ZHAO Mei-ai, LEl Zhen, PEl Yu-he, SHAO Xiao-yu, GUO Xin-mei, SONG Xi-yun

1 Key Laboratory of Plant Biotechnology in University of Shandong Province/College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, P.R.China

2 Key Laboratory of Qingdao Major Crop Germplasm Resource Innovation and Application/College of Agronomy, Qingdao Agricultural University, Qingdao 266109, P.R.China

1. lntroduction

Maize is an important feeding source and industrial raw material, which occupies an important position in the world economy. In recent years, maize rough dwarf disease(MRDD) has become a devastating disease, especially in China (Taoet al.2013). This disease has caused severe yield losses, and there are currently no effective prevention and control strategies. MRDD was firstly reported in 1954 in China (Luanet al. 2012). An epidemic outbreak occurred in the 1990s, especially on the Yellow-Huai-Hai River Plain,which are summer maize-growing regions of China (Wanget al. 2006, Liuet al. 2014). Three pathogens, maize rough dwarf virus (MRDV), rice black-streaked dwarf virus(RBSDV) and Mal de Rio Cuarto virus (MRCV), could result in its phenomenon. The RBSDV, which belongs to theFijivirusgenus in the family ofReoviridae, was identified as the main virus of MRDD in China (Wanget al. 2006; Liuet al. 2014). The genome of RBSDV is a double-strand RNA that contains 10 linear genomic segments (S1-S10),according to polyacrylamide gel electrophoresis (PAGE)migration (Zhanget al. 2008). MRDD is transmitted in a persistent manner through the insect, brown planthopper(Laodelphax striatellus). To date, preventive measures such as chemicals or alterations on the sowing date, could not completely control MRDD. Furthermore, the progress of studies has been slow, which contributed to great natural variations in the amount of insects. Moreover, there is no ideal artificial inoculation method.

In order to investigate the invasion and defense mechanism of thefijivirus, it is important to understand its virus-host interaction. The proteomics technique has been confirmed to be a powerful tool for understanding the response of the host to pathogens in many species of plants (Slykhuis 1976;Brownet al. 2006; Afrozet al. 2011; Yanget al. 2011; Wuet al.2013a). The identification of differentially expressed proteins during virus inoculation not only uncovers physiologically consistent protein patterns associated with the whole process, but also reveals the unexpected importance of some pathways. Especially for virus and other pathogens,proteomics has contributed to defining functional genes and proteins involved in plant-pathogen interactions. Wuet al. (2013a) obtained 24 new proteins with 2-dimensional electrophoresis (2-DE) analysis after sugarcane mosaic virus (SCMV) infection between resistant and susceptible maize ecotypes. After soybean mosaic virus infection, 16 proteins were potentially involved in protein degradation,defense signal transfer, reactive oxygen, and other metabolic pathways (Yanget al. 2011).

In the case of MRDD, Liet al. (2011) identified 91 different protein-related multiple metabolic/biochemical pathways between RBSDV infected and control plants using the proteomics method. Various resistance-related maize genes and cell wall- and development-related genes were found in RBSDV infected maize with oligomer-based microarrays (Jiaet al. 2012). Comparative proteomics analysis of IR64 with near-isogenic rice mutants revealed 22 proteins that may be potentially associated with rice resistance to the brown planthopper (Sanghaet al. 2013). The above research could provide an important clue for understanding the defense mechanism of insect-mediated diseases. Nevertheless,little is known on the molecular basis of the maize defense mechanism against this pathogen. The virus-host interaction of RBSDV infection remains unclear in maize.

The purpose of this study was to investigate the protein expression pattern of maize inbred lines, which had different resistances to RBSDV. Differentially expressed proteins were identified by 2-DE proteomics analysis. This enabled the investigators to explore the mechanism of disease resistance, and the interaction between the virus and plant.Furthermore, this would also enhance the understanding of the disease resistance mechanism, and provide a theoretical basis for maize resistant to MRDD breeding.

2. Materials and methods

2.1. Plant materials and inoculation with RBSDV

Two inbred maize lines (Zheng 58 and LN 287) were selected, because Zheng 58 was sensitive to RBSDV,which was the variant of Ye 478 (U8112×5003), and LN 287 was highly resistant to RBSDV，which was selected from US hybrid P78599. When the plants grew to the three-leaf stage, these plants were inoculated with small planthoppers captured from a winter wheat field where MRDD happened seriously. Each plant was inoculated by two planthoppers per seedling for 3 d, and transferred to the field in Jiaozhou Farm, Shandong Province, China. A total of 180 plants were planted for proteomics analysis,in which row spacing was 5 cm and line spacing was 60 cm. The control groups were shrouded with net sheds to prevent MRDD infection, while the treatment groups were exposed to its surroundings for a second inoculation, when planthoppers were prevalent at early June in the east of Shandong region. These field trials were repeated three times. After one month, leaves were sampled for real-time PCR to confirm the existence of RBSDV. In the field, the following phenotypes were observed in RBSDV sensitive plants: the height of the plant was significantly lower than the control; white waxy apophysis appeared behind the leaves;the color of the leaves was dark green. Leaves with obvious symptoms with Zheng 58 and leaves in the same position with LN 287 were sampled at 45, 60 and 75 d after sowing.

2.2. RNA extraction and quantitative real-time PCR(qRT-PCR)

In order to identify the inoculation degree of RBSDV in the molecular level, real-time PCR was performed. Total RNA was extracted from the leaves of inoculated and non-inoculated plants using TRIzol Reagent (TaKaRa,Japan), according to manufacturer’s instructions. Then,total RNA was treated with RNase-free DNase I (TaKaRa)to remove the possible contamination of genomic DNA.RNA concentration was determined using Nanodrop 2000 (Thermo, USA). Approximately 2 μg of total RNA was reverse-transcribed (RT) using a PrimeScriptTMRT Reagent Kit with a gDNA Eraser (TaKaRa), and RT products equivalent to 50 ng of total RNA were used as a template in qRT-PCR. The PCR reaction was performed with SYBRPremix Ex TaqTM(TaKaRa). The PCR master mix was prepared according to manufacturer’s instructions: 10 μL of 2×SYBRPremix Ex Taq(Tli RNaseH Plus), 0.4 μL of each forward and reverse primers for the target gene, and 0.4 μL of ROX Reference Dye (50×). The qRT-PCR was performed in a final volume of 20 μL for 5 min at 95°C as an initial denaturation step, followed by 35 cycles of 15 s at 95°C, 30 s at 65°C, 20 s at 72°C, and 10 min at 72°C.After the qRT-PCR, the temperature was programmed to ramp from 65 to 99°C, raising the degree by 0.1°C s–1after the extension step. A Rotor-Gene 6000 Real-time Rotary Analyzer (ver. 6.0, Corbett Life Science, Germany) created the melting curves. Ct values and qRT-PCR efficiency were computed using the ‘comparative quantitation’method in the Real-time qPCR Analysis Software (Corbett Life Science). The Ct value was defined as the point at which the fluorescence rose above the background fluorescence. The Ct of RBSDV was normalized to that of the selected reference gene (Actin). The Actin primer was: forward, 5′-GTCCATGAGGCCACGTACAA-3′;reverse, 5′-CCGGACCAGTTTCGTCATA-3′. The primers for RBSDV identification were: forward,5′-TCAGCAAAAGGTAAAGGAACG-3′; reverse,5′-AGAGCTCTTCTAGTTATTGCG-3′.

In order to confirm the expression of the genes detected in 2-DE, GlyandAPXgenes were selected for qRTPCR. The primer forGlywas: forward, 5′-ACCGAATC GAGACAGAC CCT-3′; reverse 5′-ACCTGGGTATCCCG AGTCTT-3′. The primer forAPXwas: forward, 5′-GAGTTCC CCACCCTCTCCTATG-3′; reverse, 5′-TGCTTGCCAA AGACTTGCCTC-3′. All reactions were performed in three biological replicates. Data were presented as means±SD. The statistical significance of quantitative data were determined using Student’st-test. Differences were considered statistically significant whenP＜0.05.

2.3. Protein extraction

Fresh leaf samples were grounded to fine powder with liquid nitrogen using mortar and pestle. The modified trichloroacetic acid (TCA)/acetone precipitation method for protein extraction was as follows: 0.5 g of leaf powder was homogenized sequentially until it melted in 5 mL of TCA/acetone buffer (–20°C) containing 10% TCA, 0.07%dithiothreitol (DTT), 1% polyvinyl pyrrolidone (PVP) and 1 mol L–1of phenylmethanesulfonyl fluoride (PMSF); centrifuged at 13 000 r min–1for 1 h at 4°C; and the pellet was washed with 5 mL of cold acetone twice. After air drying, the pellets were re-suspended in lysis buffer (7 mol L-1of urea, 2 mol L-1of thiourea, 4% (w/v) (3-cholamidopropyl dimethylammonio)propanesulfonic acid (CHAPS), 2% immobilized pH gradient(IPG) buffer, pH 4–7, and 40 mmol L-1of DTT) by vortexing for 4 h at 25°C. The suspension was centrifuged at 13 000 r min–1for 1 h at 4°C. Then, cold acetone (–20°C) was added with the supernatant in a new centrifuge tube with at a ratio of 1:10, placed overnight, followed by centrifugation at 13 000 r min-1for 1 h at 4°C. Next, the supernatant was discarded,and the resulting pellet was dried and re-suspended in a rehydration solution (7 mol L-1of urea, 2 mol L-1of thiourea,2% (w/v) CHAPS, 0.5% IPG buffer at pH 4–7, and 2.8 mg mL–1of DTT). The suspension was centrifuged at 13 000 r min–1for 1 h at 4°C, and the supernatant was finally collected as the protein extract. Protein concentration was determined using the Bradford method (Bradford 1976), and bovine serum albumin (BSA) was used as the standard for the calibration curve. The protein extract was subjected to 2-DE.

2.4. 2-DE and image analysis

Protein extracts were analyzed by 2-DE, as described by operating manual of GE Healthcare (USA). Then, 300 μg of protein sample in a final volume of 450 μL of rehydration buffer containing 0.5% IPG pH 4–7 (GE Healthcare) was loaded into a focusing tray. Immobilized linear strips(pH 4–7, 24 cm) (GE Healthcare) were passively rehydrated for 16 h. The isoelectric focusing was performed with an EttanTMIPGphorIITMIsoelectric Focusing System (GE Healthcare) using IPG strips (24 cm, pH 4–7). Focusing was performed at 100 V for 1 h, 300 V for 1 h, and 500 V for 1 h; gradient, 1 000 V for 2 h; gradient, 8 000 V for 3 h; and maintained at 8 000 V for 10 h. The second dimension was conducted using 12.5% SDS polyacrylamide gels through the EttanTMDALTsix System (GE Healthcare). The 2-DE gels were stained with silver nitrate for gel analysis. For matrixassisted laser desorption/ionization time-of-flight (MALDETOF) analysis, 1.3 mg of protein was used for 2-DE; and the gels were stained with Coomassie brilliant blue (CBB)G-250 (Bio-Rad, USA). A digital image of the gels was captured using a flat image scanner (UMAX Powerlook 2100XL) at 300 days post inoculation (dpi). The 2-DE was repeated three times for each sample. Digital images of the stained gels were obtained using an image scanner, and protein profiles were analyzed by the ImageMaster Platinum Software (ver. 6.0, GE Healthcare). After background subtraction and spot detection, spots were matched and normalized using the total density index in the gel image;and proteins with a change of 1.5 folds were considered differentially accumulated.

2.5. Protein identification by mass spectrometry

In order to digest the in-gel protein, the selected gel points were washed once with 500 μL of H2O, followed by washing for three times with 500 μL of 25 mmol L–1ammonium bicarbonate in 50% acetonitrile for 60 min on a mixer.The gel points were dehydrated by 500 μL of acetonitrile.Disulfide bonds were cleaved by incubating the samples with 200 μL of 10 mmol L–1of DTT in 25 mmol L–1of ammonium bicarbonate buffer for 60 min at 56°C. The alkylation of cysteines was performed by the addition of 200 μL of 55 mmol L–1of iodoacetamide in 25 mmol L–1of ammonium bicarbonate buffer, and incubated for 45 min at room temperature in the dark. Gel bands were washed twice with 25 mmol L–1of ammonium bicarbonate buffer and dehydrated with 500 μL of acetonitrile. Gel points were covered with trypsin solution (10 ng μL–1in 25 mmol L–1of ammonium bicarbonate buffer). After 30 min of incubation on ice, the remaining trypsin solution was removed; and 25 μL of 25 mmol L–1of ammonium bicarbonate were added.Proteolysis was performed overnight at 37°C, and stopped by the addition of 5% formic acid.

MALDI-TOF/TOF analysis by ultra-fleXtreme (Bruker,Germany). After digesting the sample gel point, 1 μL of peptide solution was dripped onto the AnchorChip target plate. Then, 0.1 μL of alpha-cyano-4-hydroxycinnamic acid(CHCA) as matrix was dripped onto the plate at the same place after the droplets were dried at room temperature. The plate was placed into a spectrometer and the instrument’s parameter was set for reflect mode. The mass range was from 500 to 3 500 Da, and scan resolution was 50 000.Approximate 3–5 samples among the most abundant mass spectrometry (MS) peaks were selected for MS/MS scan.

3. Results

3.1. MRDD phenotype in the field and identification with qRT-PCR

After 45 d of inoculation in the field, the susceptible line Zheng 58 revealed obvious MRDD symptoms, and the susceptibility rate was high. The diseased plants grew much shorter than the healthy plants with short internodes, stunting, dark green leaves, and waxy white galls along the veins behind the leaf blades (Fig.1). All Zheng 58 samples completely revealed MRDD symptoms at 75 d after inoculation. However, the resistant line LN 287 exhibited a high resistance to RBSDV.Furthermore, the treatment groups and controls both grew well; and the plant did not show the MRDD phenomenon.In addition, samples revealed MRDD symptoms in the field,as verified by real-time PCR. The content of RBSDV in Zheng 58 was significantly higher than that in LN 287 and controls (P＜0.01), but there was no obvious change on RBSDV content in LN 287 (Fig. 1). Based on these results,we concluded that samples on the susceptible and resistant lines were adaptable for further proteomics analysis.

3.2. 2-DE analysis

In order to study the changes in protein profiles in response to RBSDV, 2-DE was performed with infected leaves. A total of 944 spots were detected by 2-DE analysis software(ImageMasterTMplatinum 6, Switzerland). Three repeats revealed a high level of reproducibility, since the correlation coefficient for intra-groups was more than 0.822 (e.g.,correlation=0.911, 0.918, 0.850, and 0.822). Silver dying was used when identifying the protein spot with the software,and Comas dying was applied for MALDI-TOF identification.A total of 44 protein spots were identified after MALDI-TOF analysis. Over 91% of the spots were identified. The representative protein changes were shown in Figs. 2 and 3.

3.3. ldentification of RBSDV responsive proteins by MALDl-TOF/MS

Comparative analyses of 2-DE gels between different maize inbred lines on different growth stages were performed.Results revealed that 17 protein spots were up-regulated and 26 protein spots were down-regulated in treated Zheng 58 materials, compared to control (untreated Zheng 58); while 27 up-regulated and 18 down-regulated proteins were detected in treated LN 287 plants rather than in control(untreated LN 287). For example, glutathineS-transferase 4,putative glyoxalase family protein, cytochrome b6-f complex iron-sulfur subunit and putative peptidyl-prolylcis-trans isomerase family protein were up-regulated only in resistant line. At the different growth stage, the changed proteins showed the different levels in the resistant and susceptible lines. At 45 d, the responsive proteins in LN 287 were more than those in Zheng 58, including stress responsive protein, peroxiredoxin-5 and 50S ribosomal protein, it suggested that defense system has started and protected against viruses in disease-resistant materials. In addition, stress responsive protein showed down-regulated in susceptible line at 60 d. Cytochrome-C oxidase subunit and oxygen-evolving enhancer protein 3-1 showed upregulated in LN 287 but down-regulated in Zheng 58 at 60 and 75 d, respectively (Appendices A and B). The reason for the difference in gene expression and regulation might be: due to the virus-host interaction after virus infection, and maybe due to the difference in genetic background between susceptible and resistant lines.

Differential protein spots were further analyzed by mass spectrometry identification, and the proteins were divided into six categories: photosynthesis-related protein (25.0%),energy and metabolism protein (17.9%), stress-related protein (17.9%), protein synthesis and folding protein(14.3%), protein of unknown function (14.3%), and other proteins (7.1%) (Fig. 4).

Fig. 1 Plant height and leaf growth of maize inbred lines in the field. A, the healthy (left) and the infected (right) plants of Zheng 58.B, the healthy (left) and the infected (right) plants of LN 287. C, the healthy (left) and the infected (right) leaves of Zheng 58. D, the enlarged leaves parts of Fig.1-C in the rectangle. E, virus content in Zheng 58 and LN 287 analyzed by real-time PCR, respectively.F, quantitative real-time PCR (qRT-PCR) results for Zheng 58 and LN 287, respectively. 1 and 5, LN 287 control plants; 2 and 6,LN 287 infected plants; 3 and 7, Zheng 58 control plants; 4 and 8, Zheng 58 infected plants. M, DNA marker DL2000.

3.4. Photosynthesis-related proteins

Five photosynthesis-related proteins were identified after MALDI-TOF/MS analysis. The ratio to the total proteins identified was 25.0%. This mainly include ribulose biphosphate carboxylase large chain, oxygen-evolving enhancer protein 1, and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit. For example,oxygen-evolving enhancer protein 3-1 precursor and oxygen-evolving enhancer protein 1 were up-regulated in resistant line LN 287 after 60 and 75 d, but was downregulated in susceptible line Zheng 58. In addition,chlorophylla/bbinding protein 6A was up-regulated in the Zheng 58 line. The expression of many photosynthesisrelated proteins was affected during virus infection in maize.

3.5. Stress related proteins

Five stress-related proteins were identified in resistant line LN 287, including glutathioneS-transferase 4, putative glyoxalase family protein, APX1, peroxiredoxin-5, and stress responsive protein. However, a down-regulated stressrelated protein (stress responsive protein) was identified in susceptible line Zheng 58. The putative glyoxalase family protein was the protein rarely reported before with 2-DE analysis in maize. This was up-regulated in LN 287,and was not detected in Zheng 58. APX1 and glutathineS-transferase were previously reported when inoculated with SCMV (Wuet al. 2013a) or other cases (Jinet al.2015) in maize.

3.6. Energy metabolism and structure proteins

Energy metabolism related proteins were largely upregulated, such as cytochrome-C oxidase subunit, in resistant line LN 287 after 60 and 75 d. However, these proteins were down-regulated in Zheng 58. The structure protein mainly include ribosomal related-protein, and protein-associated with synthesis and folding, such as 50S ribosomal protein L21, L12-1, and putative petidyl-prolylcis-trans isomerase family protein. In addition, hypothetical proteins and unknown proteins were also identified. The function of these proteins needs to be studied in the future.

Fig. 2 Two-dimensional electrophoresis (2-DE) analysis of proteins extracted from maize leaves of Zheng 58 control group (A) and treated group (B) 60 d after sowing. The numbers in the figures mean the protein spot showed in the imaging analysis software.

3.7. ldentification of gene expression by qRTPCR

In order to confirm the expression of regulated proteins in the RNA level, qRT-PCR was conducted. Two stressrelated genes (APXandGly) were selected to compare the expression pattern. The result in Fig. 5 revealed that theAPXgene was significantly up-regulated in the susceptible line (Zheng 58) at RNA level (P＜0.01), and it was also upregulated in 2-DE. The expression of theGlygene was upregulated slightly in the susceptible line, but the difference was not statistically significant. The trend of two genes was consistent with the patterns in the proteomics.

The expression of the candidate gene in the RNA level may be different with the pattern at the protein level. Hence,this needs to be confirmed again before the next work,such as cloning; and these results could provide a clue for understanding the relationship between RBSDV and the candidate genes.

3.8. A putative RBSDV infection response network

A RBSDV infection response network was proposed that included the majority of the 44 RBSDV responsive proteins,which are involved in several functional processes such as redox homeostasis, virus defense system, photosynthesis,energy and metabolism regulation.

The infection of RBSDV could result in the increase of ROS content, and cause an imbalance of redox in maize. To maintain redox homeostasis, the synthesis of anti-oxidation enzymes such as APX and peroxiredoxin-5,increase to decrease the levels of ROS and allow the cell to achieve redox balance (Jinget al. 2015; Huanget al. 2016). Stress responsive protein and glutathioneS-transferase could have an effect on virus defense system. The photosynthetic rate of maize leaves is impaired by the up- and down-regulation of rubisco subunit,chlorophylla/bbinding protein and oxygen-evolving enhancer protein 3-1. The increase of above proteins in RBSDV resistant plant generates extra energy to avoid pathogen effector molecules (Kunduet al. 2013). The regulation of 50S ribosomal protein L21 could change the speed of transformation of proplastids to chloroplasts(Chenet al. 2014). The difference response of resistant and susceptible maize lines provides us with important information on understanding the mechanism of resistant to RBSDV, enable the enhancement of RBSDV resistance and obtain protection from virus infection.

Fig. 3 Two-dimensional electrophoresis (2-DE) analysis of proteins extracted from maize leaves of LN 287 control group (A) and treated group (B) 60 d after sowing. The numbers in the figure mean the protein spot showed in the imaging analysis software.

Fig. 4 Functional categories of the identified proteins.

Fig. 5 Expression analyses of maize rough dwarf disease(MRDD) responsive genes by quantitative real-time PCR (qRTPCR). Bars mean SD. ＊＊, significant at 0.01 level.

4. Discussion

Proteins identified after 2-DE provided more information to explain the mechanism of RBSDV resistance. Among stress-related proteins, the putative glyoxalase family protein has not been reported earlier in virus-maize interactions;and may play a role in maize resistance to RBSDV. The glyoxalase family-identified includes glyoxalase I and II. These were used to detoxify methylglyoxal (MG),which was a cytotoxic compound that increases rapidly under stress conditions. Glyoxalase I converts MG to S-D-lactoylglutathione by utilizing glutathione, while glyoxalase II converts S-D-lactoylglutathione to D-lactic acid. In addition, glyoxalase I was found to be important in cell division and proliferation, microtubular assembly,vesicle mobilization, and other processes (Wuet al.2013b). Moreover, the overexpression of the glyoxalase gene revealed an increase in ROS and MG under stress conditions by maintaining glutathione homeostasis and antioxidant enzyme levels (Yadavet al. 2005; Singla-Pareeket al. 2006); and enzyme activity and glyoxalase I transcription could be enhanced by some abiotic and biotic stresses, such as NaCl, mannitol, zinc and MG (Linet al.2010; Mustafizet al. 2011).

GlutathioneS-transferase 4 plays a role in protecting cells against the toxic product of lipid peroxidation. Each subunit of this contains active sites that bind glutathione and hydrophobic ligands. Plant glutathione S-transferases attach glutathione to electrophilic xenobiotics, which tag these for vacuolar sequestration. The role of glutathione S-transferasein metabolism remain unclear, although their complex regulation by environmental stimuli implies that these have important protective functions. For example,glutathione S-transferase was up-regulated after drought,heat, salicylic acid and abscisic acid treatment in maize(Liuet al. 2013; Wuet al. 2013b). Recent studies have shown that glutathione S-transferases catalyzed glutathionedependent isomerization and the reduction of toxic organic hydroperoxides. GlutathioneS-transferases might have non-catalytic roles as carriers for phytochemicals (Edwardset al. 2000). The same up-trend of glutathione S-transferase 4 appeared in our study, which might be related with the resistance of maize RBSDV through the glutathionedependent mechanism.

Several members of peroxiredoxin and peroxidase were induced by biotic and abiotic stresses, and cytosolic ascorbate peroxidase was the main part of the plant pathway of AsA-GSH redox, which was the key enzyme for clearing H2O2(especially in chloroplast). These were closely connected with stress, cell programmed death,and plant growth and development (Li and Wang 2008).The expression ofAPX1was both up-regulated in the susceptible and resistant lines, which could suggest thatAPX1was responsible to RBSDV infection. A similar trend was obtained on peroxidase gene expression.

One of the stress responsive proteins was down-regulated at 60 d in Zheng 58. It was up-regulated at 45 d in LN 287,and did not reveal a significant change after 45 d; which indicated that the stress responsive protein was expressed at the early stage of RBSDV infection. This could be related to the early response to virus stress in maize, and further studies would be conducted with these proteins.

The cytochrome-C oxidase subunit revealed a significant difference in protein expression. Cytochrome-C oxidase is a marker enzyme in the mitochondrial inner membrane. It transfers the proton into the (inter) membrane space, and synthesizes ATP in the mitochondria. The cytochrome-C oxidase subunit was down-regulated at 60 and 75 d in Zheng 58, while it was up-regulated at 60 d in LN 287.This suggests that the efficiency of transferring electrons in Zheng 58 is reduced, and respiration efficiency decreased.Furthermore, the ATP produced by plants was reduced.Hence, the growth of these plants was worse than that of controls. However, the level of energy metabolism increased in resistant line LN 287. Hence, the change in the cytochrome-C oxidase subunit might be consistent with the trend of RBSDV resistance.

Photosynthesis-related proteins revealed the highest proportion in differential proteins, which meant that the impact of photosynthesis on MRDD was great. In the case of ribulose bisphosphate carboxylase small unit 2, this was down-regulated; but was up-regulated in LN 287. This could be due to the decreased chlorophyll content and photosynthetic rate, indicating that rubisco was gradually degraded during leaf senescence (Minamikawaet al.2001; Khanna-Chopra 2012; Onoet al. 2013; Chenet al.2014). The ribulose bisphosphate carboxylase catalytic carboxylation between ribulose-1,5-bisphosphate and carbon dioxide in the Calvin cycle transfers the free carbon dioxide in the atmosphere into energy storage molecules in the organism. The phenomenon suggests that the resistance of LN 287 might be related with the improvement of photosynthetic efficiency.

Aspartate aminotransferase and the peptidyl-prolylcistrans isomerase family of proteins mainly participate in protein synthesis and modification. The latter protein was up-regulated in LN 287. This suggests that there were significant changes on protein synthesis and modification,and the efficiency of substance metabolism improved in the resistant line.

Cell structure-related proteins mainly include ribosomal proteins (the sites of protein synthesis in plants), which synthesize the 30S and 50S subunit of ribosomes. Several structures and folding proteins were found to be differentially expressed in response to stress (Gammullaet al. 2010;Chenet al. 2014). The expression change in ribosomal proteins prompts its relevance to ribosome characteristics.

5. Conclusion

In this study, the expression patterns of differential proteins after RBSDV infection were investigated in maize. A total of 44 differently expressed spots were successfully identified by MALDI-TOF/TOF MS, which represented 40 proteins. This revealed that up-regulated proteins were greater than the down-regulated proteins in the resistant line, while up-regulated proteins were lesser than the downregulated proteins in the susceptible line. This means that more proteins were induced and involved in the defense system for improving host resistance to plant disease.The identified proteins were related to stress responsive proteins, photosynthesis, energy metabolism, carbon fixation, protein structure and folding. RBSDV infection in maize significantly induced APX-1, Gly1, and glutathione S-transferase proteins, when the plant responded to stress.Our results provide valuable scientific clues regarding the regulatory mechanisms of RBSDV resistance.

Acknowledgements

We thank Dr. LI Jun (Qingdao Agricultural University, China)for helping in editing this manuscript. This research was funded by the National Natural Science Foundation of China(31371636), the Modern Agricultural System of Shandong Province, China (SDAIT-01-022-01), the Key Research and Development Project of Shandong Province, China(2016GNC110018), and the Applied Basic Research Project of Qingdao, China (14-2-4-13-jch).

Appendicesassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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