Eur J Plant Pathol DOI 10.1007/s10658-014-0414-9

Proteomic analysis of silicon-mediated resistance to Magnaporthe oryzae in rice (Oryza sativa L.) Min Liu & Kunzheng Cai & Yuting Chen & Shiming Luo & Zhixing Zhang & Wenxiong Lin

Accepted: 3 March 2014 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2014

Abstract Silicon can significantly enhance plant resistance against various pathogens. However, the mechanism of this resistance has not been fully revealed. In the present study, the role of Si in inducing resistance to Magnaporthe oryzae in rice (Oryza sativa L.) was elucidated by a proteomics approach. Results showed that Si supply significantly inhibited blast incidence. The contents of soluble and total protein in rice leaves were significantly increased. Through proteomic analysis by two-dimensional gel electrophoresis (2-DE) and liquid chromatography–mass spectrometry (LC-MS/MS), 61 protein spots were found significantly influenced by M. oryzae inoculation and/or Si application. Among these proteins, 43 were altered (30 increased and 13 decreased) when Si was added to M. oryzae-inoculated rice plants. These altered proteins were involved in the functional groups active in energy/metabolism, photosynthesis, redox homeostasis, protein synthesis, transcription and pathogen response. The present study provided novel insights into Si-mediated resistance of rice against M. oryzae at the proteome level. Keywords 2-DE . Magnaporthe oryzae . Plant resistance . Proteomics . Rice . Silicon M. Liu : K. Cai (*) : Y. Chen : S. Luo Key Laboratory of Tropical Agro-Environment, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642, China e-mail: [email protected] Z. Zhang : W. Lin Agroecological Institute, Fujian Agriculture and Forestry University, Fuzhou 35002, China

Abbreviations 2-DE Two-dimensional electrophoresis ABA Abscisic acid ACN Acetonitrile DMRT Duncan multiple range test GA Gibberellin IEF Isoelectric focusing M. oryzae Magnaporthe oryzae MS Mass spectrometry ROS Reactive oxygen species RuBisCO Ribulose bisphosphate carboxylase oxygenase SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis

Introduction Rice (Oryza sativa L.) blast caused by the fungus Magnaporthe oryzae is a serious problem in most ricegrowing regions of the world, being a plant pathogen that is extremely difficult to control (Valent and Chumley 1991). Silicon is the second most abundant element in the Earth’s crust. Although Si has not been listed as an essential element for higher plants, numerous studies have suggested that silicon has a positive role in promoting plant growth and in alleviating both biotic and abiotic stresses in plants (Epstein 1994; Liang et al. 2007). Studies have shown that the incidence of plant disease can be alleviated by Si supply (Datnoff et al.

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1997; Cai et al. 2008). Si-mediated resistance to a pathogen is associated with higher Si deposition in leaves. In effect, this resistance can strengthen the mechanical barrier and activate biochemical defence responses by increasing the activities of defencerelated enzymes and the accumulation of antifungal compounds (Rémus-Borel et al. 2005; Cai et al. 2008). However, most of these reports only elucidate the importance of Si in terms of specific physiological and biochemical prospects, whereas information on the overall molecular mechanisms involved in Simediated stress resistance in plants is not fully understood. Brunings et al. (2009) reported that Si treatment and M. oryzae infection resulted in the differential expression of 54 unique genes in rice, implying that Si affects rice response to blast infection at the transcriptional level. Recent studies shown that Sienhanced resistance to Ralstonia solanacearum in tomato is associated with the up-regulated expression of defence marker genes (Ghareeb et al. 2011). Proteomics is a powerful tool for elucidating the plant’s response mechanisms to biotic stress. Konishi et al. (2001) analyzed 63 proteins to understand M. oryzae infection of rice grown under different levels of nitrogen fertilizer. Kim et al. (2003) used proteomic approaches to identify proteins altered in suspensioncultured rice cells in response to M. oryzae infection and treatment with elicitor or signal molecules. Similar studies were conducted by Li et al. (2012) to identify differentially altered proteins involved in defence, redox homeostasis and signal transduction. So, it is necessary to investigate the biological function regulated by Si in plants by using proteomics. In the present study, we used 2-DE combined with mass spectrometry (MS) to identify the protein profile associated with Si-induced resistance to rice blast and to elucidate the possible molecular mechanisms involved. We hypothesized that Si increases blast resistance through altering metabolism-related proteins. The information will provide a novel insight into the role of Si in plant resistance.

sterilized with 10 % H2O2 for 10 min, rinsed thoroughly and soaked with distilled water, and germinated on moist filter paper for 48 h in Petri dishes. The germinated seeds were sown in sterilized quartz sand in a growing chamber with a 13/11 h day/night cycle, at 27 °C and 70 % humidity. At the two-leaf stage, 20 uniform seedlings were selected and transferred to a polyethylene plastic pot (9 cm in diameter and 7.5 cm in height) filled with sterilized quartz sand and 50 ml Hoagland’s nutrient solution. The final pH of solution for each treatment was adjusted to 5.6 by using 1 M KOH or HCl, and solutions were renewed every 3 days. Rice seedlings were grown in a growing chamber at 25/22 °C (day/ night) under a photoperiod of 13 h and a light intensity of 300 μmol m−2 s−1. Silicon treatments and M. oryzae inoculation

Materials and methods

Four treatments were applied in this study: (1) no Si added and no inoculation with M. oryzae (CK), (2) 2.0 mM Si added but no M. oryzae inoculation (Si), (3) no Si added but with M. oryzae inoculation (M. oryzae), and (4) 2.0 mM Si added with M. oryzae inoculation (Si+M. oryzae). Treatments were arranged in a randomized complete block design with three replications and 20 plants for each replicate. Si was added as potassium silicate (K2SiO3) to the Hoagland’s nutrient solution 10 days after transplanting. In the Sideficient treatment, potassium chloride (KCl) was used to replenish potassium in the solution. M. oryzae strain 98-288a was used to inoculate rice seedlings 18 days after transplanting (four-leaf stage). The whole plants were inoculated by spraying a suspension of conidia (20 ml suspension per 20 seedlings, containing 5×105 conidia ml−1). The non-inoculated plants were treated by spraying the same amount of distilled water. Inoculated and non-inoculated rice plants were kept in two separate chambers at 25 °C and 90 % relative humidity for 24 h to optimize pathogen growth. Leaves (the second and third fully expanded leaves) were sampled at 4 days after M. oryzae inoculation. Sampled materials were immediately frozen in liquid N2 and stored at −80 °C for protein content and protein expression analyses.

Plant materials and growth conditions

Disease assessment

Rice variety CO39 (susceptible to M. oryzae) was used throughout the experiment. Rice seeds were surface-

Disease development was evaluated 4 days after inoculation. Infected leaves were scored with a rating (r) of 0

Eur J Plant Pathol

to 9, denoting proportions of blast disease over the whole leaf area (IRRI 2002). The disease index was determined according to the following equation: hX i Disease indexð%Þ ¼ ðr  nr Þ=ð9  Nr Þ  100 where r = rating value, nr = number of infected leaves with a rating of r, and Nr = total number of leaves tested. Measurement of protein content in rice leaves The soluble, insoluble, and total protein contents in rice leaves were determined using a colorimetric assay (Zheng 2006). Leaf tissue (0.3 g) was ground and mixed in 1 M Tris–HCl buffer (pH 8.0). Subsequently, the sample was centrifuged at 10,000g for 20 min at 4 °C, and the supernatant was collected as soluble protein. The precipitates were mixed with 3 ml of 1 mol/l NaOH, incubated at 90 °C for 20 min, and centrifuged at 4,000 × g for 10 min at 4 °C. The supernatant was collected as insoluble protein. Protein content was measured at 595 nm following the method described by Bradford (1976). Total protein extraction Leaf samples (1.0 g) were ground to a fine powder in a mortar and immersed in liquid nitrogen continuously to maintain protein stability. The leaf proteins were suspended in pre-cooled 10 % tricarboxylic acid (TCA) in acetone containing 0.07 % ß-mercaptoethanol, incubated at −20 °C for 12 h, and centrifuged at 11,000 × g for 15 min at 4 °C. The top liquid was poured out, and the precipitates were washed with precooled 80 % acetone containing 0.07 % ß-mercaptoethanol. The precipitates were centrifuged and washed using the same method about every 2 h until the top liquid reached achromaticity. The protein pellets were dried by vacuum freeze-drying, and stored at −80 °C or solubilized in sample buffer for further use. 2-DE and image analysis The final protein pellet was re-suspended in Sample Buffer (7 M urea, 2 M thiourea, 5 % CHAPS, 2 % 2mercaptoethanol, and 5 % ampholines; pH 3.5 to 10). The protein concentration was quantified using the Bradford method (Bradford 1976). Bovine serum albumin was used as the standard.

The isolated leaf proteins were separated by 2-DE. Immobilized pH gradient (IPG) strips (24 cm pH 4 to 7, ReadyStrip™, Bio-Rad) were passively rehydrated overnight with 450 μl rehydration/sample buffer containing about 1,500 μg isolated protein. Isoelectric focusing (IEF) was carried out on an Ettan IPGphor 3 (GE Healthcare) at 20 °C using a maximal current of 50 μA/ strip. The settings described below. IEF was operated at 200 V for 30 min, 500 V for 30 min, 1,000 V for 6 h, 8,000 V for 3 h, 8,000 V for 4 h and 3,000 V for 30 h. For second-dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the focused IPG strips were equilibrated in two steps, first in equilibration buffer containing 30 % v/v glycerol, 50 mM Tris–HCl (pH 8.8), 6 M urea, 2 % (w/v) SDS, 1 % DTT followed by a second equilibration step in buffer containing 30 % (v/v) glycerol, 50 mM Tris–HCl (pH 8.8), 6 M urea, 2 % (w/v) SDS, and 4 % (w/v) iodoacetamide. The strips were then transferred to 12 % SDS-PAGE gels for second-dimension electrophoresis using the Ettan DALTsixLarge Vertical System (GE Healthcare). SDS-PAGE was run at 12 mA/gel until the bromphenol blue dye front reached the lower end of the gel. Thereafter, the gels were stained with 0.12 % (w/v) Coomassie brilliant blue G-250 (Sigma) overnight, destained and scanned under an Image Scanner III (GE Healthcare, Bio-Sciences, Uppsala, Sweden) and analyzed with ImageMaster 5.0 software (GE Healthcare, Bio-Sciences, Uppsala, Sweden). Protein spots showing significant changes (% vol varied more than 1.5-fold, p<0.05 by paired Student’s t-test) in each of the three replicate gels were selected for further identification. The gels were stored at 4 °C temperature and wrapped with clingfilm to prevent contamination after scanning.

In-gel protein digestion and mass spectrometry The differentially expressed CBB-stained protein spots were excised manually and digested from gels for mass spectrometric analysis (Rosenfeld et al. 1992). Gel pieces were first destained in 25 mM ammonium bicarbonate and 50 % methanol several times until the gel pieces turned colourless. Gel pieces were then rinsed and dehydrated with 100 % acetonitrile (ACN). Following vacuum-drying, gel pieces were digested with 20 μg ml−1 sequencing grade trypsin in 25 mM ammonium bicarbonate (pH 8.0), for 16 h at 37 °C. Tryptic

Eur J Plant Pathol

peptides were extracted with 2.5 % trifluoroacetic acid and 50 % ACN. Mass spectrometric analysis was carried out using LC-MS/MS. Tryptic digested spots from the gel-based protein samples were injected into a LC (Thermo Scientific Accera System) coupled to a spray LTQ-XL (Thermo Scientific). Peptides (10 μl) were loaded in 0.1 % formic acid onto a 100×0.18 mm C18 Column at a 2.5 μl/min flow rate. The peptides were eluted and separated from the trap column using 0.1 % formic acid in ACN with a spray voltage of 3.5 kV. Full scan mass spectra were acquired in Orbitrap over 400–2,000 m/z. The MS/MS data were searched with the software Proteome Discoverer 1.2 and the database of UNIPROT (http://www.uniprot.org/). The resulting peptide sequence data were used to search the database using the MASCOT search engine (ver.2.2.04, Matrix Science, London, UK). The minimal requirement for accepting a protein as identified was at least two peptide sequence matches above the identity threshold. Database searching and hierarchical cluster analysis The putative functional annotation for the identified proteins were assigned based on the National Center for Biotechnology Information (NCBI) (http://www. ncbi.nlm.nih.gov/) protein–protein BLAST results obtained with an embedded conserved domain search option. The assignment of the proteins into metabolic pathways was done by the Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/ kegg2.html) database in Blast2GO (v. 2.4.3) software. Hierarchical clustering of protein expression patterns was performed using Cluster software version 3.0 (Eisen et al. 1998). Input data was calculated by dividing vol % of each protein spot at Si and M. oryzae treatment by vol % of the same protein spot at control (CK). The clustering image was visualized by Treeview software version1.1.4 (Eisen et al. 1998). Statistical analysis A randomized block design was used to set up all the experiments. The data presented are means ± standard errors of three biological replicates examined by analysis of variance. Replications were used to determine the significance of differences in the volume of individual protein spots. Significant differences among treatments (p<0.05) were determined using the Duncan multiple

range test (DMRT) through the SPSS software (Statistical Analysis Systems Institute, version 13.0; SPSS Inc., Chicago, IL).

Results and discussion Pathogen incidence and protein content Si application significantly inhibited M. oryzae infection. Compared with the non-Si-treated plants, both the incidence of disease and disease index of rice blast were significantly decreased after 2.0 mM Si application to rice plants (Table 1). Si supply reduced blast incidence by 35 % and disease index by 55.3 %. Si alone decreased both soluble and total protein contents in rice leaves, whereas these contents evidently increased with Si treatment in M. oryzae-infected plants (Table 2). This finding indicates that Si might be involved in processes that control protein synthesis or turn-over and, consequently, enhance pathogen resistance of rice. Another study also showed that salicylic acid could increase leaf protein content of Vigna mungo upon Mungbean yellow mosaic India virus infection (Kundu et al. 2011). Protein identification and functional categorization The protein samples of rice leaves under different M. oryzae and/or Si treatments were isolated by 2-DE (Fig. 1). Fig. 2 shows the typical gel images of the treatments, the pI value and Mr of these protein spots ranged from 4 to 7 and from 10 to 250 kDa, respectively. A total of 1,439 protein spots were detected in the Coomassie-stained gels. Given the differences of relative change in abundance of 73 proteins were greater than 1.5 times (Fig. 2), they were considered Table 1 Effects of silicon application and Magnaporthe oryzae inoculation on incidence of disease and disease index (%) of blast in rice susceptible cultivar CO39. +Si and -Si indicate treatment with or without 2.0 mM silicon in culture solution, respectively. Values are means ± standard error from three replicates (n=3). Different letters denote statistical difference using a least significant difference test (P<0.05) Treatment

Incidence of disease (%)

Disease index (%)

−Si

99.2±4.8a

75.4±5.2a

+Si

65.0±3.9b

33.7±4.7b

Eur J Plant Pathol Table 2 Effects of silicon application and Magnaporthe oryzae inoculation on protein contents (μg.g−1) in rice leaves. CK indicates treatment with no Si added and no M. oryzae inoculation. Si indicates treatment with 2.0 mM silicon in culture solution but no M. oryzae inoculation. M. oryzae indicates treatment with M. oryzae inoculation but no Si added. Si+ M. oryzae indicates treatment with Si added and M. oryzae inoculation. Protein samples were treated with silicon and M. oryzae. Values are means ± standard error from three replicates (n=3). Different letters denote statistical difference using a least significant difference test (P<0.05) Treatment

Soluble proteins

Insoluble proteins

Total proteins

CK

7.4±0.2b

6.1±0.6b

13.5±0.4b

Si

5.5±0.3c

6.9±0.3b

12.4±0.3c

M. oryzae

6.6±0.1b

6.2±0.1b

12.8±0.2bc

Si+ M. oryzae

8.7±0.1a

8.1±0.1a

16.8±0.2a

pH 4 CK

M. oryzae

Fig. 1 2-DE maps of rice leaves protein spots revealed by CBB staining under Magnaporthe oryzae inoculation and silicon (Si) supply. CK indicates treatment with no Si added and no M. oryzae inoculation. Si indicates treatment with 2.0 mM silicon in culture solution but no M. oryzae inoculation. M. oryzae indicates

significantly altered by various treatments. In this study, 61 proteins were successfully identified by LC-MS/MS. The 61 identified proteins can be divided into seven functional categories (Fig. 3A): (1) Energy metabolism (23 %), (2) Photosynthesis (23 %), (3) Redox homeostasis (8 %), (4) Protein Synthesis (8 %), (5) Signal transduction/Transcription (15 %), (6) PathogenResponse (3 %), (7) Others (20 %). The abundances of 43 out of 61 proteins were significantly changed by Si (Table 3), while another 18 proteins were not affected by Si. Among the identified protein spots, only eight were significantly decreased by Si treatment alone, while 60 were altered by the inoculation of M. oryzae without Si. Forty-three protein spots were significantly altered by the addition of Si to the M. oryzae-inoculated plants, among which 30 were increased and 13 decreased (Fig. 3B). Microarray analysis of Si effects in tomato additionally indicated that Si had a limited effect on the 7 Si

Si+ M. oryzae

treatment with M. oryzae inoculation but no Si added. Si+ M. oryzae indicates treatment with Si added and M. oryzae inoculation. Protein samples were treated with silicon and M. oryzae. A protein sample of 1,500 μg was loaded on each IPG strip (pH 4–7)

Eur J Plant Pathol

pI 4.5

5.0

5.5

6.0

6.5

250 150 75

Mr (kDa)

50 37

25 20

proteins were all up-accumulated. Similarily, a large part of the proteins involved in signal transduction/ transcription (spot nos. 27, 28, 36 and 71), Redox homeostasis (spot nos. 20, 22 and 70), protein susynethese (spot nos. 21, 35 and 37) and pathogen-response(spot no. 42) were up-accumulated under M. oryzae inoculation, but when added Si, these proteins all downaccumulated. These results suggest that Si affects biochemical process and induces a resistance response. Proteins involved in energy metabolism

15

10

Fig. 2 Typical 2-DE gel image showing the matched protein spots from total leaf proteins of rice leaves grown under Magnaporthe oryzae inoculation and silicon (Si) supply. A sum of 1,500 μg protein was loaded on pH 4−7 IpG strip and protein spots were visualized by staining with Coomassie Brilliant Blue (CBB). The relative Mr (on the left) and pI (on the top) are given. A total of 61 spots were successfully identified

transcriptome in the absence of pathogen infection (Ghareeb et al. 2011). These findings imply that silicon treatment does not directly induce immunity but rather exerts a priming of defence pathways. Clustering analysis of protein expression patterns To acquire a comprehensive overview of the expression dynamics of proteins in rice leaves that were coexpressed under silicon and M. oryzae inoculation treatments, hierarchical clustering was performed, in which proteins with similar expression patterns were grouped together. As shown in Fig. 4, there were minor protein abundance changes with Si application alone (Si treatment) compare to CK, and majority of the identified proteins were down-accumulated under M. oryzae treatment, and most of these proteins were up-accumulated when Si was added to M. oryzae infected plants (Si+ M. oryzae). The results showed that many proteins related to photosynthesis were down-accumulated under M. oryzae treatment, such as Chlorophyll a/b-binding protein (spot no. 40), Chloroplast putative thylakoid lumenal 16.5 kDa protein (spot no. 49), Sedoheptulose-1,7-bisphosphatase (spot no.2), Ribulose bisphosphate carboxylase large chain (spot no.23) and so on. But when Si was added to M. oryzae–infected plants (Si+M. oryzae), these

The abundance of five protein spots related to energy metabolism were significantly altered (two increased and three decreased) due to the addition of Si to M. oryzae-inoculated rice plants (Table 3). Among the five proteins, only one protein (spot no. 51) was altered by Si application alone (Si). The two increased proteins were fructokinase-2 (spot no. 1) and glutamate dehydrogenase (spot no. 41), whereas the three decreased proteins were vacuolar ATPase (spot no. 54), chloroplastic fructose-bisphosphate aldolase (spot no. 7) and chloroplastic ATP synthase epsilon chain (spot no. 51). Glutamate dehydrogenase is an enzyme involved in N metabolism, which was found decreased in Sinorhizobium meliloti 1,021 infected seedlings (Chi et al. 2010). Chloroplastic fructose-bisphosphate aldolase as a protein involved in pentose-phosphate shunt (Pawlowski 2009) was increased in rice seedlings by cold and salt stress. Our results showed that chloroplastic fructose-bisphosphate aldolase was increased in M. oryzae infected rice leaves, which is similar to a previous study illustrating that M. oryzae stress resulted in an increase of this protein (Liao et al. 2009). The chloroplastic ATP synthase participates in producing ATP thylakoid membrane (Yuan et al. 2011), indicating the important function in energy generation and transmission. Our results indicated that the addition of Si to M. oryzae-infected rice plants significantly influenced the aforementioned proteins associated with energy and metabolism, with increasing pathogen resistance. Proteins involved in photosynthesis Our study revealed that the abundance of most proteins involved in photosynthesis was decreased under M. oryzae infection. Si treatment significantly induced higher amounts of ten proteins (Table 3), such as ribulose bisphosphate carboxylase (spot no. 23),

Eur J Plant Pathol Energy/Metabolism

A 20%

23%

Photosynthesis

B a

-M. oryzae/-Si

b

3%

22 38

Redox homeostasis

Regulation/Protein Synthesis

+M. oryzae /-Si

8%

8%

-M. oryzae / +Si 30 13

Signal transduction/Transcription

15%

23%

0 8

18 12

Pathogen-Response

Others

c

+ M. oryzae / +Si

d

Fig. 3 Differentially regulated rice leaf proteins due to Magnaporthe oryzae inoculation and/or silicon treatments. A Functional category distribution of the 61 identified rice leaf proteins that were differentially produced in response Si and/or M. oryzae treatments. B Venn diagram with intersections a, b, c and d, showing the number of identified protein spots that were

significantly up- (▲) or down- (▼) accumulated in + M. oryzae/Si plants compared to - M. oryzae/-Si plants (a), - M. oryzae/+Si plants compared to - M. oryzae/-Si plants (b), + M. oryzae/+Si plants compared to + M. oryzae/-Si plants (c), + M. oryzae/+Si plants compared to –M. oryzae/+Si plants (d)

chloroplastic ferredoxin-1 (spot no. 32), chlorophyll a/bbinding protein (spot no. 40) etc. Konishi et al. (2001) found that the protein abundance of ribulose-1,5bisphosphate carboxylase/oxygenase (RuBisCO LSU) was increased after nitrogen application in M. oryzaeinfected plants, which implied the role of nitrogen in photosynthetic carbon assimilation. Chlorophyll a/b-binding protein (spot no. 40) was significantly up-accumulated after Si added to M. oryzae-inoculated plants. Xu et al. (2012) found that chlorophyll a/b-binding protein was decreased in response to drought stress in Arabidopsis thaliana. Ribulose bisphosphate carboxylase is a key enzyme in the Calvin cycle, that was found to be decreased during senescence of rice flag leaves (Zhang et al. 2010). Similar results were observed in this study, this protein showed a decrease in relative abundance under M. oryzae inoculation, but increased after Si application. The abundance of thioredoxin was found to be decreased in response to selenium in rice (Wang et al. 2012). 50S ribosomal protein L21 presumably functions in chloroplast protein biosynthesis, which may affect photosynthesis in rice plants. The protein was also found to be decreased in the incompatible interaction between soybean and Pseudomonas syringae (Zou et al. 2005). In the present study, Sedoheptulose-1,7bisphosphatase (Spot no. 2) and Chloroplast putative thylakoid lumenal 16.5 kDa protein (spot no. 49) proteins were increased by Si application under M. oryzae inoculation. These results suggested that Si application induced the accumulation of photosynthesis-related proteins to increase resistance to M. oryzae, possibly

explaining our previous observation of Si-enhanced photochemical efficiency after M. oryza infection (Gao et al. 2011). Proteins involved in redox homeostasis Reactive oxygen species (ROS) have an important role in regulating leaf development (Gapper and Dolan 2006). ROS, such as hydrogen peroxide (H2O2), are toxic by-products of respiration and photosynthesis. Proteins associated with redox homeostasis are usually involved in the prevention of oxidative stress. In the present study, we identified a group of antioxidant enzymes including cytosolic L-ascorbate peroxidase 2 (spot no. 19), cytosolic L-ascorbate peroxidase 1 (spot no. 20), dehydroascorbate reductase (spot no. 22), 4hydroxyphenylpyruvate dioxygenase and superoxide dismutase [Cu-Zn] (spot no. 70). Three proteins (spot no. 20, 22 and 70) were significantly suppressed after M. oryzae infection, but increased by Si supply. These findings strongly suggest that Si may be actively involved in the inhibition of oxidative stress in plants, and possibly explain our previous results of Sienhanced defense related enzyme activity following M. oryza infection (Cai et al. 2008). Alvarez et al. (2011) found that dehydroascorbate reductase was decreased in Brassica juncea roots under oxidative stress. Therefore, the change may increase ascorbate regeneration. The decrease in the levels of superoxide dismutase (Cu/Zn), is a key enzyme involved in ROS scavenging, may indicate damage to cells under M. oryzae infection. Our results showed

Eur J Plant Pathol Table 3 Identified rice leaf protein spots by MS/MS that were significantly regulated by silicon. Proteins were isolated from rice leaves under Magnaporthe oryzae inoculation and silicon supply. Mass spectrometric analysis was carried out using LC-MS/MS Spot no.

a

ASVb A B C D

Protein function/namec Source is italicized

Accessiond

Coveragee

Peptidesf

no.

MWg

calch

[kDa]

pI

Scorei

Energy Metabolism 1

Fructokinase-2

A2YQL4

63.9

20

35.5

5.17

406.73

Q40677

50.52

11

42.0

6.80

238.48

Q852M0

10.46

2

44.3

6.60

28.08

P0C2Z1

29.93

2

15.2

5.10

67.68

Q93W07

27.05

10

54.0

5.19

88.71

Q84JG8

48.98

14

42.2

6.09

372.67

P0C510

30.19

14

52.8

6.68

245.50

Q7M1Y7

72.97

2

4.0

9.52

260.08

A2YQD9

57.55

3

14.9

4.56

58.25

Q6K241

12.62

6

23.3

6.60

65.47

Q5ZA98

13.69

5

26.2

6.10

70.30

Q8LNF2

25

4

24.5

7.55

56.18

Q9ZP20

15.7

2

18.5

7.93

190.99

Q5Z8V3

10.04

2

24.8

8.56

17.96

Q8S6F2

12.16

6

52.9

6.92

60.62

A2Y886

40.91

2

15.6

5.87

109.85

Oryza sativa subsp. japonica

7

Chloroplastic fructose-bisphosphate aldolase Oryza sativa subsp. japonica

41

Glutamate dehydrogenase Oryza sativa subsp. japonica

51

Chloroplastic ATP synthase epsilon chain Oryza sativa subsp. japonica

54

Vacuolar ATPase B subunit Oryza sativa subsp. japonica

Photosynthesis 2

Sedoheptulose-1,7-bisphosphatase Oryza sativa subsp. indica

23

Ribulose bisphosphate carboxylase Oryza sativa subsp. japonica Photosystem II oxygen-evolving complex

26 protein 2 Oryza sativa subsp. japonica

32

Chloroplastic ferredoxin-1 Oryza sativa subsp. japonica

38

Chloroplast putative 50S ribosomal protein L21 Oryza sativa subsp. japonica

40

Chlorophyll a/b-binding protein Oryza sativa subsp. japonica

45

Hypothetical protein (Os10g0502000 protein) Oryza sativa subsp. japonica

48

Chloroplastic thioredoxin M5 chloroplastic Oryza sativa subsp. japonica

49

Chloroplast putative thylakoid lumenal 16.5 kDa protein (Os06g0705100 protein) Oryza sativa subsp. japonica Putative rbcL; RuBisCO large subunit from

67 chromosome 10 chloroplast insertion Oryza sativa subsp. japonica

72

Chloroplastic plastocyanin Oryza sativa subsp. japonica

Eur J Plant Pathol Table 3 (Continued) Spot no.

a

ASV b A B C D

Protein function/namec Source is italicized

d

Accession

e

Coverage

f

Peptides

no.

MW

g

[kDa]p

h

calc

i

Score

I

Redox homeostasis Cytosolic L-ascorbate peroxidase 2

19

Q9FE01

12.2

2

30.7

5.68

7.74

A2XFC7

19.6

4

27.1

5.49

94.79

Q65XA0

15.49

2

23.6

6.21

21.33

Q0D3U5

13.16

3

15.1

6.42

3.39

Q10CX6

10.15

2

34.3

5.41

24.43

Q6H443

35.51

9

26.7

8.41

91.23

Q10 I42

31.25

6

33.7

8.07

201.89

Q10NM5

7.74

2

34.5

6.38

27.06

Q6H852

21.28

3

25.8

5.48

29.05

6Z L3 1

4.55

2

31.5

8.10

1.92

5SMU8

2.31

2

51.8

5.96

2.52

Q53R J3

3.95

2

97.2

8.73

2.77

Q5VMF2

2.24

21

11.0

7.09

3.89

Q6Z I53

27.84

10

50.4

6.64

96.52

Q5Z 6X2

1.29

2

58.3

6.25

5.06

94GP6

2.53

2

46.9

6.39

5.86

Q2Q LR 2

16.05

3

16.1

6.74

21.67

Q8S718

11.2

3

25.7

5.52

61.64

Oryza sativa subsp. japonica

20

Cytosolic L-ascorbateperoxidase 1 Oryza sativa subsp. japonica

22

Dehydroascorbate reductase Oryza sativa subsp. japonica

70

Superoxide dismutase [Cu-Zn] Oryza sativa subsp. japonica

Protein Synthesis 21

Cysteine synthase Oryza sativa subsp. japonica

35

Putative plastid-specific ribosomal protein 2 Oryza sativa subsp. japonica

37

HAD-superfamilyhydrolase, subfamily IA, variant 3 containing protein, expressed Oryza sativa subsp. japonica

60 expressed Oryza sativa subsp. japonica

66

Proteasome subunit alpha type Oryza sativa subsp. japonica

Transcription 27

F-box domain containing protein-likeQ Oryza sativa subsp. japoni ac

28

Putative DNA helicaseQ Oryza sativa subsp. japonica Protein kinase domain containing protein,

29 expressed Oryza sativa subsp. japonica

36

PHD zinc finger protein-like Oryza sativa subsp. japonica

58

Elongation factor Tu Oryza sativa subsp. japonica

68

MYB transcription factor-like Oryza sativa subsp. japonica

69

Putative WD repeat proteinQ Oryza sativa subsp. japonica Glycine-rich RNA-binding protein GRP1A,

71 putative, expressed Oryza sativa subsp. japonica

Pathogen-Response Glutathione S-transferase GSTU6, putative,

42 expressed Oryza sativa subsp. japonica

Eur J Plant Pathol Table 3 (Continued) Spot no.

a

ASVb A B C D

47

Protein function/namec Source is italicized Root specific pathogenesis-related protein

Accessiond

Coveragee

Peptidesf

no.

MWg

calch

[kDa]

pI

Scorei

Q75T45

45.63

6

16.9

5.01

49.33

Q69LC1

17.81

3

25.2

7.11

24.68

A2YVG3

50.39

10

28.8

4.84

129.05

Q75HU7

2.93

2

42.0

5.62

6.10

Q0E040

16.79

4

29.7

4.77

22.52

Q6Z6B5

12.2

2

30.7

5.68

7.74

Q8GTK4

45.67

10

26.9

8.50

248.24

A2XF65

34.12

5

22.7

5.85

99.72

Q0DI00

6.08

2

16.4

4.97

11.45

Oryza sativa subsp. japonica

Others 4

Os07g0171100 protein Oryza sativa subsp. japonica

14

Putative uncharacterized protein Oryza sativa subsp. japonica

15

Os05g0220500 protein Oryza sativa subsp. japonica

16

Os02g0580300 protein Oryza sativa subsp. japonica

17

Os02g0328300 protein Oryza sativa subsp. japonica

25

Os07g0141400 protein Oryza sativa subsp. japonica

43

Putative uncharacterized protein Oryza sativa subsp. japonica

44

Os05g0427800 protein Oryza sativa subsp. japonica

a

The spot no. corresponds to the no. given in Fig. 2

b

Average spot volume per treatment group (n=12); column A: treatment with no Si added and no M. oryzae inoculation; column B: treatment with 2.0 mM silicon in culture solution but no M. oryzae inoculation. column C: treatment with M. oryzae inoculation but no Si added. column D: treatment with Si added and M. oryzae inoculation c

Protein function/name was determined by http://www.ncbi.nlm.nih.gov/BLAST/

d

Accession no: Accession number in NCBI database

e

Coverage: Sequence coverage of the peptides matched against the translated sequence

f

Peptides: Number of peptides that matched with the identified protein in mass analyses

g

MW (KDa): Molecular weight

h

caIc.pI: Theoretical isoelectric point

i

Score: Score of Mascot Search Results

the abundance of this enzyme was enhanced by Si application. Superoxide dismutase (Cu/Zn) was also verified as an SA (salicylic acid)-mediated protein. SA treatment of Mungbean yellow mosaic India virusinfected leaves increased abundance of superoxide dismutase by 3.25 fold (Kundu et al. 2011). The protein was also negatively influenced by Cd stress in rice (Nwugo and Huerta 2011). Li et al. (2012) reported that three proteins related to ROS in rice leaves were altered

after fungal infections and SA treatment, including Lascorbate peroxidase 1 (APX), 2-Cys per-oxiredoxin and superoxide dismutase [Cu-Zn] (SOD). These proteins play important roles in stress resistance, and their accumulation can alleviate damage of plants under stress conditions. Our results showed that Si may activate the metabolic pathways associated with plant redox homeostasis to alleviate oxidative damage induced by pathogen infection.

Eur J Plant Pathol Fig. 4 Hierarchical clustering analysis of the 61 differentially expressed proteins. Protein samples were treated with silicon and M. oryzae are shown on the top of the heat map. Si indicates treatment with 2.0 mM silicon but no M. oryzae inoculation. M. oryzae indicates treatment with M. oryzae inoculation but no Si added. Si+ M. oryzae indicates treatment with Si added and M. oryzae inoculation. Colours ranging from green to red represent protein expression from the lowest level of downaccumulation to the highest level of up-accumulation, respectively. Black colour indicates no change compared to CK (Control). The similarities of protein expression patterns represented by the distance of tree branches are shown on the left side. The spot sample numbers with putative protein identification are indicated on the right side of the heat map

Proteins involved in protein synthesis In non-inoculated rice seedlings with Si application alone (Si), the abundance of proteins involved in regulation/protein synthesis was not altered. However, M. oryzae inoculation and Si application (Si+M. oryzae) significantly influenced the abundance of these proteins. Proteins including cysteine synthase (spot no.21), putative plastid-specific ribosomal protein 2 (spot no.35) and HAD (haloacid dehalogenase) hydrolase subfamily IA as well as variant 3-containing protein (spot no.37) were markedly decreased by M. oryzae infection without Si (M. oryzae). However, the abundance of all these proteins was increased when Si was added (Si+M. oryzae). In addition, Si treatment induced a decrease of putative chloroplast 50S ribosomal protein L4 expressed (spot no.60) and a putative proteasome subunit alpha (spot no.66) under M. oryzae infection conditions. As an important amino acid required for the biosynthesis of redox-active peptides such as glutathione and proteins such as thioredoxins, Cysteine is involved in protection against abiotic and biotic stresses (Pandey et al. 2010). Moreover, it was decreased by Cd stress in rice leaves (Nwugo and Huerta 2011). Similar to our

Si

M. oryzae

Si+M. oryzae

results, Wang et al. (2012) reported that proteasome subunit alpha was enhanced in rice seedlings under selenium (Se) treatment. The abundance of this protein was also increased in soybean hypocotyls under PEG treatment and drought stress (Mohammadi et al. 2012). As the main component of the ribosomal, plastidspecific ribosomal protein plays an important role in protein biosynthesis. Proteins involved in signal transduction/transcription Seven proteins involved in signal transduction/ transcription (spot nos. 6, 27, 28, 36, 58, 71, and 29), were influenced by M. oryzae infection. Five of them were decreased by M. oryzae infection, including putative SHOOT1 protein (spot no. 6), F-box domain containing protein-like (spot no. 27), putative DNA helicase (spot no. 28), PHD zinc finger protein-like (spot no. 36) and glycine-rich RNA-binding protein GRP1A (spot no. 71). However, four proteins (spot nos. 27, 28, 36 and 71) of them exhibited higher protein amounts after the addition of Si (Si+M. oryzae). Moreover, we found that elongation factor Tu (spot no.58) was increased by M. oryzae infection alone but decreased by Si application

Eur J Plant Pathol

(Si+M. oryzae). Only one protein (spot no. 29) was not influenced by M. oryzae inoculation but was increased by Si application in M. oryzae-inoculated plants. Another two proteins were influenced by Si application regardless of M. oryzae inoculation, including MYB transcription factor-like (spot no. 68) and putative WD repeat protein (spot no. 69). Li et al. (2012) identified only one protein NDPK1, which was increased in the resistant cultivar C101LAC at 24 h after SA treatment in rice leaves with M. oryzae infection, but no significant changes in susceptible cultivar CO39, which plays a significant role in signal transduction pathways. With the same rice material CO39, we did not find this protein. Kim et al. (2003) reported JA might active pathogen and elicitor responsive proteins through common signal transduction pathways. Elongation factor Tu was also found increased in rice leaves under M. oryzae infection (Liao et al. 2009). Fbox domain-containing proteins play a key role in plant defence as putative photoreceptors and clock-related proteins. Ye et al. (2012) found that silencing COI1 as an F-box protein increased the susceptibility to defence. Similar to the present study, Brunings et al. (2009) studied the gene expression of rice in response to Si in M. oryzae inoculated plants, and found the cyclin-like Fbox domain-containing protein was increased after Si treatment. PHD finger containing proteins are associated with chromatin-mediated transcriptional regulation (Shi et al. 2010). The zinc finger was found involved in defence response to bacterial blight in rice (Chen et al. 2007). Glycine-rich RNA-binding protein has a regulatory role in RNA-binding. Our results showed that the abundance of this protein was decreased by M. oryzae infection, but increased by Si application. Glycine-rich RNA-binding protein was identified as down-accumulated in inoculated root avocado with p. cinnamomi (Wei et al. 2009). Pawlowski (2009) reported that glycine-rich RNA binding proteins were decreased by gibberellin acid (GA) but increased by abscisic acid (ABA), indicating that the expression of the proteins may be associated with a specific plant hormone. The WD domain is involved in a wide variety of functions, especially in signal transduction (Dooki et al. 2006). Our study showed that the WD domain repeat protein (spot no. 69) and protein kinase domain containing protein (spot no. 29) were significantly altered by Si with M. oryzae inoculation.

Proteins involved in pathogen-response Pathogen-response (PR) proteins are generally known to be responsive to biotic stress. Our study identified two proteins correlated with pathogen response, including root specific pathogenesis-related protein 10 (spot no. 47) and a putative glutathione S-transferase GSTU6 ortholog expressed (spot no. 42). Si application alone did not change the abundance of the two proteins (Si). However, M. oryzae infection alone induced the higher amount of root specific pathogenesis-related protein 10 (spot no. 47), and repressed the putative glutathione Stransferase GSTU6 ortholog expressed (spot no. 42). Si application significantly altered these proteins under M. oryzae infection (Si+M. oryzae). Zhao et al. (2007) also identified root specific pathogenesis-related protein 10 in rice leaf, suggesting that this protein may be involved in stressresponse processes. Glutathione S-transferase plays an important role in the detoxification and metabolism of many xenobiotic and endobiotic compounds (Ahsan et al. 2007), which was also found to be decreased in rice leaf under Cd stress, but increased by Si application (Nwugo and Huerta 2011). It was consistent with this study. Si application increased the glutathione S-transferase abundance levels, which may suggest that Si has a beneficial role in the protection against pathogen stress. Others There is a large group with several unknown proteins. Eight proteins (Table 3) were altered by Si treatment under M. oryzae infection. If the function of these proteins were identified, we may be able to gain deeper understanding of the function of Si in enhancing plant resistance.

Conclusion Proteomic analysis of Si treated plants revealed the involvement of complex metabolic interactions among several biochemical pathways leading to increased resistance against M. orzyae. The higher abundances of photosynthesis-related proteins mediated by Si may function as a light receptor or play a role in protein biosynthesis in chloroplast, thereby affecting the photosynthesis of rice plants. Differential expression of

Eur J Plant Pathol

energy metabolism-related proteins involved in the TCA cycle or the pentose phosphate pathway may increase pathogen resistance. Si-mediated-resistance to M. oryzae is also indicated by several proteins involved in protein synthesis, defence responses, and redox homeostasis which have important functions in alleviating blast damage. The abundance of signal/transcriptionrelated proteins involved in modulating secondary metabolism may directly act as regulatory molecules or increase susceptibility to defense. The present study thus provides important revelations on the mechanism of Si in increasing the resistance of rice against blast. Further bioinformatics and metabolomics-based studies should be conducted to obtain detailed understanding of the metabolism pathway related to these Si influenced proteins.

Acknowledgments This study was financially supported by grants from the National Key Basic Research Funds of China (2011CB100400), the National Natural Science Foundation of China (31070396, 31370456), Doctoral Foundation of the Ministry of Education of China (20094404110007) and the Natural Science Foundation of Guangdong Province (S2012010010331).

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