebase (www.uniprot.org) or the NCBI (www.ncbi.nlm.nih.gov) database with BLASTP. The results of the identification of the 69 spots were shown in Table 1. The corresponding homologues with annotations were listed in Table S2 and the peptide sequences identified by MOLDI-TOF/TOF-MS were shown in Table S3. According to the annotations, the identified proteins were involved in the following 12 categories according to their functional features, including defense and stress related proteins, photosynthesis, redox homeostasis, energy pathway, amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, transcription and translation, cell wall modifications, plant hormone responses, signal transduction and virus proteins (Table 1, Figure 3A). About 80% different proteins were related to defense and stress response, photosynthesis, redox homeostasis, energy pathway and amino acid metabolism. The virus infection seems to have triggered the defense and stress response, and impaired the photosynthesis, as most of the DEPs related to defense and stress response were found up-regulated, and most of those proteins related to photosynthesis were down-regulated (Table 1).

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Figure 3. Classification of the differentially expressed proteins (DEPs).

A, functional classification of the 69 DEPs in RBSDV-infected rice plant. Function, (number of DEP spots in each functional classification/ total DEP spots) % and the number of DEP spots in each classification were shown. B, functional classification of the 37 DEP spots co-regulated by RBSDV infection and by H₂O₂ stress. Function, (number of co-regulated protein spots in each functional classification/total DEP spots) % and the number of the co-regulated protein spots/total DEP spots in each classification were shown.

https://doi.org/10.1371/journal.pone.0081640.g003

The overlapping responsive proteins under H₂O₂ stress and RBSDV infection

An H₂O₂ stress-responsive protein network in rice seedling had been revealed by Wan et al. [25]. To obtain the overlapping proteomic profile of rice under RBSDV infection and H₂O₂ stress, we compared the DEPs identified in this study with the responsive proteomic profile of rice under H₂O₂ treatment published previously [25]. Under RBSDV infection, 45 proteins corresponding to 69 identified spots was differentially expressed (Table 1). Of these proteins, 19 proteins corresponding to 37 spots were also differentially expressed under H₂O₂ stress. Moreover, in comparison with previously published proteomic and microarray data [21,22], nine of 19 proteins were also detected differentially expressed under RBSDV infection in maize. The expression patterns of these RBSDV and H₂O₂ co-regulated proteins were shown in Table 2 and Figure 3B. Among these co-regulated proteins, some proteins had both up- and down- regulated change patterns in abundance. Likewise, many similar phenomena were also observed in other previously reported proteomics studies [32-34].

Transcriptional profiles of DEPs under RBSDV infection

To assess the validity of the alterations in protein expression during RBSDV infection, the transcriptional levels of the mRNA corresponding to the identified proteins were analyzed by quantitative real-time PCR. Nine genes, including four genes related to defense and stress response and five genes related to redox homeostasis, were selected for analysis. Transcription of the genes associated with defense and stress, including thaumatin-like protein (TLP, P01), putative chitinase (CHT, spot 08), beta-1,3-glucanase (BGL, spot 17) and chloroplast heat shock protein 70 (HSP70, spot 39), were elevated after virus infection (Figure 4). The changes in mRNA abundance of these genes were similar to those of their corresponding proteins in the 2-DE gels. The expression patterns of five genes related to redox homeostasis including L-ascorbate peroxodase 1 (APX1, spot 04), glutathione S-transferase (GST, spot 05), ferredoxin-NADP(H) oxidoreductase (FNR, spot 14), catalase (CAT, spot 21) and protein disulfide isomerase (PDI, spot 40) have the same changes as their corresponding proteins (Figure 4). The results suggested that the changes in protein expression detected by proteomic analysis reflect the actual alterations in mock-inoculated and RBSDV-infected plants.

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Figure 4. The changes in transcription of the differentially expressed proteins in RBSDV-infected rice.

TLP, thaumatin-like protein; CHL, putative chitinase; BGL, beta-1,3-glucanase precursor; HSP70, chloroplast heat shock protein 70; APX1, L-ascorbate peroxodase 1; GST, glutathione S-transferase; FNR, ferredoxin-NADP(H) oxidoreductase; CAT, catalase; PDI, protein disulfide isomerase.

https://doi.org/10.1371/journal.pone.0081640.g004

The photosynthetic response under RBSDV infection stress

Photosynthesis is a process that converts carbon dioxide into organic compounds and plays a pivotal role in plant growth. In this study, 15 differentially expressed spots (21.7%) were associated with a photosynthetic response, and most of them were down-regulated (Table 1). To investigate the effects of virus infection on rice photosynthesis, the photosynthetic capacity was measured. The net photosynthetic rate (Pn) of the mock-infected rice was 4.68 μmol of CO₂ m^-2 s^-1 at 50 dpi, and the rate for the RBSDV-infected rice was 0.86 μmol of CO₂ m^-2 s^-1 (Figure 5). The stomatal conductance (Cond), the intercellular CO₂ concentration (Ci) and the transpiration speed (Tr) were also significantly decreased after RBSDV infection (Figure 5). The data above indicated that the RBSDV infection results in a decrease of photosynthesis in rice.

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Figure 5. Effects of RBSDV infection on the Pn, Cond, Ci and Tr in rice plant at 50 dpi.

https://doi.org/10.1371/journal.pone.0081640.g005

The overproduced H₂O₂ and plant growth deficiency

With both reducing and oxidizing properties, H₂O₂ at high concentrations could cause oxidative stress and cell damage. H₂O₂ can cross membranes via aquaporin-mediated transport to chloroplasts, mitochondria and cytosol [35] and damage a large variety of biomolecules such as lipids, proteins, and nucleic acids that are essential to the activity and integrity of the cell [36]. Electron microscopy of ultrathin sections data, detected in maize plant infected with Maize rough dwarf virus which was later proved to be RBSDV [15,16,37], showed that RBSDV infection destroyed the membrane systems of various cell organelles including chloroplast, mitochondria, nucleus and vacuole, albeit no virus was discovered in these organelles [37]. The accumulated H₂O₂ in RBSDV infected plant may harm the membrane structures of various organelles and result in the cell membrane rupture. The H₂O₂ content in the virus-susceptible plants was observed increased much higher than in the resistant cultivar [14]. Only in the susceptible plants was the increase in apoplastic H₂O₂ levels accompanied by an increase in electrolyte leakage [14]. The H₂O₂ at a high level in the virus-susceptible plants probably resulted in the cell damage and electrolyte leakage.

Hydroxyproline-rich glycoprotein-like protein (HRGP) is one of the major classes of structural cell wall proteins and is involved in cell elongation [38,39]. The ROS could limit cell elongation by oxidative crosslink of HRGPs [39]. Previously report showed that cells infected by RBSDV in maize were shorter than those in the mock-inoculated plants [40]. The expression of HRGP was down-regulated by both H₂O₂ stress and RBSDV infection (Table 2), suggesting that the H₂O₂ overproduced in RBSDV infection may crosslink the HRGP, inhibit the cell elongation, and finally result in the dwarf plant.

The cross-talk on photosynthesis

The long-term RBSDV accumulation in rice leads to the increase of endogenous H₂O₂ and the decrease of photosynthetic parameters, including Pn, Cond, Ci, and Tr (Figure 1 and Figure 5). These phenomena are similar to that obtained in rice under H₂O₂ stress. An increase in the chloroplastic hydrogen peroxide levels and an alteration in chloroplast ultrastructure was also observed after Plum pox virus infection [13]. It is presumed that the increased H₂O₂ may do damage to the chloroplast ultrastructure and result in the decreased photosynthesis.

Besides, among the seven DEPs related to photosynthesis, six proteins were also responsive to H₂O₂ treatment in rice (Table 2). These data indicated that the overproduction H₂O₂ had a pivotal role in regulating the process of photosynthesis. The increased H₂O₂ under long-term virus infection may affect photosynthesis by regulating proteins in three subgroups, including the light-harvesting reaction, the activation reaction of Rubisco large subunit, and the Calvin cycle.

In the first subgroup, chlorophyll a/b binding protein was down regulated in response to RBSDV infection and H₂O₂ stress. Chlorophyll a/b binding protein is a component of the light-harvesting complex of photosystem I and II and facilitates light absorption and energy transfer [41,42]. The decrease in abundance of the protein may inhibit the light absorption and energy transfer and further affect the photosynthetic process in the RBSDV-infected plant.

In the second subgroup, the Rubisco activase small isoform precursor was up- and down-regulated under both RBSDV infection and H₂O₂ stress (Table 2). Rubisco activase was engaged in the activation reaction of Rubisco large subunit. It binds to the inactive Rubisco, facilitates the ATP-dependent removal of sugar phosphates from Rubisco active sites, and maintains Rubisco in its active configuration [43]. The expression change of this protein may severely affect the activation of Rubisco.

In the third subgroup, four proteins involved in Calvin cycle were co-regulated by RBSDV infection and H₂O₂ stress (Table 2). Of them, the abundance of glyceraldehyde-3-phosphate dehydrogenase A, fructose-bisphosphate aldolase and phosphoribulokinase (PRKA) dropped in virus infected plant, implying that the CO₂ assimilation might be slowed down by RBSDV infection. Glyceraldehyde-3-phosphate dehydrogenase A and fructose-bisphosphate aldolase had the same expression patterns when they were under H₂O₂ stress and RBSDV infection. In contrast, the expression patterns of PRKA and Rubisco large subunit were opposite under these two stresses. It suggested that the Calvin cycle was probably disturbed by factors other than H₂O₂. PRKA catalyzes the ATP-dependent phosphorylation of ribulose 5-phosphate to form ribulose-1,5-bisphosphate (RuBp) [44]. It was reported that a decrease in Rubisco activity and RuBp regeneration rate were associated with the decreased photosynthesis [45,46]. The decline in Rubisco activity and RuBp regeneration, yet not in the quantity of Rubisco, may result in the decreased in CO₂ assimilation of rice under RBSDV infection.

Taken together, our results suggested that the overproduction of H₂O₂ may diminish light absorption, CO₂ assimilation and Rubisco activity by modulating the expression of the proteins pertaining to photosynthesis, and finally impaired the photosynthesis. The finding reported above provided new insights into the relationship between the decreased photosynthesis and the overproduction of H₂O₂ in RBSDV-infected rice.

The redox reaction under virus infection and H₂O₂ stress

The level of H₂O₂ was enhanced after virus accumulation, indicating that an oxidative stress was caused by virus infection. Besides, a series of redox related proteins were differentially expressed under RBSDV infection (Table 1). The significant increase in abundance of APX, CAT and peroxidase (POD) suggested that the H₂O₂-scavenging system was activated to reduce the intracellular H₂O₂ levels and the oxidative damage. Consistent with this observation, the increase in catalase activity, peroxidase activity and SOD activity was observed after virus inoculation [12,14]. However, the upregulation of these H₂O₂-scavenging enzymes seemed to fail in regulating the H₂O₂ homeostasis, as an increase of H₂O₂ could be observed in virus infected rice (Figure 1).

Under RBSDV infection, 13.0% (nine proteins) of the DEPs were related to redox homeostasis. Three of these proteins were also differentially expressed under H₂O₂ stress (Table 2), indicating that the overproduced H₂O₂ has a direct effect on plant antioxidative response. APX, a major ROS-scavenging enzyme, could efficiently decompose hydrogen peroxide in various subcellular compartments [47]. GST catalyzes the reduction of toxic organic hydroperoxides with glutathione as a cosubstrate or coenzyme [48]. PDI is participated in the thioredoxin-based redox pathway and the antioxidative defense system [49]. The upregulation of these proteins implied that the host plant provoked the antioxidative response to reduce the toxicity of the accumulated H₂O₂ after virus infection.

The effect of overproduced H₂O₂ on the other processes

Proteins involved in carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, energy pathway, and transcription and translation were regulated by both H₂O₂ and RBSDV stresses (Table 2), suggesting that the enhanced H₂O₂ in RBSDV-infected plant may disturb these processes.

Fructokinase catalyzes the transfer of a phosphate group from ATP to fructose and plays a role in the glycolysis, in particular, sucrose and fructose metabolism. Fructokinase 2 could inhibit the growth of stems and roots in tomato when it was suppressed [50]. The abundance of fructokinase 2 was decreased under H₂O₂ stress, but was up- and down-regulated under RBSDV infection (Table 2). The multiple expression patterns indicated that fructokinase 2 may impair the glycolysis and inhibit the plant growth in disease plant.

ADP-glucose pyrophosphorylase (AGPase) activates the first, rate limiting step in starch biosynthesis. UDP-glucose pyrophosphorylase could catalyze UDP-glucose into glucose-1-phosphate (Glc-1-P),which can be converted into ADP-glucose by AGPase and finally stored as starch [51]. The upregulation of AGPase and UDP-glucose pyrophosphorylase suggested that more starch might be accumulated under these stresses, which may explain why starch granules boomed in the maize leaves infected with RBSDV [37].

The RNA binding protein play a major role in post-transcriptional regulation, including pre-mRNA splicing, capping, mRNA transport and translation of functional mRNAs [52]. Under RBSDV infection and H₂O₂ stress, some responsive proteins had different isoelectric points and different expression patterns (Table 1). We reasoned that mRNA binding protein is likely involved in posttranslational regulation and results in the opposite expression patterns of the responsive proteins.