Open Access

iTRAQ-based quantitative proteomics analysis of rice leaves infected by Rice stripe virus reveals several proteins involved in symptom formation

  • Biao Wang1,
  • Jamal-U-Ddin Hajano1,
  • Yingdang Ren2,
  • Chuantao Lu2 and
  • Xifeng Wang1Email author
Virology Journal201512:99

https://doi.org/10.1186/s12985-015-0328-y

Received: 1 May 2015

Accepted: 18 June 2015

Published: 26 June 2015

Abstract

Background

Rice plants infected by Rice stripe virus (RSV) usually leads to chlorosis and death of newly emerged leaves. However, the mechanism of RSV-induced these symptoms was not clear.

Methods

We used an iTRAQ approach for a quantitative proteomics comparison of non-infected and infected rice leaves. RT-qPCR and Northern blot analyses were performed for assessing the transcription of candidate genes.

Results

As a whole, 681 (65.8 % downregulated, 34.2 % upregulated infected vs. non-infected) differentially accumulated proteins were identified. A bioinformatics analysis indicated that ten of these regulated proteins are involved in chlorophyll biosynthesis and three in cell death processes. Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis. Three aspartic proteases expressed higher in RSV-infected leaves than those in the control leaves, which were also implicated in RSV-induced cell death. Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results.

Conclusions

The magnesium chelatase and aspartic proteases may be associated with RSV-induced leaf chlorosis and cell death, respectively. The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.

Keywords

Rice Proteome iTRAQ Magnesium chelatase Peptidase Plant defense

Background

Rice stripe virus (RSV), a member of the genus Tenuivirus, is one of the most economically important viruses in eastern Asia including China, Korea, and Japan [1]. In 1964, RSV was reported for the first time in Zhejiang Province [2] and then spread to 18 provinces in rice-growing areas of China [3]. From 2000 to 2005, 1,700,000 ha of rice fields were affected by this virus in Jiangsu Province, including 1,000,000 ha area where incidence was so severe that yield losses exceeded 50 %, and in some places no rice was harvested [4].

RSV is transmitted predominantly in a persistent propagative manner by the small brown planthopper (SBPH; Laodelphax striatellus Fallen) [5] and can be transmitted transovarially for more than 40 generations [6]. RSV has four single-stranded RNA segments, named RNA 1, 2, 3 and 4 in order of their molecular weight. Among these, RNA 3 encodes a nucleocapsid protein (NCP) from the viral complementary RNA [7], while RNA 4 encodes a disease specific protein (SP) from the viral RNA [8]. RSV-induced symptoms of rice typically are chlorotic stripes and mottlings on the leaves. Newly emerged leaves exhibit yellow stripes or necrosis, then folding and twisting; plants are stunted and finally dead [1].

Leaf chlorosis in general is widely accepted as a sign of reduction in chlorophyll [9, 10], and leaf chlorosis upon virus infection is also related to decreased chlorophyll [11]. Subsequent studies have shown that various molecular mechanisms are involved in leaf chlorosis during virus infection. For example, during Cucumber mosaic virus (CMV) infection, the expression of the genes encoding magnesium chelatase is regulated by CMV satellite RNA, thus blocking chlorophyll biosynthesis [12, 13]. In addition, chlorotic symptoms induced by African cassava mosaic virus (ACMV) are linked to the expression level of chlorophyll-related genes encoding proteins such as chlorophyllide a and chlorophyllide b [14]. However, the chlorosis on tobacco leaves during the flavum strain of Tobacco mosaic virus (TMV) infection not resulted from the reduction of chlorophyll biosynthesis, but was reduction of the core complexes of photosystem II and the oxygen evolving complex [15]. In a recent report, RSV SP interacted with PsbP (an oxygen-evolving complex protein) resulting in the downregulation of PsbP in chloroplasts, and then modulating RSV symptoms through disruption of chloroplast structure and function [16]. Whether other chlorophyll relation proteins are modulated during RSV infection has not been known.

In addition, if the cultivar is susceptible to RSV infection, newly emerged rice leaves usually exhibit necrosis [1]. Previous report indicated that a vacuolar processing enzyme that has caspase protease activity was indispensable for the TMV-induced hypersensitive response, which involves programmed cell death in tobacco [17]. Even in an uninfected healthy plant, the expression of aspartic proteases induces programmed cell death, and then involves in senescence [18]. Nevertheless, we still need to elucidate how the expression of aspartic proteases is regulated after RSV infection. Therefore, the key rice protein(s) involved in RSV-induced disease symptom formation require(s) further exploration.

Some techniques have been shown as powerful tools for understanding plant-pathogen interactions, including yeast two-hybrid system [1921], glutathione-S transferase pull-down assay [22, 23], immunofluorescence laser scanning confocal microscopy [24, 25], 2D gel-based technology [26, 27], and iTRAQ (isobaric tag for relative and absolute quantitation) LC-MS/MS (liquid chromatography tandem mass spectrometry) technology [28]. iTRAQ LC-MS/MS technology adopted stable isotope labeling strategies of proteins or peptides for measurement and allowed relative quantitation comparison using an internal reference, and could simultaneously label and accurately quantify proteins from multiple samples [29, 30]. In this study, by using an iTRAQ-based quantitative proteomics approach, we analyzed protein accumulation profiles of RSV-infected leaves in comparison with healthy leaves to explore symptom formation and to understand rice-RSV interactions.

Results

Symptom formation and RT-PCR confirmation of infection

There were 10 viruliferous SBPH allowed to feed on each plant of cv. Aichiasahi for 2-day inoculation access period. Newly emerged leaves on the initially inoculated plant developed pale-yellow stripes, which then collapsed in the form of blotches at 21 days post inoculation (dpi) (Fig. 1a). At 23 dpi, severe necrosis resulted in plant death (Fig. 1b). No disease symptoms were observed on mock plants. Samples of RSV-infected plants and control plants that were collected at 21 dpi to confirm infection by RT-PCR yielded an expected 969-bp fragment that was also found in a previously confirmed-positive sample (Fig. 1c). The 969-bp fragment was not present in the mock control or no-template control (NTC).
Fig. 1

Typical chlorotic stripes and necrosis symptoms in rice plants induced by Rice stripe virus (RSV) and infection confirmation by RT-PCR detection. Both RSV-infected and mock rice plants were shown at (a) 21 days post inoculation (dpi) and (b) 23 dpi; (c) RT-PCR confirming that 969-bp fragment for RSV nucleocapsid gene (NCP) was absent in mock and present in symptomatic RSV-infected plants. Mock rice had typical healthy growth, but RSV-infected leaves showed typical chlorosis at 21 dpi (corner of panel a), and the entire plant was dead at 23 dpi (b). M, DL2000 DNA marker; P, RSV infected sample as positive control; N, healthy sample as negative control; 1-3, mock leaves; 4-6, RSV-infected leaves

Protein identification and quantification

When the iTRAQ approach was used to analyze proteins obtained from RSV-infected leaves and mock leaves which were collected at 21 dpi, 128,144 spectra were totally obtained from an ABI-5600 system and then approximately 59,824 MS spectra identified matched known spectra. Overall, 3687 different proteins were identified when a false discovery rate (FDR) <1 % was applied to the dataset (Fig. 2). A total of 681 proteins were differentially accumulated, with a fold-change >1.5 (P < 0.05); 448 were downregulated, and 223 had a fold-change <0.67 (P < 0.05) (Table 1).
Fig. 2

Statistics for total spectra for reversed-phase HPLC and LC-MS/MS, identified proteins and differentially accumulated proteins from iTRAQ proteomics by searching and analysis of NCBI database. Spectra scan ranged from 350 to 1800 m / z. Number refer to statistics of different parts: total spectra were generated from the iTRAQ experiment using the materials (RSV-infected and mock leaves); spectra identified matched known spectra; proteins identified analyses were determined by spectra identified upon the NCBI database; differentially accumulated proteins analysis based on the fold-change >1.5 or <0.667 (P < 0.05); downregulation proteins with fold-change >1.5 (P < 0.05); upregulation proteins with fold-change <0.667 (P < 0.05)

Table 1

Summary of the proteins identified by iTRAQ as being differentially accumulated in RSV-inoculated plants compared with mock-inoculated rice plants at 21dpi

Regulation

No. of proteins

David

GOa

Categoriesb

Percentagec

No. of functional groups

Down

448 (65.8 %)

332

317

203 BP

61.1

53

    

154 CC

46.4

20

    

233 MF

70.2

33

    

129 KEGG

38.9

16

  

unknown 116

    

Up

233 (34.2 %)

178

175

112-BP

62.9

17

    

64 CC

36.0

13

    

125 MF

70.2

16

    

7 -KEGG

39.9

13

  

unknown 55

    

Total

681

     

Note: Using the David platform, 332 downregulated and 178 upregulated proteins were analyzed, and 317 and 175 proteins were annotated by GO, respectively. Annotated proteins were clustered by groups based on the BP, CC, MF and KEGG analyses

aGO annotation: BP, biological process; CC, cellular component; MF, molecular function

bCategories based on BP, CC, MF and KEGG

cPercentage of total proteins annotated

Bioinformatics analysis

The identified and quantified proteins were then analyzed for function, pathway and interaction network. In the GO analysis, 358 proteins were involved in molecular function, 233 (70.2 %, 35 functional groups) were downregulated and 125 (70.2 %, 16 functional groups) were upregulated (Table 1, Additional file 1: Table S1). The molecular function of downregulated proteins was mainly in cofactor binding (14.2 %), electron carrier activity (10.7 %), coenzyme binding (10.3 %), calcium ion binding (6.0 %), antioxidant activity (5.6 %), magnesium ion binding (4.7 %), peroxidase activity (3.9 %), vitamin B6 binding (3.4 %), FAD (flavin adenine dinucleotide) binding (3.4 %), and primary active transmembrane transporter activity (3.0 %) (Fig. 3a, Additional file 1: Table S1). Upregulated proteins were involved in cofactor binding (15.2 %), peptidase activity (13.6 %), coenzyme binding (12.0 %), electron carrier activity (12.0 %), endopeptidase activity (8.8 %), threonine-type peptidase activity (5.6 %), antioxidant activity (5.6 %), unfolded protein binding (4.8 %), FAD binding (4.8 %), and disulfide oxidoreductase activity (4.0 %) (Fig. 3b, Additional file 1: Table S1). Peptidase activity, the largest group within the catalytic activity group, comprised metallopeptidase activity, aspartic-type endopeptidase, cysteine-type peptidase activity, serine-type peptidase activity. Biological process was influenced by 315 proteins, 203 (61.1 %, 53 functional groups) downregulated proteins which mostly were involved in oxidation reduction (23.2 %), nitrogen compound biosynthesis (16.3 %), photosynthesis (12.3 %), generation of precursor metabolites and energy (11.8 %), cofactor metabolism (10.8 %), translation (9.9 %), monosaccharide metabolism (9.4 %), hexose metabolism (8.4 %), carboxylic acid biosynthesis (8.4 %), glucose metabolism (7.9 %) (Fig. 3a, Additional file 1: Table S1). The other 112 (62.9 %, 17 groups) upregulated proteins were mostly involved in oxidation reduction (25.0 %), proteolysis (17.0 %), generation of precursor metabolites and energy (12.5 %), macromolecule catabolism (11.6 %), protein catabolism (10.7 %), cellular protein catabolism (8.9 %), cofactor metabolism (8.0 %), cellular homeostasis (8.0 %), protein folding (6.3 %), and carbohydrate catabolism (6.3 %) (Fig. 3b, Additional file 1: Table S1). Cellular components that were downregulated included 154 proteins (46.4 %, 20 component groups), located in the plastid (70.8 %), chloroplast (31.8 %), thylakoid (12.3 %), photosynthetic membrane (9.1 %), organellar membrane (9.1 %), thylakoid part (7.8 %), plastid part (7.8 %), photosystem (6.5 %), chloroplast part (5.2 %), extrinsic to membrane (5.2 %), and oxygen evolving complex (4.5 %) (Fig. 3a, Additional file 1: Table S1). The 64 (36.0 %, 13 component groups) upregulated proteins were located in the cytosol (17.2 %), proteasome complex (15.6 %), organelle membrane (12.5 %), proteasome core complex (10.9 %), endoplasmic reticulum (9.4 %), Golgi apparatus (9.4 %), envelope (7.8 %), mitochondrial membrane (6.3 %), ribosomal subunit (4.7 %), membrane coat (4.7 %), and cell junction (3.1 %) (Fig. 3b, Additional file 1: Table S1).
Fig. 3

Gene Ontology enrichment analysis of differentially accumulated proteins from RSV-infected leaves compared with mock leaves. a Downregulated differentially accumulated proteins were annotated among 33 groups for molecular function (MF), 53 for biological process (BP) and 20 for cellular components (CC), respectively; b, Functional grouping of upregulated differentially accumulated proteins: 16 for MF, 17 for BP and 13 for CC

The KEGG pathway analyses indicated that among the downregulated proteins, 13 % were involved in the biosynthesis of plant hormones; 9 % in photosynthesis, carbon fixation in photosynthetic organisms, biosynthesis of terpenoids and steroid; and 4 % in porphyrin and chlorophyll metabolism (Fig. 4a). However, among the upregulated proteins, 16 % were involved in biosynthesis of plant hormones, 11 % in biosynthesis of alkaloids derived from shikimate pathway, 10 % in biosynthesis of phenylpropanoids, and 9 % in proteasome, starch and sucrose metabolism, citrate cycle, tryptophan metabolism, fatty acid metabolism, propanoate metabolism, and pentose and glucuronate interconversions (Fig. 4b). When the identified proteins were analyzed with the STRING software, the results showed that 547 proteins were interacting with each other. In the constructed interaction network (Additional file 2: Figure S1), the proteins were roughly divided into three groups: metabolism (B), chloroplast (C) and defense (D).
Fig. 4

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of differentially accumulated proteins that are identified from mock leaves and RSV-infected leaves for (a) downregulated and (b) upregulated. a downregulated proteins were annotated and participated in 16 pathways; (b) upregulated proteins were classified 13 pathways

Proteins differentially accumulated in response to RSV infection

Metabolism group

Functions of the down- and up-regulated differentially accumulated metabolism group of proteins included monosaccharide metabolism, disaccharide metabolism, polysaccharide metabolism, generation of precursor metabolites and energy, amino acid metabolism, fatty acid metabolism, phosphorus metabolism, and sulfur metabolism. Basically, carbohydrate metabolism provided more suitable source of energy and carbon for plant development. For example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 115458768, 115450493) and fructose-bisphosphate aldolase (115484401, 115468886, 115434198) were two important metabolic enzymes in glycolysis and gluconeogenesis [31]. Notable, evidences increasingly support the nonglycolytic functions of GAPDH, including apoptosis, DNA and RNA replication, DNA repair, RNA exportation, RNA synthesis, immunity response to various pathogens [3238]. GAPDH strong binding of negative strand Tomato bushy stunt virus (TBSV) was key regulatory step to promote asymmetric RNA synthesis, so GAPDH played a role in viral RNA replication and RNA synthesis [34]. However, GAPDH preferentially binds positive strand Bamboo mosaic virus (BaMV), and it negatively regulated the accumulation of BaMV [35]. Additionally, GAPDH negatively regulate autophagy interaction with host protein and immunity-associated cell death and defense on TMV infection [38]. GAPDH may be involved in viral replication and defense during RSV infection. Proteins that decreased in expression belonged to the vitamin, nucleotide, isoprenoid, phosphorus, sulfur and cofactor metabolism groups, suggesting that RSV infection inhibited their expression (Table 2). Thus, numerous biological processes helped rice to counteract RSV invasion.
Table 2

Differentially accumulated proteins between mock-inoculated leaves and RSV-infected leaves

Accession number

Protein name categorized by process

Cov (95)

Number of Matching Peptides

Ratio

P-Value

 

Chlorophyll biosynthetic process

    

115453785

Magnesium-chelatase subunit ChlI, chloroplastic

46.7

29

17.5

4.50 × 10-8

115438661

Uroporphyrinogen decarboxylase 1, chloroplastic

14.9

8

13.4

2.85 × 10-2

115444475

Porphobilinogen deaminase, chloroplastic

51.1

21

11.7

4.50 × 10-4

115456135

Magnesium-chelatase subunit ChlD, chloroplastic

27.8

23

9.4

5.19 × 10-7

115477483

Glutamate-1-semialdehyde 2,1-aminomutase, chloroplastic

34.7

26

5.3

2.85 × 10-2

115452897

Uroporphyrinogen decarboxylase 2, chloroplastic

36.4

21

5.0

1.83 × 10-2

115436038

Protoporphyrinogen oxidase, chloroplastic

21.3

12

4.8

3.07 × 10-4

115435974

Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase

42.2

25

3.7

1.16 × 10-5

115469822

Delta-aminolevulinic acid dehydratase, chloroplastic

29.1

16

3.5

4.11 × 10-4

115482796

Glutamyl-tRNA reductase, chloroplastic

16.4

9

3.4

1.34 × 10-3

 

Photosynthesis

    

109156602

Ribulose bisphosphate carboxylase large chain

82.2

508

44.1

1.71 × 10-5

115472625

Oxygen-evolving enhancer protein 3

41.5

57

31.9

3.46 × 10-5

115436780

Putative 33 kDa oxygen evolving protein of photosystem II

59.2

119

28.3

2.30 × 10-10

115470529

Probable photosystem II oxygen-evolving complex protein 2

58.3

62

21.1

8.92 × 10-4

115488344

Photosystem I reaction center subunit XI, chloroplast

30.8

13

19.1

3.57 × 10-2

115472753

Chlorophyll a/b-binding protein

49.0

39

18.7

8.07 × 10-4

115477831

Chloroplast photosystem I reaction center subunit II-like protein

59.1

50

18.4

3.46 × 10-7

115476576

Putative chlorophyll a/b-binding protein

36.5

27

14.5

2.05 × 10-2

115458738

OSJNBa0036B21.6 protein

38.5

19

13.7

2.74 × 10-4

115484899

Chlorophyll a/b-binding protein

63.2

67

13.3

1.17 × 10-5

115470199

PsbQ domain protein family, putative-like protein

28.4

11

10.9

1.53 × 10-3

115472785

Putative chlorophyll a/b-binding protein of LHCII type III, chloroplast

50.4

20

10.4

4.90 × 10-3

115446893

Putative Oxygen-evolving enhancer protein 3-2, chloroplast

26.2

6

10.1

2.74 × 10-2

115487694

Photosystem I reaction centre subunit N, chloroplast

28.2

8

10.0

2.12 × 10-2

115450991

Ribulose-phosphate 3-epimerase, chloroplastic

50.0

28

7.8

8.74 × 10-4

115467828

Chlorophyll a/b-binding protein

31.1

27

7.8

6.31 × 10-3

115452127

Fructose-1,6-bisphosphatase, chloroplastic

38.9

45

6.8

5.54 × 10-6

115482366

PsbP family protein

18.1

16

5.6

1.14 × 10-3

115465942

Ferredoxin--NADP reductase, leaf isozyme, chloroplastic

49.7

66

5.4

5.64 × 10-3

115447507

Putative ferredoxin-thioredoxin reductase

20.1

4

2.5

2.91 × 10-2

 

Defense response

    

115458852

Bet v I allergen family protein

29.9

5

0.3

1.89 × 10-3

115452513

Pathogenesis-related protein 1

49.4

7

0.1

7.92 × 10-4

115489022

Pathogenesis-related protein

29.8

5

0.04

9.16 × 10-4

115489014

Pathogenesis-related protein PR10

25.6

4

0.03

1.88 × 10-2

 

Proteolysis

    

115470052

ATP-dependent zinc metalloprotease FTSH 1, chloroplastic

42.6

50

9.9

3.31 × 10-8

115453893

Membrane-associated zinc metalloprotease family protein

17.6

7

7.7

8.99 × 10-4

115489316

Eukaryotic aspartyl protease family protein

25.1

10

6.8

1.04 × 10-2

115447609

ATP-dependent zinc metalloprotease FTSH 7, chloroplastic

4.3

4

6.5

3.77 × 10-2

115480844

Serine carboxypeptidase family protein

13.3

8

5.6

3.69 × 10-2

115435898

ATP-dependent Clp protease proteolytic subunit

18.5

4

3.5

2.12 × 10-2

115450022

Oligopeptidase A-like

24.2

20

3.2

1.35 × 10-4

115452585

Probable glutamyl endopeptidase, chloroplastic

15.0

18

2.9

6.00 × 10-3

115488046

Serine carboxypeptidase 1

11.2

4

2.7

4.20 × 10-2

115444859

Peptidase aspartic

24.1

10

0.5

4.99 × 10-2

115437452

Ubiquitin carboxyl-terminal hydrolase

16.2

7

0.4

3.60 × 10-2

115482252

Ubiquitin-conjugating enzyme E2-23 kDa

20.1

3

0.4

1.14 × 10-2

115483755

Ubiquitin-activating enzyme E1 2

22.6

25

0.4

3.41 × 10-3

115463349

Putative DNA-binding protein GBP16

26.0

15

0.4

1.26 × 10-2

115454751

Proteasome subunit beta type-2

30.2

9

0.4

2.65 × 10-2

115465685

Putative serine carboxypeptidase

24.9

12

0.4

4.19 × 10-3

115451123

Proteasome subunit alpha type-6

43.1

13

0.3

1.22 × 10-3

115456219

Leukotriene A-4 hydrolase homolog

20.8

12

0.3

6.69 × 10-3

115480143

Proteasome subunit beta type

36.3

7

0.3

1.63 × 10-2

115444057

Proteasome subunit alpha type-1

40.0

13

0.3

1.08 × 10-2

115440299

Putative insulin degrading enzyme

3.0

2

0.3

2.91 × 10-2

115440617

Proteasome subunit alpha type-3

39.4

10

0.3

4.04 × 10-2

115480019

Proteasome subunit beta type-1

28.1

6

0.2

7.50 × 10-3

115448935

Proteasome subunit beta type

40.7

12

0.2

6.34 × 10-4

115476300

Aminopeptidase M1-B

22.7

19

0.2

8.85 × 10-5

115461973

Aspartic proteinase

23.8

11

0.2

3.50 × 10-3

115445047

Aminopeptidase M1-A

18.8

17

0.2

1.04 × 10-5

115451209

Eukaryotic aspartyl protease family protein

11.4

4

0.2

2.37 × 10-3

 

Protein transport

    

115475569

Preprotein translocase subunit SECY, chloroplastic

4.1

3

5.5

1.76 × 10-3

115454153

SEC1 family transport protein SLY1

8.0

4

5.3

1.93 × 10-2

115451815

Translocase of chloroplast

20.2

7

3.1

1.42 × 10-2

115452177

Protein TOC75, chloroplastic

34.4

25

2.9

3.10 × 10-6

115435528

Importin-alpha re-exporter

5.5

2

0.5

7.40 × 10-4

115435714

GTP-binding protein

21.2

3

0.3

4.33 × 10-2

115463933

Putative GDP dissociation inhibitor

30.8

16

0.3

3.53 × 10-5

115454911

Coatomer subunit alpha-1

22.6

25

0.3

4.93 × 10-5

115461356

Clathrin light chain 1

14.6

3

0.2

4.25 × 10-2

115463119

Coatomer subunit delta-1

11.5

7

0.2

1.90 × 10-3

 

Translation

    

115480611

Cysteinyl-tRNA synthetase

11.8

6

8.7

1.93 × 10-3

115450395

50S ribosomal protein L11, chloroplast

38.1

13

7.2

2.71 × 10-2

115488938

Elongation factor Ts

25.6

45

6.7

1.06 × 10-9

115436768

Tyrosine--tRNA ligase

22.3

12

6.6

3.46 × 10-3

115472897

Ribosome-recycling factor, chloroplastic

31.2

17

6.6

1.13 × 10-3

115449027

Putative isoleucyl-tRNA synthetase

8.2

8

6.3

1.95 × 10-6

115470767

Probable polyribonucleotide nucleotidyltransferase 1, chloroplastic

9.6

10

6.1

4.16 × 10-4

115445399

Putative 50S ribosomal protein L21, chloroplast

25.7

7

5.8

1.12 × 10-2

115489150

60S ribosomal protein L2

30.6

11

4.8

6.71 × 10-3

115486501

Peptide chain release factor 1

17.8

7

4.7

4.88 × 10-4

50233964

30S ribosomal protein S2, chloroplastic

25.9

10

4.5

8.95 × 10-4

115438779

Peptide deformylase 1B, chloroplastic

16.4

5

4.1

4.50 × 10-2

115458788

OSJNBa0072F16.12 protein

21.3

5

3.9

4.05 × 10-2

115450427

50S ribosomal protein L5, chloroplastic

42.6

17

3.9

1.43 × 10-3

115448755

Putative histidine-tRNA ligase

6.6

4

3.8

4.60 × 10-2

115451609

50S ribosomal protein L15, chloroplast

29.7

11

3.5

3.81 × 10-2

115446545

Putative threonyl-tRNA synthetase

14.1

9

2.9

1.25 × 10-6

115439267

Met-tRNAi formyl transferase-like

20.7

6

2.8

2.49 × 10-3

115465593

Translation initiation factor IF-2

14.3

7

2.5

4.04 × 10-2

115463659

Putative chloroplast ribosomal protein L1

30.6

24

2.4

1.55 × 10-3

115487526

60S ribosomal protein L3

29.3

17

2.1

1.93 × 10-2

115447385

Lysine--tRNA ligase

14.5

9

1.5

4.36 × 10-2

115488928

Tryptophanyl-tRNA synthetase

21.8

7

0.5

4.03 × 10-2

115453877

40S ribosomal protein S3

44.7

14

0.5

2.32 × 10-3

115487104

40S ribosomal protein S16

27.5

6

0.4

1.16 × 10-2

115434960

Putative tRNA-glutamine synthetase

11.2

8

0.3

7.99 × 10-3

115473889

Elongation factor 1-beta

39.7

21

0.3

1.27 × 10-2

115486179

40S ribosomal protein S9

26.2

6

0.3

2.61 × 10-3

115475427

Putative 60S ribosomal protein L7

22.5

9

0.2

2.22 × 10-2

 

Protein folding

    

115444001

Putative uncharacterized protein P0576F08.31

16.7

6

22.9

1.64 × 10-4

115458444

GrpE protein homolog

26.6

9

18.2

1.12 × 10-2

115476198

Putative peptidyl-prolyl cis-trans isomerase, chloroplast

34.3

21

14.9

5.61 × 10-5

115449059

Putative 20 kDa chaperonin, chloroplast

46.3

9

8.2

1.49 × 10-2

115461585

Peptidyl-prolyl cis-trans isomerase

39.2

23

7.7

3.33 × 10-3

115460872

OSJNBb0079B02.1 protein

4.6

3

6.1

2.96 × 10-2

115467746

Trigger factor-like

39.5

27

4.8

1.77 × 10-4

115472829

Putative peptidyl-proly cis-trans isomerase protein

29.2

20

4.7

5.14 × 10-5

115448437

Putative protease IV

14.5

10

4.7

6.37 × 10-3

115472151

Peptidyl-prolyl cis-trans isomerase

23.3

5

4.6

4.48 × 10-2

115488160

60 kDa chaperonin alpha subunit

55.5

64

3.8

5.70 × 10-5

115473507

Receptor protein kinase

11.7

8

3.8

1.55 × 10-2

115466004

Putative chaperonin 60 beta

48.2

63

3.7

1.65 × 10-3

115475740

Putative uncharacterized protein OSJNBb0075O18.114

23.2

6

3.6

6.47 × 10-3

115465267

Serine/threonine-protein kinase SNT7

13.6

8

3.4

1.46 × 10-2

115448713

Peptidyl-prolyl cis-trans isomerase

34.3

11

3.1

8.95 × 10-4

115484731

ABC-1 domain containing protein

9.0

7

2.9

1.57 × 10-2

115441683

ABC1-like

5.3

3

2.8

4.11 × 10-2

115477014

Putative heat-shock protein

21.0

17

2.5

1.11 × 10-2

115463261

Putative DnaJ protein

25.3

14

2.5

4.82 × 10-3

115487998

70 kDa heat shock protein

45.4

60

2.3

1.13 × 10-2

115469982

Endoplasmin homolog precursor

26.7

28

0.5

1.62 × 10-2

115456045

T-complex protein 1, theta subunit

34.1

17

0.4

1.77 × 10-2

115462083

Chaperonin protein

19.4

11

0.3

3.37 × 10-2

115471369

Calreticulin

19.8

9

0.2

1.11 × 10-2

115477393

Putative 70 kDa peptidylprolyl isomerase

15.3

9

0.2

3.70 × 10-4

115468394

T-complex protein 1 subunit gamma

21.3

12

0.2

1.36 × 10-3

115458184

Calnexin

26.6

15

0.2

4.69 × 10-4

 

Monosaccharide metabolism

    

115458768

Glyceraldehyde-3-phosphate dehydrogenase

63.4

120

22.5

1.98 × 10-4

115484401

Fructose-bisphosphate aldolase, chloroplastic

74.0

126

22.1

4.10 × 10-7

115468886

Fructose-bisphosphate aldolase

57.3

49

20.5

8.04 × 10-7

115455637

Malate dehydrogenase

67.0

35

12.6

8.32 × 10-4

115450493

Glyceraldehyde-3-phosphate dehydrogenase

57.2

91

7.7

2.32 × 10-5

115466256

Putative enolase

46.0

32

7.3

1.48 × 10-2

115470849

Putative ribose-5-phosphate isomerase

52.5

32

5.6

1.19 × 10-2

115477891

PfkB type carbohydrate kinase protein family-like

12.1

4

5.3

1.34 × 10-2

115434516

Triosephosphate isomerase, cytosolic

69.2

29

5.0

4.75 × 10-2

115462281

Fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase

22.9

20

5.0

8.62 × 10-8

115479643

Glucose-6-phosphate isomerase

29.9

19

4.8

3.56 × 10-4

115457638

OSJNBa0023J03.8 protein

31.3

8

4.5

2.00 × 10-2

115455133

4-hydroxy-3-methylbut-2-enyl diphosphate reductase, chloroplastic

35.9

19

3.5

7.98 × 10-4

115437808

Oxidoreductase-like

24.3

8

2.3

3.35 × 10-2

115464965

Hexokinase-5

24.1

10

2.2

2.59 × 10-2

115439869

Hexokinase-6

27.1

14

2.1

1.22 × 10-2

115452337

L-ascorbate peroxidase 1, cytosolic

49.2

29

0.4

9.02 × 10-3

115467370

Putative pyrophosphate-dependent phosphofructokinase beta subunit

31.2

17

0.4

9.90 × 10-3

115484175

Pyruvate kinase

31.3

23

0.3

2.35 × 10-2

115465974

6-phosphogluconate dehydrogenase, decarboxylating 1

44.8

29

0.3

6.70 × 10-7

115434198

Fructose-bisphosphate aldolase

24.5

10

0.3

1.53 × 10-2

115441963

Putative transaldolase

42.6

22

0.2

2.29 × 10-3

115473973

Xylose isomerase

34.5

17

0.2

1.06 × 10-5

 

Disaccharide metabolism

    

115439937

Putative trehalose-6-phosphate synthase/phosphatase

5.0

5

4.1

2.00 × 10-2

115452927

Sucrose synthase 4

9.4

9

0.5

1.24 × 10-2

115466896

Sucrose synthase 2

36.1

32

0.4

1.10 × 10-3

115453437

Sucrose synthase 1

43.3

36

0.1

2.55 × 10-5

 

Polysaccharide metabolism

    

115471703

Granule binding starch synthase II

22.2

14

25.6

7.88 × 10-5

115474235

Putative uncharacterized protein P0034A04.101-1

26.4

30

17.4

5.07 × 10-5

115451283

Inositol-3-phosphate synthase

23.7

11

9.2

4.04 × 10-5

115476014

Glucose-1-phosphate adenylyltransferase small subunit, chloroplastic/amyloplastic

36.1

23

6.4

7.94 × 10-3

115455167

Glucose-1-phosphate adenylyltransferase

42.7

30

4.4

4.71 × 10-8

115460666

Soluble starch synthase III-1

11.5

16

3.8

3.09 × 10-2

115461086

Probable UDP-arabinopyranose mutase 2

10.7

4

0.3

1.00 × 10-2

115470060

1,4-alpha-glucan-branching enzyme, chloroplastic/amyloplastic

7.3

6

0.1

1.93 × 10-3

115454033

UDP-arabinopyranose mutase 1

52.2

24

0.1

1.53 × 10-6

115459168

Chitinase 4

11.8

3

0.1

1.53 × 10-2

 

Fatty acid metabolism

    

115444801

Lipoxygenase

16.3

12

17.1

4.99 × 10-6

115489048

Lipoxygenase

17.6

15

7.0

7.04 × 10-3

115441871

Acyl-[acyl-carrier-protein] desaturase 2, chloroplastic

11.5

4

4.6

1.66 × 10-2

115436430

Putative tetrafunctional protein of glyoxysomal fatty acid beta-oxidation

17.3

13

0.3

2.76 × 10-4

115445513

Peroxisomal fatty acid beta-oxidation multifunctional protein

21.9

18

0.1

7.07 × 10-8

 

Amino acid metabolism

    

115455221

Serine hydroxymethyltransferase

57.1

73

22.1

4.47 × 10-12

115461066

Glutamine synthetase, chloroplastic

61.0

69

20.1

5.47 × 10-4

115460656

Aminomethyltransferase

57.1

51

19.8

4.34 × 10-5

115442595

Cysteine synthase

51.3

60

14.6

1.19 × 10-4

115439533

Glycine dehydrogenase P protein

60.8

157

12.8

1.08 × 10-4

115457070

Cysteine synthase

43.0

18

9.7

3.31 × 10-5

115478398

Aspartate kinase-homoserine dehydrogenase

10.9

11

5.8

2.85 × 10-3

115476972

Putative 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

23.6

12

5.2

2.76 × 10-4

115433966

Os01g0101200 protein

19.0

10

3.1

2.48 × 10-2

115480417

Putative dehydroquinate synthase

37.9

20

2.8

7.39 × 10-3

115450561

ATP phosphoribosyltransferase, chloroplastic

22.8

10

2.7

1.57 × 10-2

115448201

Carbamoyl-phosphate synthase small chain, chloroplastic

20.7

9

2.7

4.08 × 10-2

115445929

Probable diaminopimelate decarboxylase, chloroplastic

30.4

14

2.5

2.14 × 10-3

115486343

Phosphoserine phosphatase

17.6

4

2.5

4.98 × 10-2

115468570

Cysteine synthase

11.2

5

2.3

4.85 × 10-2

115482324

Glutamine synthetase family

4.9

4

0.6

3.26 × 10-2

115461214

Methylthioribose kinase 1

14.2

6

0.4

4.30 × 10-2

115449517

Glutathione reductase, cytosolic

20.8

9

0.4

2.44 × 10-2

115456165

Probable methylenetetrahydrofolate reductase

36.4

24

0.4

9.95 × 10-6

115466226

3-phosphoshikimate 1-carboxyvinyltransferase

22.7

12

0.4

3.98 × 10-2

115434790

Phospholipase D alpha 1

28.5

23

0.3

6.75 × 10-4

115454997

Glutamate decarboxylase

22.4

10

0.3

7.16 × 10-3

115447403

Phenylalanine ammonia-lyase

45.6

36

0.1

2.39 × 10-2

 

Generation of precursor metabolites and energy

    

115472339

Putative ATP synthase gamma chain 1, chloroplast

44.4

70

24.9

1.72 × 10-9

115472727

Cytochrome b6-f complex iron-sulfur subunit, chloroplastic

56.0

37

23.1

1.34 × 10-4

115457390

ATP synthase B chain

50.3

23

11.7

2.97 × 10-3

115435200

Putative phosphoenolpyruvate carboxylase 1

29.0

34

7.4

1.51 × 10-4

115452259

ATP synthase B chain, chloroplast

34.6

30

5.8

8.67 × 10-4

115448701

Putative H(+)-transporting ATP synthase

26.3

25

5.1

8.73 × 10-4

115469362

Putative vacuolar proton-ATPase

43.4

36

0.6

1.69 × 10-2

115435934

NAD-dependent isocitrate dehydrogenase a

29.3

11

0.6

3.96 × 10-2

115474559

Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial

24.9

8

0.5

1.16 × 10-2

115438975

Putative H + -exporting ATPase

40.0

11

0.5

6.50 × 10-3

115444791

Citrate synthase

26.9

13

0.4

2.19 × 10-3

115447367

Succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial

31.0

14

0.3

1.77 × 10-2

115470583

Ferredoxin--NADP reductase, embryo isozyme, chloroplastic

16.4

6

0.3

5.45 × 10-3

115470493

Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial

13.2

9

0.2

2.91 × 10-3

115469332

Glutaredoxin-C8

36.4

3

0.1

4.59 × 10-2

115459340

Glutaredoxin-C6

43.8

7

0.1

9.69 × 10-3

115470941

Thioredoxin H1

40.2

11

0.1

7.29 × 10-3

 

Vitamin metabolism

    

115472485

Thiamine thiazole synthase, chloroplastic

49.8

29

6.7

5.04 × 10-3

115454593

Thiamine biosynthesis protein thiC

25.7

14

5.4

3.81 × 10-7

115446113

Riboflavin biosynthesis protein RibD family protein

9.2

4

3.9

1.81 × 10-2

115482032

GDP-mannose 3,5-epimerase 1

42.6

26

2.7

4.02 × 10-2

 

Nucleotide metabolism

    

115475007

Putative uncharacterized protein OJ1590_E05.35-1

10.5

4

9.5

7.33 × 10-3

115455473

WRKY DNA binding domain containing protein

4.9

5

5.1

1.59 × 10-2

115450117

(RAP Annotation release2) Formyltetrahydrofolate deformylase family protein

13.2

4

4.2

1.28 × 10-2

115462253

Probable GTP diphosphokinase CRSH2, chloroplastic

15.7

9

3.8

4.78 × 10-2

115480339

Deoxyribodipyrimidine photolyase family protein-like

8.5

6

3.5

1.70 × 10-2

115488968

Nucleoside diphosphate kinase

31.8

11

3.3

9.20 × 10-3

115454773

Adenylosuccinate synthetase 2, chloroplastic

34.0

21

3.1

8.43 × 10-4

115464251

Putative uracil phosphoribosyltransferase

28.9

9

3.0

7.42 × 10-4

115451155

SAP-like protein

13.1

4

2.9

4.14 × 10-2

 

Isoprenoid metabolism

    

115472641

Putative isopentenyl pyrophosphate:dimethyllallyl pyrophosphate isomerase

12.6

3

15.6

7.33 × 10-3

115447171

4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, chloroplastic

28.2

21

9.2

1.37 × 10-8

115471093

Zeta-carotene desaturase

26.8

18

7.9

4.21 × 10-6

115458652

Zeaxanthin epoxidase, chloroplastic

16.2

10

5.9

3.86 × 10-5

115434044

1-Deoxy-D-xylulose 5-phosphate reductoisomerase, chloroplastic

24.7

15

4.5

3.59 × 10-2

115451171

Phytoene dehydrogenase, chloroplastic/chromoplastic

15.4

9

2.8

2.84 × 10-2

 

Phosphorus metabolism

    

115463815

Pyruvate, phosphate dikinase 1, chloroplastic

40.4

51

7.4

1.85 × 10-11

115448919

Chloroplast inorganic pyrophosphatase

42.2

19

6.3

2.57 × 10-2

115488252

Phosphoglucan, water dikinase, chloroplastic

12.9

15

3.8

2.37 × 10-6

115468200

Alpha-glucan water dikinase

13.0

18

3.0

8.67 × 10-5

 

Sulfur metabolism

    

115456862

ATP sulfurylase

55.6

17

7.0

2.17 × 10-3

115472303

Probable 5′-adenylylsulfate reductase 1, chloroplastic

20.6

11

3.9

5.43 × 10-4

115450913

Glutathione reductase, chloroplast

31.0

20

3.3

1.25 × 10-3

 

Macromolecule catabolic process

    

115444937

26S proteasome regulatory particle triple-A ATPase subunit 6

30.9

16

0.4

3.81 × 10-3

115466690

Putative 26S proteasome regulatory particle triple-A ATPase subunit 5a

20.3

12

0.2

2.09 × 10-3

 

Response to reactive oxygen species

    

115446663

Probable L-ascorbate peroxidase 8, chloroplastic

27.2

31

6.7

2.50 × 10-2

115450521

Catalase

47.2

38

6.2

1.09 × 10-2

115477837

Superoxide dismutase [Cu-Zn], chloroplastic

54.0

28

5.4

1.30 × 10-2

115473833

Thioredoxin reductase NTRC

33.0

12

4.4

1.38 × 10-5

115477687

L-Ascorbate peroxidase

34.4

24

3.2

1.48 × 10-2

 

Cofactor metabolism

    

115479433

Formate-tetrahydrofolate ligase

29.4

25

3.0

4.12 × 10-3

115440827

ABC transporter subunit-like

13.2

8

2.7

1.62 × 10-2

115434288

Putative SufD

18.1

9

2.6

4.64 × 10-2

 

Regulation of nitrogen utilization

    

115477733

Putative NADPH-dependent reductase

41.2

18

7.4

2.14 × 10-7

115445203

Putative UOS1

30.3

19

6.9

1.72 × 10-6

115469824

Putative UOS1

23.3

13

5.5

6.31 × 10-4

115453029

Divinyl chlorophyllide a 8-vinyl-reductase, chloroplastic

24.2

11

4.6

6.54 × 10-3

 

Cellular homeostasis

    

115472057

Thioredoxin-like protein CDSP32, chloroplastic

29.9

13

10.1

2.89 × 10-5

115444771

Peroxiredoxin-2E-2, chloroplastic

63.1

34

7.7

6.91 × 10-5

115466906

Peroxiredoxin Q, chloroplastic

45.2

22

7.6

5.32 × 10-4

115446541

2-Cys peroxiredoxin BAS1, chloroplastic

56.3

36

5.2

3.44 × 10-3

115477793

Putative auxin-regulated protein

32.8

13

4.5

3.11 × 10-2

115436320

Dihydrolipoyl dehydrogenase

56.3

47

3.9

4.04 × 10-5

115435536

Peptide transporter protein-like

10.7

3

2.8

1.39 × 10-2

115471449

Putative uncharacterized protein OJ1370_E02.126

39.3

10

1.8

2.24 × 10-2

115464793

Thioredoxin

14.9

3

0.5

3.06 × 10-2

115479475

Protein disulfide isomerase-like 2-3

15.7

5

0.3

2.01 × 10-2

115462193

Protein disulfide isomerase-like 2-1

17.2

6

0.3

1.99 × 10-3

115455973

Thioredoxin H2-2

14.2

2

0.2

3.38 × 10-2

115484585

Protein disulfide isomerase-like 1-1

28.1

20

0.1

9.78 × 10-8

 

Oxidation reduction

    

115484891

Rieske [2Fe-2S] domain

35.0

18

13.7

3.63 × 10-5

115459670

NAD(P)H-quinone oxidoreductase subunit M, chloroplastic

39.1

14

11.5

6.11 × 10-3

115481490

Flavonoid 3′-hydroxylase

6.1

3

7.8

3.64 × 10-2

115476190

Putative oxidoreductase, zinc-binding

51.0

34

6.6

3.97 × 10-6

115476820

Nitrate reductase [NADH] 1

6.3

5

6.0

1.29 × 10-2

115477461

Moco containing protein

34.5

13

5.1

1.02 × 10-3

115482950

Aldo/keto reductase family protein

9.3

3

5.1

2.94 × 10-3

115454109

Oxidoreductase, aldo/keto reductase family protein

38.5

16

4.9

2.69 × 10-4

115476618

Glyceraldehyde-3-phosphate dehydrogenase

36.5

29

4.7

8.09 × 10-3

115443657

Putative ferredoxin-NADP(H) oxidoreductase

55.1

51

4.3

2.52 × 10-3

115484125

L-galactono-1,4-lactone dehydrogenase 1, mitochondrial

6.7

3

3.9

1.56 × 10-3

115446723

Glucose/ribitol dehydrogenase family protein

19.1

4

2.6

1.50 × 10-2

115477843

Putative malate dehydrogenase [NADP], chloroplast

21.5

13

2.5

1.35 × 10-2

115438082

Cytosolic aldehyde dehydrogenase

21.5

11

2.1

4.10 × 10-2

115487892

NADP-dependent oxidoreductase P2

17.9

6

1.8

2.31 × 10-2

115456131

Putative alcohol dehydrogenase

26.7

6

0.6

4.09 × 10-2

115443911

NADPH-dependent mannose 6-phosphate reductase

26.9

12

0.6

1.66 × 10-2

115482810

Malic enzyme

20.2

11

0.5

2.47 × 10-3

115460254

OSJNBa0009P12.34 protein

12.4

4

0.5

1.82 × 10-2

115478070

Putative NADPH-dependent retinol dehydrogenase/reductase

26.1

8

0.4

3.40 × 10-2

115484519

Aldehyde dehydrogenase

12.0

5

0.4

7.24 × 10-3

115479375

Aldehyde dehydrogenase

29.9

15

0.4

6.28 × 10-3

115463191

Superoxide dismutase [Mn], mitochondrial

32.9

13

0.3

3.01 × 10-2

115464645

Hypothetical protein

5.7

3

0.3

3.11 × 10-2

115434810

NADH-cytochrome b5 reductase

22.8

7

0.3

2.15 × 10-2

115451245

Oxidoreductase, zinc-binding dehydrogenase family protein

16.1

5

0.3

1.48 × 10-2

115478148

Isopenicillin N synthase family protein

5.2

2

0.2

7.89 × 10-3

115462115

Putative 1-aminocyclopropane-1-carboxylate oxidase

11.0

3

0.2

1.34 × 10-2

 

Response to oxidative stress

    

115445243

Class III peroxidase 29

38.9

20

39.8

3.19 × 10-3

115460338

Haem peroxidase family protein

32.9

20

4.5

4.36 × 10-5

115436084

Class III peroxidase 11

26.2

8

4.4

2.78 × 10-2

115474059

Peroxidase

47.0

19

0.3

1.30 × 10-2

115436300

Class III peroxidase 16

23.1

10

0.3

2.50 × 10-2

115456523

Salt tolerance protein

27.5

7

0.2

5.25 × 10-4

115459848

Glutathione peroxidase

33.9

10

0.2

4.64 × 10-2

115442403

Putative peroxidase

37.9

19

0.1

3.85 × 10-4

 

Others

    

115450080

Cell division inhibitor-like

20.9

14

5.4

2.53 × 10-2

115450329

Peroxisomal membrane protein 11-1

21.9

5

4.8

2.94 × 10-2

115452321

Ribosomal protein L10 containing protein

50.9

15

4.1

3.30 × 10-4

115439157

Two pore calcium channel protein 1

2.0

1

3.8

3.56 × 10-2

115457630

Phototropin-2

17.0

12

2.9

1.21 × 10-4

115474273

Phosphoinositide phospholipase C

27.3

15

0.5

4.60 × 10-2

115446411

RNA binding protein Rp120

29.6

29

0.5

1.61 × 10-2

115448225

GTPase activating protein-like

5.2

4

0.3

5.92 × 10-3

115453079

Villin-3

20.7

17

0.3

5.15 × 10-3

115451401

Mitochondrial outer membrane protein porin 5

49.1

21

0.3

4.79 × 10-3

115441759

Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2

10.3

6

0.3

1.12 × 10-4

297601526

Probable linoleate 9S-lipoxygenase 4

22.4

15

0.2

2.78 × 10-3

115434036

Putative isoflavone reductase

19.8

5

0.2

1.38 × 10-2

115486998

Non-specific lipid-transfer protein 2B

57.3

17

0.1

4.26 × 10-3

115444635

Response regulator

2.1

3

0.02

2.08 × 10-2

Note: “Peptides (95 %)” indicates distinct peptides were identified with at least 95 % confidence (protein score cutoff > 1.5); “Cov (95)” means percentage of matching amino acids from identified peptides with confidence over 95 %; Ratio and P-value represents tag labeled for mock leaves: tag labeled for RSV-infected leaves. Ratio >1.5 is considered as downregulated and <0.67 is upregulated

Chloroplast group

The 30 annotated significantly downregulated proteins in the chloroplast group process were involved in chlorophyll biosynthesis and photosynthesis (Table 2). For chlorophyll biosynthesis, 10 proteins involved in the chlorophyll contents in RSV-infected leaves were more than 3 times lower than in the mock leaves: magnesium chelatase subunit I (CHLI) and subunit D (CHLD), magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase, uroporphyrinogen decarboxylase 1, uroporphyrinogen decarboxylase 2, protoporphyrinogen oxidase, porphobilinogen deaminase, delta-aminolevulinic acid dehydratase, glutamate-1-semialdehyde 2,1-aminomutase, glutamyl-tRNA reductase (Table 2; Fig. 5). Twenty photosynthesis proteins were also annotated as enriched, whereas four oxygen-evolving enhancer proteins and a type protein involved in the chloroplast biosynthesis were over 10 times lower upon RSV infection than those in the mock control. Meanwhile, five chlorophyll a/b-binding proteins were downregulated in RSV-infected leaves compared with mock leaves (Table 2). Thus, the accumulation of 30 proteins in the chlorophyll metabolism was apparently reduced by RSV infection.
Fig. 5

a Enzymes of chlorophyll biosynthetic pathway that decreased in accumulation during RSV infection. Selected steps are from KEGG pathways map (map 00860) for metabolism and enzymes. Bold words represent enzymes: glutamyl-tRNA synthetase, uroporphyrinogen III synthase, Mg-protoporphyrin IX methyltransferase, coproporphyrinogen III oxidase; boxed words represent enzymes: glutamyl-tRNA reductase, glutamate-1-semialdehyde aminotransferase, delta-aminolevulinic acid dehydratase, porphobillinogen deaminase, Mg-protoporphyrin IX monomethyl ester oxidative cyclase, magnesium-chelatase, protoporphyrinogen IX oxidase, uroporphyrinogen III decarboxylase. Eight enzymes at first stage of chlorophyll biosynthetic process were found and comprised 10 differentially accumulated proteins that were identified in RSV-induced leaves compared with the mock control leaves. b Two pathways could lead to programmed cell death including normal and RSV-induced plant. OsAP25 (radc1, Os03g0186900), OsAP37, rap, and p0026h03.19 were aspartic proteases genes

Defense group

Leaves are the primary tissue for RSV infection and colonization, so not surprisingly, four defensive proteins in RSV-infected leaves were identified as being altered in accumulation. Three pathogenesis-related proteins and a Bet v 1 allergen family protein were significantly more abundant in RSV-infected leaves than those in mock leaves: pathogenesis-related protein 1, pathogenesis-related protein 10, pathogenesis-related protein and Bet v I allergen family protein (Table 2). The upregulation of those proteins indicated that defensive reactions were induced after inoculation with RSV. From the 70 kDa heat shock protein (HSP70) family, ubiquitous in plants in response to diverse DNA and RNA viruses [39, 40], HSP70 and HSP (putative heat shock protein) were expressed at high levels in RSV-infected leaves compared with mock leaves, indicating that RSV activates the expression of the genes encoding HSP. In addition, superoxide dismutase [Mn] and four peroxidases expressed were upregulated in response to RSV (Table 2).

Of 28 annotated proteins involved in proteolysis, 19 proteins increased in response to RSV infection: 7 proteasome subunits, 3 ubiquitin type proteins, 3 aspartic type proteins, 2 aminopeptidase M1 subunits, 1 DNA-binding protein, 1 leukotriene A-4 hydrolase,1 serine carboxypeptidase and 1 insulin degrading enzyme. Three aspartic type proteins (eukaryotic aspartyl protease family protein, aspartic proteinase and peptidase aspartic) were expressed at a high level in the RSV-infected leaves (Table 2).

Validation of changes in RNA level by RT-qPCR and Northern blotting

Based on a proteomics analysis, the proteins differentially accumulated during RSV infection, key proteins for chlorophyll biosynthesis and an aspartic-type endopeptidase were identified as involved in the formation of RSV induced symptoms, and their presence was quantitatively confirmed using RT-qPCR and Northern blot to evaluate the correlation between mRNA and protein levels. Total RNA extracted from RSV-infected and mock leaves was analyzed to measure mRNA transcription levels of putative target proteins. The RT-qPCR results demonstrated that expression of the genes for CHLI and CHLD (magnesium chelatase) in RSV-infected leaves was downregulated more than three times the level of the control (Fig. 6a), and transcription of genes encoding radc1, rap and p0026h03.19 in RSV-infected leaves were upregulated 14, 2, 3 times higher than the level of the control leaves, respectively (Fig. 6a), verifying the iTRAQ results. Similarly, this trend for mRNA levels of the genes for CHLI and p0026h03.19 by Northern blotting analyses also supported the transcription of genes encoding respective protein by RT-qPCR (Fig. 6b). Whereas, elevated levels of five genes were different between transcription and proteins levels that may be due to posttranscription and posttranslational regulatory processes.
Fig. 6

Validation of rice gene expression levels by real time RT-PCR and Northern blotting. a Comparison of protein and mRNA expression levels of mock leaves and RSV-infected leaves using RT-qPCR. Blue represents mock leaves; red represents RSV-infected leaves. The averaged readings from the three biological replicates normalized against endogenous gene OsEF1α; error bar denoted SD. Statistics were analyzed using the Student’s t-test. An asterisk indicated a significant difference from the corresponding control (P < 0.01). b Northern blot of two differentially expressed genes selected for verifying RT-qPCR results. Mock, mock-inoculated leaves; RSV-infected, RSV-infected leaves. Equal loading of total RNA was assessed by staining rRNA with ethidium bromide. Marker contained 2000 bp, 1500 bp, 1000 bp and 750 bp

Discussion

In the present study, iTRAQ-based experiments were implemented to identify proteins that were differentially accumulated between the RSV-infected and mock-inoculated leaves, then to determine which proteins may be involved in symptom formation. During RSV infection, 681 differentially accumulated proteins were found (Fig. 2; Table 1); 492 of these proteins were annotated by GO and located mostly in plastids, including the chloroplast, and participating in chlorophyll metabolism (Fig. 3, 4; Table 2). Chloroplast proteins was degraded by chloroplast vesiculation [41]. Upon RSV infection, the chloroplast vesiculation possibly targeted and destabilized the chloroplast for protein degradation, which resulted in cell death and induced the formation of vesicle containing many plastid proteins. According to the String database, protein-protein interaction networks were clustered in the chloroplast, defensive and metabolism groups (Additional file 2: Figure S1). Based on the functional analysis and RSV-induced disease symptoms, several proteins were associated with leaf chlorosis, cell death and plant defense during RSV invasion (Fig. 1, 3, 4). Additionally, the transcription of genes encoding selected proteins using RT-qPCR and Northern blot analyses matched with iTRAQ results (Fig. 6). We will discuss these various changes in proteins with regard to their significance to disease symptoms.

RSV induced a decrease in chlorophyll

At 21 dpi, chlorotic stripes on newly emerged leaves are typical on rice plants infected by RSV (Fig. 1). Chlorosis is correlated with a reduction in chlorophyll during infection with a virus [11]. Recently, chlorophyll structure was also confirmed to be altered by accumulation of RSV SP, and PsbP (oxygen-evolving complex protein) was shown to participate in the interaction between rice and RSV [16]. Similarly, we used iTRAQ to determine that the accumulation of four oxygen-evolving enhancer proteins in RSV-induced leaves was lower than in the control plants (Table 2); thus, reduced accumulation of oxygen-evolving enhancer protein is involved in interrupting chlorophyll production.

Chlorophyll production is also influenced independently by chlorophyll anabolic and catabolic reactions [42]. Here, eight enzymes involved in early steps of chlorophyll biosynthesis were identified as being lower in RSV-infected leaves than in the mock-inoculated leaves (Fig. 5a; Table 2), again implicating RSV infection in significantly inhibiting chlorophyll biosynthesis. One of these eight, magnesium chelatase, comprising three subunits (CHLI, CHLD, CHLH), is an important synthetic enzyme for chlorophyll a and chlorophyll b [43]. Specifically, subunits CHLI and CHLD were downregulated in RSV-infected leaves (Table 2, Fig. 3a) and had decreased mRNA levels (Fig. 6) compared with the control. These subunits are AAA+ proteins (ATPases associated with various cellular activities) and form a motor unit, which provides a structure for the functioning of magnesium chelatase [44, 45]. The reduced accumulation of CHLI and CHLD thus indicates that the function of magnesium chelatase in chlorophyll biosynthesis is also limited. These results suggest that the reduction of chlorophyll is associated with downregulation of magnesium chelatase during infection with RSV. Previous studies of CMV have shown that the yellow mosaic symptoms are induced by a domain of satellite RNA [46, 47]. Recently, small interfering RNA (siRNA) derived from this domain of satellite RNA was shown to mediate RNA silencing of the chlorophyll biosynthetic gene CHLI (magnesium protoporphyrin cheltase subunit I) and that CHLI mRNA is downregulated in the infected tobacco [12, 13]. The yellowing domain of CMV satellite RNA induces RNA silencing of chlorophyll biosynthetic gene by small interfering RNA [12, 13]. Unlike CMV, RSV does not have satellite RNA; so how does RSV regulate and alter the chlorophyll biosynthetic pathway and induce chlorosis? In addition, a reduction of chlorophyll a/b-binding protein was shown to cause a downregulation of chlorophyll accumulation [14]. Here, the level of five chlorophyll a/b-binding proteins was reduced during RSV infection (Table 2). Therefore, RSV infection disrupts chlorophyll biosynthesis.

Proteases coincided with cell death

The ubiquitin-26S proteasome system targets intercellular regulators that have a central role in battling pathogens [4851] and in leaf senescence [52]. Several of the 26S proteasome units rose in accumulation in RSV-infected leaves compared with mock leaves (Table 2), suggesting it might promote host defense, then induce cell death in rice to restrict pathogen spread.

At the end stage of RSV infection, rice leaves developed chlorotic stripes, then the whole leaf died (Fig. 1b). Cell death requires a series of appropriate proteases. For example, over-expression of OsAP25 (Os03g0186900) and OsAP37 encoding aspartic proteases induces programmed cell death [18]. Similarly, in this study aspartic proteases encoded by radc1 (Os03g0186900), rap, and p0026h03.19 in RSV-infected leaves were sharply upregulated compared with the control leaves (Figs. 4 and 5b), indicating that the expression of the genes encoding aspartic protease was induced by RSV infection and participated in programmed cell death. However, we found that the aspartic protease pathway in RSV-infected leaves contained three proteins (radc1, rap, and p0026h03.19) that differed from the aspartic proteases (OsAP25 and OsAP37) in the normal plant. The aspartic protease pathway induced by a pathogen might thus be a new biological process.

Defense reaction during RSV infection

Pathogenesis-related protein is associated with systemic acquired resistance of plant against diverse pathogens [53]. RSV infection induced a plant defense response, as noted by the upregulation of the expression of the genes encoding rice pathogenesis-related proteins. Bet v1 allergen, a member of the ubiquitous family of pathogenesis-related plant proteins, acts as a plant steroid carrier and has ribonuclease activity, suggesting it might play a key role in the plant defense response against pathogens [5456]. In RSV-infected leaves, three pathogenesis-related proteins belonging to the Bet v1 allergen family of proteins (OSJNBb0048E02.12) accumulated at a higher level than in mock leaves (Table 2). So the upregulation of Bet v1 allergen family proteins might improve the transport of a steroid such as a brassinosteroid and enhance ribonuclease activities against virus infection. In addition, the heat-shock protein HSP70 was more abundant in the RSV-infected leaves than in mock leaves (Table 2); thus RSV can induce HSP70 accumulation, as can various other RNA and DNA viruses [39, 40]. The expression of the genes encoding superoxide dismutase [Mn], superoxide dismutase [Cu-Zn] and peroxidase was also altered in response to RSV invasion (Table 2). Superoxide dismutase and peroxidase in plant were also identified as upregulated in response to TMV infection [57]. However, superoxide dismutase [Cu-Zn] was identified as downregulated during Sugarcane mosaic virus infection, showing that the regulation of superoxide dismutase can differ depending on the virus [58]. RSV infection thus clearly activated the accumulation of rice defense-related proteins, similar to the defense-related proteins such as PR10, HSP70 and peroxidase induced in rice infected by Rice yellow mottle virus (RYMV) that were identified using the 2-D method [59].

Conclusions

In summary, comparative proteomics analysis using iTRAQ LC-MS/MS technology identified 448 downregulated proteins and 233 upregulated proteins in many metabolic pathways during RSV infection. Several pathways potentially involved in RSV-induced symptom were found, including chlorophyll biosynthesis, proteolysis and defense response. Although our investigation provides knowledge of key proteins associated with the RSV-induced symptom, gene function analysis is needed to further understand the roles of these proteins in symptom formation. Therefore, our findings may provide new clues for elucidating the molecular mechanisms underlying RSV-induced symptom formation.

Methods

Insect population, plant materials and inoculation

A SBPH (small brown planthopper) population was maintained on susceptible rice (Oryza sativa var. japonica) cultivar (cv.) Wuyujing 3 in a climate chamber at 26 °C and a photoperiod of 14 h light and 10 h dark [60]. Third instar SBPH nymphs were allowed to feed on RSV-infected rice plants for a 3-day acquisition access period (AAP), then maintained in the climate chamber through the 10-day latent period. Ten viruliferous SBPH were then allowed to feed for a 2-day inoculation access period on three-leaved seedlings of Oryza sativa cv. Aichiasahi that had been grown in plastic pots containing a greenhouse soil mixture (40 % soil, 30 % vermiculite, 30 % straw powder). Subsequently, seedlings infested with non-viruliferous SBPH were used in the same way as a mock control. After the inoculation access period, seedlings were sprayed with insecticide and were transferred to insect-free greenhouse at 28 °C to observe symptom formation daily.

Sampling and RT-PCR (reverse transcription-polymerase chain reaction)

Samples were collected from both RSV-infected leaves and mock leaves at 21 dpi and immediately immersed in liquid nitrogen. Total RNA was extracted using Trizol reagent (Invitrogen Trading, Shanghai, China). M-MLV reverse transcriptase (Promega, Madison, USA) was used to reverse-transcribe 2 μg of the total RNA with gene-specific primers (Additional file 1: Table S1). PCR was performed in a final volume of 50 μL at 95 °C for 5 min, 32 cycles of 95 °C for 30 s, 57 °C for 45 s, 72 °C for 50 s. Amplified products were fractionated in a 1 % agarose gel.

Protein extraction, digestion and iTRAQ labeling

To extract total proteins from the RSV-infected leaves and control leaves, the samples were homogenized in lysis buffer (7 M urea, 2 M thiourea, 0.1 % CHAPS), and the mixture was then incubated at 30 °C for 30 min, and centrifuged at 15,000 × g for 20 min at 4 °C. The supernatant was collected and the proteins concentration was determined by the Bradford protein assay (Bio-Rad Laboratory, Hercules, CA, USA). Bovine serum albumin (BSA) was performed as the standard for the calibration curve. Approximately 200 μg proteins were reduced with 1 M dithiothreitol, alkylated with 1 M iodoacetamide, dissolved in the dissolution buffer, and digested with trypsin (AB Sciex, Foster City, USA) at 1:50 (w/w) for 37 °C overnight, which were then labeled using the iTRAQ Reagents 4-plex kit (AB Sciex) according to the manufacturer’s instructions. The peptides from RSV-infected leaves and mock leaves were labeled with 117 and 116 tags, respectively (Fig. 7).
Fig. 7

Strategy for iTRAQ LC-MS/MS analysis of comparative proteomics in rice infected with Rice stripe virus (RSV). At 21 days after inoculation, mock leaves (inoculated with healthy small brown planthoppers [SBPH]) and RSV-inoculated leaves (inoculated via RSV-viruliferous SBPH) were collected to extract total proteins. After prepared proteins were digested with trypsin, the peptides were labeled with the iTRAQ reagent and pooled. Pooled peptides were fractioned using the reversed-phase HPLC system, then individual fractions were analyzed using LC-MS/MS. MS raw data were processed using the NCBI protein database. Identified proteins were then analyzed using the DAVID platform and STRING software. Finally, key proteins were selected to validate their expression

Fractionation by reversed-phase high-performance liquid chromatography (HPLC)

Using the RIGOL L-3000 HPLC Pump system, the iTRAQ-labeled samples were reconstituted with mobile phase A (98 % H2O, 2 % acetonitrile, pH 10 adjusted by ammonia water) and mobile phase B (98 % acetonitrile, 2 % H2O adjusted by ammonia water), then fractionated on a Durashell-C18 column (4.6 mm × 250 mm, 5 μm, 100 Å; Agela, USA) at a speed of 0.7 mL min-1 using the gradient 0-5 min, 5-8 % buffer B; 5-35 min, 8-18 % buffer B; 35-62 min, 18-32 % buffer B; 62-64 min, 32-95 % buffer B; 64-68 min, 95 % buffer B; 68-72 min, 95-5 % buffer B. The chromatograms were recorded at 214 nm.

Mass spectrometric (MS) analysis

The fractionated peptides, dissolved in 2 % methyl alcohol and 0.1 % formic acid were analyzed using an ABI-5600 system (Applied Biosystems). After equilibration of the column with solvent A (100 % H2O, 0.1 % formic acid), the peptides eluted from the column (EASY-Spray column, 12 cm × 75 μm, C18, 3 μm) with a 90-min mobile phase gradient using solvent B (100 % acetonitrile, 0.1 % formic acid) with a flow rate of 350 nL min-1, a spray voltage of 2.1 kV and ionization interface temperature of 250 °C. Scan range was from 350 to 1800 m/z. The charge states of peptides were set to +2 to +4.

Protein identification and quantification

Proteins were identified and quantified by Protein Pilot Software 4.0 using the NCBI rice protein database (http://www.ncbi.nlm.nih.gov/protein/?term=oryza+sativa) search algorithm (Applied Biosystems). Proteins were identified based on various parameters such as trypsin digestion; dynamic modification; false discovery rate (FDR) determination for all peptide and protein identifications < 1 %; precursor ion mass tolerance, ±15 ppm; fragment ion mass tolerance, ±20 mmu; max missed cleavages, 2. Proteins were quantified as a change in relative expression; proteins with a fold-change >1.5 (P < 0.05) were considered to have decreased in level and those with fold-change <0.67 (P < 0.05) as increased.

Bioinformatics analysis

The Gene Ontology (GO) annotation for functional analysis was done using the DAVID resources 6.7 (http://david.abcc.ncifcrf.gov/) [61], and proteins were classified based on the molecular function, biological process, and cellular components. The Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.kegg.jp/) annotation was also done for a pathway analysis [62], and we assessed the interaction network for differentially accumulated proteins using STRING software (http://string-db.org/) [63].

Analysis of gene expression by RT-qPCR (reverse transcription quantitative polymerase chain reaction)

RT-qPCR primers were designed by Primer Premier Version 5.0 based on the ORF (open reading frame) sequence of candidate genes cloned from rice (Additional file 1: Table S1), and a primer set for endogenous gene OsEF1α designed for another study [64] was also used. About 2 μg total RNA was reverse-transcribed using the FastQuant RT kit (Tiangen Biotech-Beijing Co.) according to the manufacturer’s instructions and then its concentration was measured by NanaDrop-1000 [65]. The RT-qPCR was done in final volume of 20 μL using the SupperReal PreMix Plus (SYBR Green) kit and the manufacturer’s instructions (Tiangen Biotech-Beijing Co.) in a ABI 7500 Real Time PCR thermal cycler and the following conditions: 95 °C for 15 min; 40 cycles of 95 °C for 10 s, 55 °C for 32 s, and 72 °C for 32 s. The experiment was repeated three times. Data for the melt curve were collected at 95 °C for 15 s, 60 °C for 1 min, 95 °C for 30 s, and 60 °C for 15 s. Relative gene expression was calculated by the 2-ΔΔCT method [66].

Northern blot analysis

Fifteen micrograms of the total RNA extracted was electrophoresed in a 1.5 % formaldehyde agarose gel and transferred to a Hybond-N+ membrane (GE Healthcare Bio-Scienes Corp., USA) [67]. The membrane was then baked at 80 °C for 2 h, then probed with α-32P-dCTP- randomly primer labeled probe at 65 °C overnight in a perfect hyb™ plus hybridization buffer (Sigma-Aldrich, St. Louis, USA). After the hybridization, the membrane was washed twice with 2× SSC (sodium chloride-sodium citrate), 1× SDS (sodium dodecyl sulfate); 1× SSC, 1× SDS and 0.5× SSC, 0.5× SDS at 65 °C, and the radioactive signals were detected using phosphor imaging.

Declarations

Acknowledgments

Financial support was provided by the National Key Basic Research of China (2010CB126203), the Special Fund for Agro-scientific Research in the Public Interest (201303021), and the Plan for Scientific Innovation Talent of Henan Province (144100510018).

Authors’ Affiliations

(1)
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences
(2)
Institute of Plant Protection, Henan Academy of Agricultural Sciences

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© Wang et al. 2015

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