Exploring Pathogenic Mechanisms of Botrytis cinerea Secretome

Article pubs.acs.org/jpr

Exploring Pathogenic Mechanisms of Botrytis cinerea Secretome under Different Ambient pH Based on Comparative Proteomic Analysis Boqiang Li,† Weihao Wang,†,‡ Yuanyuan Zong,†,‡ Guozheng Qin,† and Shiping Tian*,† †

Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China Graduate School of Chinese Academy of Sciences, Beijing, 100039, China

‡

S Supporting Information *

ABSTRACT: Botrytis cinerea causes gray mold rot on over 200 plant species worldwide, resulting in great economic loss every year. Cooperation of proteins secreted by B. cinerea plays an important role in its successful infection to host plants. The ambient pH, as one of the most important environmental parameters, can regulate expression of secreted proteins in various fungal pathogens. In the present study, we mainly investigated the effect of ambient pH on secretome of B. cinerea strain B05.10 with a comparative proteomic method based on 2-DE. Distinct differences in secretome of B. cinerea were found between pH 4 and 6 treatments, and 47 differential spots, corresponding to 21 unique proteins, were identified using MALDI-TOF/TOF. At pH 4, more proteins related to proteolysis were induced, whereas most of up-accumulated proteins were cell wall degrading enzymes at pH 6. Analysis of gene expression using quantitative real-time PCR suggests that production of most of these proteins was regulated at the level of transcription. These findings indicate that B. cinerea can adjust protein profile of secretome responding to different ambient pH values and provide evidence to deeply understand the complicated infecting mechanisms of B. cinerea on a wide range of plant hosts. KEYWORDS: ambient pH, Botrytis cinerea, cell wall degrading enzymes, proteomics, secretome

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INTRODUCTION Botrytis cinerea Pers. ex. Fr. is a necrotrophic phytopathogenic fungus belonging to the Ascomycetes. The pathogen causes gray mold rot on different organs (fruits, legumes, flowers and leaves) of over 200 plant species worldwide, particularly on many economically important crops such as tomato, berries, chickpeas, French beans, and grapes as well as cut flowers.1 In the past few years, B. cinerea attracted the attention of a large number of researchers and has become an important model system in molecular phytopathology. Genomic information of two B. cinerea strains, B05.10 and T4, and its neighbor species Sclerotinia sclerotinium has become available.2 The availability of the genomic information promotes the development of “-omics” techniques such as genomics, transcriptomics and proteomics in B. cinerea.3 Like many other fungal pathogens, B. cinerea secretes a large set of extracellular enzymes to degrade plant cell wall polymers and infects plant tissues by the cooperation of these enzymes, such as polygalacturonases, pectin methylesterases, proteases and laccases.4 Part of these enzymes are encoded by gene family and differentially expressed according to various hosts and environmental factors,5−7 which may be one of the reasons that B. cinerea is able to infect a broad range of hosts. To date, these © 2012 American Chemical Society

enzymes have predominantly been studied on a protein-byprotein basis using biochemical or molecular genetics techniques. However, successful infection by pathogen needs the cooperation of various extracellular proteins, though only a few of them may be the “real” virulence factor. Application of “-omics” techniques opens a new gate to deeply explore the infecting mechanisms of pathogens to plants. Proteomic analysis, as a complement to genome and transcriptome analysis, has been proven to be a powerful method for offering a more-direct analysis of cellular response, and showed increasing importance in the quest for virulence factors in plant pathogenic fungi.8 In recent years, the secretomes of several fungal pathogens, including Penicillium expansum, Fusarium graminearum, Aspergillus f lavus, and B. cinerea, have been analyzed.3,9−14 More recently, a secretome analysis of B. cinerea during the interaction between the pathogen and tomato fruit was reported.15 pH, as an important environmental factor, has a significant effect on physiological and pathogenic aspects of fungi. Meanwhile, fungi develop a complicated regulatory system to Received: April 17, 2012 Published: July 3, 2012 4249

dx.doi.org/10.1021/pr300365f | J. Proteome Res. 2012, 11, 4249−4260

Journal of Proteome Research

Article

equal volume of Tris-HCl (pH 7.8) buffered phenol for 30 min. After centrifugation (10000g, 40 min, 4 °C), proteins in the phenol phase were precipitated with 5 volumes of ice-cold saturated ammonium acetate in methanol overnight at −20 °C. Proteins were collected after centrifugation at 15000g for 30 min at 4 °C and washed twice with cold saturated ammonium acetate in methanol and acetone each. The precipitate was airdried for 1 h at 4 °C and solubilized in 250 μL of focalization buffer containing 2 M thiourea, 7 M urea, 4% (w/v) CHAPS, 1% (w/v) DTT and 2% (v/v) carrier ampholytes pH 3−10. Protein samples were kept at −80 °C until use. The protein concentration was determined according to Bradford’s method using bovine serum albumin as standard.24

sense and respond to ambient pH signal within the long term interaction with environment.16 The system has been best documented in Aspergillus nidulans, which includes seven genes: pacC, palA, palB, palC, palF, palH and palI.17 Similar regulatory modes have also been found to operate extracellular virulence factors in several plant pathogens, such as Colletotrichum gloeosporioides,18 F. oxysporum,19 S. sclerotiorum,20 and Alternaria alternata.21 In B. cinerea, Menteau et al.22 found that ambient pH could influence the activities of extracellular protease, polygalacturonase and laccase. It has also been reported that six genes encoding endopolygalacturonase of B. cinerea, Bcpg1−6, were differentially regulated by ambient pH both in vitro and in vivo.5,6 Understanding the changes of secretome under different ambient pH values will be helpful for discovering pathogenic mechanism of fungal pathogens. In the present study, we compared the changes of secretome of B. cinerea grown at culture media buffered at pH 4 and 6. Forty seven differential spots, corresponding to 21 unique proteins, were identified by the technique of MALDI-TOF/TOF. Out of 21 proteins, 15 proteins were predicted as secreted and related to carbohydrate metabolism or proteolysis. Analysis of gene expression suggests that expressions of most of these proteins were regulated at the level of transcription. It is the first report on ascertaining the mechanism of ambient pH regulating secretome of plant pathogens.

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2-DE and Gel Analysis

Two-dimensional gel electrophoresis (2-DE) was performed as described by Li et al.23 Aliquot of 350 μg of protein sample resolved in 250 μL of focalization buffer with 0.001% [w/v] bromphenol blue was used to rehydrate gel strips (Immobiline DryStrip pH 4−7, 13 cm; GE Healthcare, Piscataway, USA.) for 16 h with a passive manner. IEF was performed at 20 °C for a total of 20 kVh on an Ettan IPGphor unit (GE Healthcare). SDS-PAGE was conducted using 5% stacking gels and 15% polyacrylamide gels at a constant 30 mA per gel. Proteins in the gel were stained with Coomassie Brilliant Blue (CBB) R-250 solution containing 50% (v/v) methanol, 15% (v/v) acetic acid and 0.1% (w/v) CBB R-250. Gel images were obtained using a flatbed scanner (Amersham Biosciences, Uppsala, Sweden) and saved in TIF format. Comparison of protein accumulation between samples was performed using Image Master 2D Elite software version 5.0 (Amersham Biosciences). To account for experimental variation, four independent biological replicate gels were analyzed for each pH treatment. Only those spots detected in all replicates within the same pH treatment were considered as valid spots. The amount of a protein spot was calculated on the basis of the volume of that spot, which was normalized against total volume of all valid spots. The normalized intensity of spots on four replicate 2D gels was averaged, and a two-tailed nonpaired Student’s t-test was used to determine whether the relative change was statistically significant between samples using SPSS software 11.0 (SPSS Inc., Chicago, IL, USA).

MATERIALS AND METHODS

Organism and Culture Conditions

Botrytis cinerea strain B05.10 was kindly provided by Dr. Tudzynski (Westfälische Wilhelms-Universität Münster, Germany). The pathogen was maintained on potato dextrose agar (PDA) at 4 °C, and reisolated on tomato fruit before the experiment was carried out. For collection of spores, the strain of B. cinerea B05.10 was first cultured on PDA plates for 1 week at 22 °C. Then spores were collected and suspended in sterile distilled water at the concentration of 1.5 × 107 spores mL−1. Aliquots of 1 mL of spore suspension were added to 99 mL of PDB medium in 250-mL conical flasks and cultured at 22 °C with shaking at 180 rpm. After 24 h, the mycelium was harvested using 4 layers of cheesecloth and washed thoroughly with sterile distilled water. Wet mycelium (about 3.0 g) was transferred to 100 mL of modified Czapek’s medium buffered with 100 mM of citric acid−sodium citrate buffer at pH 4.0 or 6.0, respectively. The modified Czapek’s medium contained 1% pectin from apple (Sigma, Saint Louis, USA), 0.2% NaNO3, 0.1% K2HPO4, 0.05% KCl, 0.05% MgSO4·7H2O, 0.036 mM FeSO4·7H2O. This liquid culture was incubated at 22 °C with shaking at 180 rpm for indicated time. The pH value of liquid medium was adjusted every 12 h with 2 M of NaOH or HCl to maintain their pH with the range of 4.0 ± 0.2, and 6.0 ± 0.2, respectively. After 12, 24, 48, and 72 hpi (hours postinoculation), mycelium and medium were separated by filtering with 4 layers of cheesecloth. The mycelium was washed thoroughly with cold sterile distilled water, quickly frozen with liquid nitrogen, and stored in −80 °C for RNA extraction. The harvested medium was used for secreted protein isolation.

In-Gel Digestion, Mass Spectrometry (MS), and Database Searching

Protein digestion was performed according to the method described by Qin et al.25 Briefly, protein spots were excised from the stained gels and destained with 50 mM NH4HCO3 in 50% (v/v) methanol for 1 h at 40 °C. After being completely destained and dried in a vacuum centrifuge, the gel particles were digested at 37 °C with 5 ng μL−1 of trypsin for 16 h. Digested peptides extracted with 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile were lyophilized and used for MS analysis by a MALDI-TOF/TOF mass spectrometer (4800 Proteomics Analyzer, Applied Biosystems, Framingham, MA, USA). Before being spotted onto the MALDI target plates, the peptides were resuspended in 5 mg mL−1 of α-cyano-4hydroxycinnamic acid solution in 50% acetonitrile containing 0.1% TFA. MS spectra were gathered with 1600 laser shots per spectrum, and MS/MS spectra were acquired with 2500 laser shots per fragmentation spectrum. The 10 strongest peaks from each MS spectra were selected as precursor ions for the acquirement of the MS/MS fragmentation spectra. The 4000 Series Explorer software (Applied Biosystems) was used for

Secreted Protein Isolation

The harvested media (72 hpi) were first centrifuged at 20000g for 30 min at 4 °C for three times to remove any residual mycelium and any other debris. The secreted proteins in the supernatant were isolated according to the method described by Li et al.23 Briefly, the supernatant was extracted with an 4250



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Figure 1. 2-DE gels from secretome of B. cinerea and classification of identified proteins. (A) and (B) 2-DE gels from secretome of B. cinerea after 72 hpi under pH 4 and 6 conditions. Arrows indicate protein spots identified by MALDI-TOF/TOF. Numbers of these protein spots correspond to those in Tables 1 and 2. (C) and (E) Numbers of up and specifically accumulated protein spots under pH 4 or 6 condition, respectively. (D) and (F) Function classification of up and specifically accumulated protein spots under pH 4 or 6 condition, respectively. Spot number and percentage in each group were shown.

spectra analyses and generation of peak list files. The parameters were set as a signal-to-noise threshold of 10 and a minimum area of 100. The peak list files were searched in NCBI nonredundant database (http://www.ncbi.nlm.nih.gov, version 20120204, 17 172 511 sequences and 5 891 506 632 residues in total, and 1 212 449 sequences for “Fungi”) using MASCOT (http://www. matrixscience.com). Search parameters were set as taxonomy, Fungi; proteolytic enzyme, trypsin; max missed cleavages, 1; fixed modifications, carbamidomethyl (C); variable modifications, oxidation (M); peptide mass tolerance, 0.2 Da; fragment mass tolerance, 0.3 Da.

RNase-free DNase I (Fermentas, Canada) to remove genomic DNA, first-strand cDNAs were generated using EasyScript First-Stand cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China). qRT-PCR was performed on an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, CA, USA) and was carried out in a total volume of 20 μL, containing 10 μL of 2 × Ultra SYBR Mixture (with ROX) (CWBIO), 2 μL of the 10× diluted cDNA, 0.4 μL of each primer (10 mM) and 7.2 μL of RNase-free water. The primers used for amplification were designed using Primer Express software 3.0 (Applied Biosystems), and the sequences were listed in Supplementary Table 1. Transcript levels were normalized against the Bcactin gene (GenBank accession number AJ000335), and the relative expression levels were measured according to the 2−ΔΔCT method.27

Prediction of Extracellular Location of Identified Proteins

To predict whether the identified proteins are secreted proteins, first SignalP 4.0 (http://www.cbs.dtu.dk/services/ SignalP/) was used to analyze the presence of signal peptide in the protein sequences. For those proteins without signal peptide as predicted by SignalP, SecretomeP 1.0b (http:// www.cbs.dtu.dk/services/SecretomeP/) was used to predict whether a nonclassical protein secretion mechanism was involved.

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RESULTS AND DISCUSSION

Organism Culture, Sample Isolation, and 2-DE

B. cinerea has a wide range of plant hosts and can infect various tissues, such as leaves, stems, flower petals and fruits. Menteau et al.22 investigated the pH values of different tissues from some important hosts of B. cinerea, and found that pH of these tissues ranged from acidic (pH 3.32) to nearly neutral (pH 6.30). Fruits, such as apple, tomato, cherry, and strawberry, usually presented lower pH ranging from 3.32 to 4.39, and leaves, legumes, stems and roots showed higher pH ranging from 5.81

RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA was isolated from aliquots of 0.2 g of mycelia using the method described by Moore et al.26 After being treated with 4251



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Table 1. Proteins Identified from B. cinerea That Were up or Specifically Accumulated at pH 4 spota

gene IDb

putative function

Carbohydrate Metabolism U1 BC1G_00455 Mannosyl-oligosaccharide alpha1,2-mannosidase U5 BC1G_04994 Alpha-L-arabinofuranosidase S4

BC1G_03983

Glucan 1,3-beta-glucosidase

Proteolysis U2 BC1G_02944

Family S53 protease

U3

BC1G_02944

Family S53 protease

U4

BC1G_03070

Aspartic protease BcAP8

U7

BC1G_03070


S5

BC1G_03070


S6

BC1G_03070


S7

BC1G_03070


S9

BC1G_03070


S10

BC1G_03070


S11

BC1G_03070


S12

BC1G_03070


S13

BC1G_03070


S14

BC1G_03070


S15

BC1G_03070


S16

BC1G_03070


S1

BC1G_07068


S2

BC1G_07068


S3

BofuT4_P119560

Serine-type carboxypeptidase

U6

BC1G_09180

Metalloprotease (Merops M35)

Unknown Function S8 BC1G_01393

Predicted protein

NCBI accession

theor Mr (kDa)/pIc

expt Mr (kDa)/pId

SignalPe

protein scoref

NPg

SCh (%)

pH 4 vs pH 6i

gi| 154324112 gi| 154314102 gi| 154314548

58.21/4.55

55.45/4.52

Y

465

6

18

16.0

52.56/5.97

35.96/6.21

Y

281

6

18

13.0

85.93/5.57

43.78/6.73

N

213

6

13

Newj

gi| 154317922 gi| 154317922 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154317437 gi| 154310297 gi| 154310297 gi| 347830326 gi| 154302873

62.23/4.81

43.78/4.65

Y

207

5

17

3.9

62.23/4.81

43.50/4.76

Y

363

5

17

3.7

39.82/5.43

35.03/4.44

Y

540

6

26

18.2

39.82/5.43

21.70/4.68

Y

226

4

16

13.0

39.82/5.43

35.03/4.36

Y

591

7

33

New

39.82/5.43

34.57/4.53

Y

606

7

33

New

39.82/5.43

27.30/4.13

Y

243

3

12

New

39.82/5.43

20.93/4.21

Y

220

4

14

New

39.82/5.43

20.45/4.43

Y

441

5

24

New

39.82/5.43

21.00/4.59

Y

295

4

16

New

39.82/5.43

20.32/4.59

Y

359

4

16

New

39.82/5.43

17.59/4.16

Y

229

2

9

New

39.82/5.43

17.36/4.39

Y

227

3

13

New

39.82/5.43

16.69/4.24

Y

190

2

9

New

39.82/5.43

16.26/4.14

Y

258

2

9

New

42.34/4.96

58.44/4.14

N

501

5

20

New

42.34/4.96

55.81/4.14

N

226

4

18

New

50.04/4.64

53.66/4.58

Y

409

6

23

New

37.65/4.66

21.56/4.43

Y

170

1

6

20.89/4.42

28.40/4.58

Y

326

4

33

gi| 154321037

6.4

New

a Spot names corresponding to spots in Figure 1. Spot name with initial of “U” represents the protein is up-accumulated at pH 4, and “S” represents the protein is specifically accumulated at pH 4. bGene ID refers to B. cinerea B05.10 database released by the BROAD institute or B. cinerea T4 database released by the French National Sequencing Center, Genoscope. cTheoretical molecular mass and isoelectric point based on amino acid sequence of the identified protein. dExperimental molecular mass and isoelectric point estimated from the 2D gels. eResults returned by the online version of SignalP 4.0 software. Y or N means that the protein sequence includes or does not include a signal peptide. fProtein scores reported by Mascot MS/MS Ion Search. gThe number of matched peptides based on MS/MS data searching, excluding peptides of duplicate matches. hAmino acid sequence coverage for the identified proteins. iAverage fold change of relative abundance of corresponding spot at pH 4 versus pH 6 from four biological repeats. jCorresponding spot appeared in the 2D gels at pH 4 but not at pH 6.

processes against plant hosts.4 Studying on secretome of plant pathogens based on proteomic technology may give a great contribution to understanding the role of secreted proteins in fungal pathogenicity. Some research focused on the secretome of B. cinerea have been documented since 2009.3,12−14 Recently, Shah et al.15 first reported the analysis of B. cinerea secretome in a plant-fungus interaction. On the basis of 2-DE, Espino et al.14 reported their findings of early secretome of B. cinerea and

to 6.30. In this experiment, we selected buffered culture media at pH 4 and 6 to mimic pH values of different tissues, and investigated the effects of different ambient pH values on secretome of B. cinerea. Pectin from apple fruit was used as the sole carbon resource to induce secretion of extracellular proteins. Extracellular proteins secreted by fungal pathogens (secretome) are very important during their complicated infecting 4252



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Table 2. Proteins Identified From B. cinerea That Were up or Specifically Accumulated at pH 6 spota

gene IDb

NCBI accession

theor Mr (kDa)/pIc

expt Mr (kDa)/pId

SignalPe

protein scoref

NPg

SCh (%)

gi| 154312003 gi| 154312003 gi| 154312003 gi| 154312003 gi| 154312003 gi| 154312003 gi| 154312003 gi| 154312003 gi| 154312003 gi| 154314564 gi| 154319343 gi| 154319343 gi| 154319343 gi| 154293875 gi| 154293875 gi| 154289225 gi| 154294331 gi| 154305175

54.4/5.6

59.60/5.23

Y

174

4

15

2.4

54.4/5.6

56.92/4.84

Y

68

3

10

2.6

54.4/5.6

56.92/4.96

Y

363

8

26

2.9

54.4/5.6

56.92/5.09

Y

162

4

12

2.8

54.4/5.6

60.78/4.84

Y

276

6

17

Newj

54.4/5.6

60.78/4.96

Y

285

6

17

New

54.4/5.6

60.78/5.09

Y

334

6

20

New

54.4/5.6

55.45/5.09

Y

71

2

6

New

54.4/5.6

58.06/5.22

Y

382

7

23

New

41.08/6.12

51.25/6.26

Y

288

5

20

2.9

47.65/5.9

47.37/5.57

Y

78

3

15

5.1

47.65/5.9

30.92/4.86

Y

168

3

7

8.6

47.65/5.9

32.81/5.00

Y

48

2

7

New

26.88/6.18

32.81/6.13

Y

72

2

16

5.3

26.88/6.18

32.81/5.72

Y

157

2

16

New

33.59/6.06

34.35/5.89

Y

244

2

12

10.1

41.47/5.36

50.25/5.41

Y

267

4

13

New

27.22/5.52

27.48/5.83

N

108

2

14

New

gi| 154310724 gi| 154310724 gi| 154305136 gi| 154313005

55.37/5.49

35.73/5.94

Y

217

3

9

6.8

55.37/5.49

35.73/4.14

Y

56

1

5

New

46.49/5.88

36.44/6.95

Y

187

5

21

New

68.25/5.5

30.52/5.14

N

52

3

8

New

Cerato-platanin family protein BcSpl1

gi| 154320365

14.16/4.66

13.78/4.65

Y

91

3

35

7.7

Oxidoreductase, putative

gi| 154309175

51.78/5.04

46.14/4.53

Y

551

6

21

4.4

putative function

Carbohydrate Metabolism U8 BC1G_06035 Exocellulase U9

BC1G_06035

Exocellulase

U10

BC1G_06035

Exocellulase

U11

BC1G_06035

Exocellulase

S17

BC1G_06035

Exocellulase

S18

BC1G_06035

Exocellulase

S19

BC1G_06035

Exocellulase

S20

BC1G_06035

Exocellulase

S21

BC1G_06035

Exocellulase

U12

BC1G_03991

U13

BC1G_02623

Arabinogalactan endo-1,4-betagalactosidase Alpha-amylase

U15

BC1G_02623

Alpha-amylase

S24

BC1G_02623

Alpha-amylase

U18

BC1G_14009

S25

BC1G_14009

U17

BC1G_16209

S22

BC1G_13938

Rhamnogalacturonan acetyl esterase Rhamnogalacturonan acetyl esterase Arabinogalactan endo-1,4-betagalactosidase Exoarabinanase

S27

BC1G_08882

Triosephosphate isomerase

Proteolysis U16 BC1G_06836

Proteases - Subtilase Family

S23

BC1G_06836

Proteases - Subtilase Family

S26

BC1G_08658

Carboxypeptidase A1 precursor

S28

BC1G_05504

Aminopeptidase

Pathogenicity Factor U19 BC1G_02163 Unknown function U14 BC1G_07482

pH 6 vs pH 4i

Spot names corresponding to spots in Figure 1. Spot name with initial of “U” represents the protein is up-accumulated at pH 6, and “S” represents the protein is specifically accumulated at pH 6. bGene ID refers to B. cinerea B05.10 database released by the BROAD institute. cTheoretical molecular mass and isoelectric point based on amino acid sequence of the identified protein. dExperimental molecular mass and isoelectric point estimated from the 2D gels. eResults returned by the online version of SignalP 4.0 software. Y or N means that the protein sequence includes or does not include a signal peptide. fProtein scores reported by Mascot MS/MS Ion Search. gThe number of matched peptides based on MS/MS data searching, excluding peptides of duplicate matches. hAmino acid sequence coverage for the identified proteins. iAverage fold change of relative abundance of corresponding spot at pH 6 versus pH 4 from four biological repeats. jCorresponding spot appeared in the 2D gels at pH 6 but not at pH 4. a

found that high amount of polysaccharides were secreted during the incubation of the pathogen that resulted in a big colored pellet difficult to dissolve when the proteins were precipitated. They developed a double precipitation method with TCA and methanol−chloroform and obtained 2D gel with

a maximum of 56 protein spots. Using DOC/TCA precipitation plus a phenol-based purification, FernándezAcero et al.3 improved quality of isolated proteins secreted by B. cinerea and increased quantity of detected protein spots (about 100 spots) in the 2D gels. In the present study, certain 4253



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amount of unknown dark and colloidal pellets was found when the culture media were collected by centrifugation at both pH 4 and 6 treatments (Supporting Information Figure S1). The dark color of the pellets might be caused by the secondary metabolites produced by the fungus during the growth, as the color of culture medium became deeper along with the incubation time. To reduce the negative effects of the pellets on protein isolation, harvested media were first centrifuged at 20000g for three times to remove most part of these components. After that, proteins in the supernatant were isolated with Tris-HCl buffered phenol and precipitated with saturated ammonium acetate in methanol. About 1200 and 500 μg of protein could be obtained from 100 mL of harvested medium after 72 hpi for pH 4 and 6 treatments, respectively. The method was previously used for isolating proteins from spores of P. expansum.23 The resulting samples were separated using 2-DE in which the first dimension was performed with pH 4−7 IPG strips. More than 300 protein spots were detected in each gel after ignoring spots with undefined shapes and areas using Image Master 2D Elite software (Figure 1A and B). Part of these spots were very faint or did not appear in all of replicated gels. After excluding these spots, there were about 180 spots in each 2D gel.

spot) (Figure 1E and F). Most of proteins in the group of carbohydrate metabolism belong to cell wall degrading enzymes (CWDEs). We found that multiple differential spots were identified as the same protein by MS/MS analysis. For example, 13 spots corresponded to aspartic protease BcAP8 encoded by BC1G_03070 (Table 1), and 9 spots corresponded to exocellulase encoded by BC1G_06035 (Table 2). Similar results were also reported by Fernández-Acero et al.3 In their study, 10 spots were found corresponding to BC1G_03070 and BC1G_06035, respectively. These series of spots may lead by post-translational modifications (PTMs) of proteins, such as glycosylation and truncation. Glycosylation is one of the most important PTMs for secreted proteins, which causes a series of spots with different molecular weights and pI values in gels depending on the nature of glycan structure.28 In addition, we noted that 10 spots (U7, S7, and S9−16) corresponding to protein BcAP8 showed much smaller molecular weight than theoretical value, which may be caused by a protein truncation event, such as protein cleavage or other unknown reasons.29 Prediction of Secreted Protein in Silico

Secretion by endoplasmic reticulum (ER)−Golgi secretory pathway, also called classical secretory pathway, is the most important mechanism to translocate proteins out of cells in eukaryotes.30 In general, a protein secreted through the classical pathway has a signal peptide within the N-terminal region of amino acid sequence, which can be predicted by software. In the present study, with the newly released software of SignalP 4.0,31 42 spots (corresponding to 17 unique proteins) were predicted to possess signal peptide within the identified 47 spots. For spot U12, encoding gene ID BC1G_03991, we obtained a negative result for prediction of signal peptide with the sequence information from the database of BROAD institute. However, protein encoded by its homologous gene BofuT4_P035980 in strain T4 was predicted to have a signal peptide. After comparing the genome sequence of BC1G_03991 with BofuT4_P035980, we concluded that there may be an uncorrected initiation codons assignment for BC1G_03991. Referring to the sequence of BofuT4_P035980, we manually moved the initiation codons of BC1G_03991 toward upstream for 42 bp (Supporting Information Figure S3), resulting in a positive return from SignalP 4.0. A similar case has also been reported in B. cinerea by Espino et al.14 Besides classical secretory pathway, proteins can be secreted out of cells by nonclassical secretory pathway, in which signal peptide may be not required.30 Software of SecretomP is widely used to predict this kind of proteins.32 With the software, Shah et al.12 reported that 19 proteins out of 29 proteins lacking a signal peptide sequence were predicted to have a nonclassical targeting signal sequence in B. cinerea secretome. In our study, 5 spots (corresponding to 4 unique proteins) lacking a signal peptide sequence were analyzed with SecretomP 1.0b, and one of them (S4, gene ID: BC1G_03983) showed a positive result, indicating that the protein may be secreted through nonclassical pathway. Other 4 spots (S1, S2, S27, S28) showed negative result from both SignalP and SecretomP, slightly suggesting a cell lysis might happen. However, there may be an alternative explanation that these proteins were secreted through unknown pathways. For example, aspartic protease BcAP1 (spots S1 and 2) has been predicted as a secreted protein by ten Have et al.7 on the basis of phylogeny and its conformation.

Protein Identification

As shown in Figure 1, distinct differences between the protein profiles of pH 4 and 6 treatments were observed from a general view. Using Image Master 2D Elite software, in further, we ascertained that the differences were from two aspects: changes in abundance of common spots between the two pH treatments and appearance or absence of spots specifically accumulated at certain pH conditions. There were 151 common spots that appeared in all of gels (8 gels, 4 biological replications for each pH treatment). Among them, protein spots showing statistically significant (p < 0.05) changes of more than 2-fold in relative abundance between the two treatments were selected as upaccumulated spots. Seven up-accumulated protein spots at pH 4 (Table 1, U1−7) and 12 up-accumulated spots at pH 6 (Table 2, U8−19) were positively identified through MALDITOF/TOF and NCBInr database searching. In addition, we also identified 16 spots specifically accumulated at pH 4 (Table 1, S1−16) and 12 spots specifically accumulated at pH 6 (Table 2, S17−28). The MS/MS peptide sequence of the identified proteins was summarized in Supporting Information Table S2. For identifications based on assignment of less than two peptides with ion score above the threshold, sampled spectra were manually inspected, and annotated spectra were shown in Supporting Information Figure S2. Through the MS/MS Ion Search with generated peak list files, except spot S3, the best match of all the identified protein spots was from B. cinerea B05.10. Spot S3 was matched to a protein encoded by a gene (BofuT4_P119560) from B. cinerea strain T4, which has a 95% sequence homology with BC1G_14591 of B05.10. In pH 4 treatment, 23 identified protein spots (7 upaccumulated, 16 specifically accumulated spots) were matched to 9 unique proteins and were categorized into 3 groups, including carbohydrate metabolism (3 spots), proteolysis (19 spots), and unknown function (1 spot) (Figure 1C and D). In pH 6 treatment, 24 identified protein spots (12 upaccumulated, 12 specifically accumulated spots) were matched to 12 unique proteins and were categorized into 4 groups, including carbohydrate metabolism (18 spots), proteolysis (4 spots), pathogenicity factor (1 spot), and unknown function (1 4254



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Figure 2. Abundance variance of predicted secreted proteins related to carbohydrate metabolism and proteolysis after 72 hpi under pH 4 and 6 conditions. (A) and (B) A close-up view in 2D gels of up and specifically accumulated protein spots under pH 4 or 6 condition, respectively. Circles with dotted line indicate the hypothetical position of specifically accumulated protein spots under certain pH conditions. (C) and (D) Abundance variance of up-accumulated protein spots under pH 4 or 6 condition, respectively. The graph represents an average of four biological replicates. Bars represent standard deviations of the mean.

Up and Specifically Accumulated Proteins at pH 4

may be utilized by pathogens to degrade structural plant cell wall proteins and/or antifungal proteins produced by the host.33 Proteases are divided into four major groups according to the character of their catalytic active site: serine proteinases, cysteine (thiol) proteinases, aspartic proteinases, and metalloproteinases.34 Aspartic protease is one of the most important protease species in fungal pathogens.7,33,35−37 In Candida albicans, an important fungal pathogen of humans, aspartic protease (AP) gene family has 10 genes (SAP1−10) and plays a crucial role during the pathogenesis.38 Movahedi and Heale39 demonstrated that the specific AP inhibitor pepstatin could reduce disease development of B. cinerea on carrot disks. In B. cinerea, the AP family includes 14 genes (Bcap1−14).7 Among them, Bcap8 is the most important one, whose product, BcAP8, was defined to constitute up to 23% of the total secreted protein and contribute to about 70% of total AP activity. Similar results were also documented by Espino et al.14 and

Compared with proteins at pH 6, 23 spots (corresponding to 9 unique proteins) showed differential accumulation under pH 4 condition, including 7 common spots and 16 specifically accumulated spots (Table 1). All spots have the putative functions of carbohydrate metabolism or proteolysis, except 1 spot (S8) with unknown function. With the exception of spots S1 and 2, all spots were predicted as extracellular location by software of SingalP or SecretomeP. A close-up view of the gels and changes in abundance of these spots are shown in Figure 2A and C. It is noteworthy that functions of 17 out of 23 protein spots were related to proteolysis. Among them, 13 spots (upaccumulated: 2, specifically accumulated: 11) were matched to one protein, aspartic protease BcAP8. Proteases have been suggested to be involved in plant−pathogen interactions and 4255



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Figure 3. Time-course changes in expression of genes under pH 4 and 6 conditions. Expression analysis of genes, which encode predicted secreted proteins related to carbohydrate metabolism and proteolysis in Tables 1 and 2, was performed using qRT-PCR. BcpacC was used as the positive control. Values are expressed as the increase in the ratio of corresponding mRNA versus BcpacC mRNA at 12 hpi in pH 4 treatment.

Fernández-Acero et al.,3 when they separated the extracellular proteins of B. cinerea with 2-DE. The protein was also identified through LC−MS/MS by Shah et al. from secretome collected from B. cinerea grown on a solid substrate of cellophane membrane with or without Arabidopsis leaf extract.12 In our study, BcAP8 has a very high abundance at pH 4 but a lower abundance at pH 6 (Figures 1 and 2). Meanwhile, the AP activity of culture medium at pH 4 was higher than that at pH 6 in each sampling time and showed a 30-fold difference after 72 hpi (Supporting Information Figure S4). These results indicate

that BcAP8 could be induced under acidic ambient pH. Recently, ten Have et al.7 reported there was no significance in virulence between the Bcap8 knockout mutant and the wild type strain B05.10 in tomato leaves and fruit. The result indicated that BcAP8, the most abundant protein in the secretome, might not be responsible for virulence in B. cinerea. As a successful plant pathogen, it seems unreasonable that B. cinerea secretes a useless protein with such high abundance. We propose that the loss of BcAP8 activity in ΔBcap8 mutant may be partially compensated by expression of other genes from the 4256



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Bcap family, or BcAP8 activity plays other important roles in the life cycle of the pathogen. Besides aspartic protease, other 3 up or specifically accumulated proteases under pH 4 condition were identified as Family S53 protease (spot U2, 3), serine-type carboxypeptidase (spot S3), metalloprotease (Merops M35) (spot U6), which belong to another two group of protease, i.e., serine protease and metalloprotease. The cooperation of these proteases can enhance the proteolytic ability of B. cinerea to proteins from plant hosts at acidic condition. Moreover, 3 proteins, mannosyl-oligosaccharide alpha-1, 2-mannosidase (U1), alpha-L-arabinofuranosidase (U5), and glucan 1, 3-betaglucosidase (S4), were up or specifically accumulated under pH 4 condition. Spots U5 and S4 belong to CWDEs and are involved in the hydrolysis of hemicelluloses and glucan in plant cell wall.40,41 Spot U1, alpha-1, 2-mannosidase, is usually reported as an ER or Golgi-targeted protein and plays an important role in protein glycosylation.42 There are few reports about the function of secreted alpha-1, 2-mannosidase.43,44

Besides the exocellulase, other 5 secreted proteins involved in carbohydrate metabolism were identified. Of them, arabinogalactan endo-1,4-beta-galactosidase (BC1G_03991, BC1G_16209), rhamnogalacturonan acetyl esterase (BC1G_14009) and exoarabinanase (BC1G_13938) belong to CWDEs and are involved in the hydrolyzation of hair region of pectins.48−50 The hairy region of pectin is comprised of xylogalacturonan and rhamnogalacturonan to which side chains of neutral sugars arabinan, galactan, and arabinogalactan were attached.51 Moreover, two secreted proteases, subtilase family proteases (BC1G_06836) and carboxypeptidase A1 precursor (BC1G_08658) were also up-accumulated or specifically accumulated under pH 6 condition. Besides the proteins involved in cell wall degradation and proteolysis, another spot U19 also attracted our notice. The spot was matched to a cerato-platanin family protein BcSpl1 and was up-accumulated for 7.7 folds at pH 6 (Table 2). First protein in the family was identified in Ceratocystis f imbriata that induced cell necrosis in tobacco leaves.52 In B. cinerea, BcSpl1 causes a fast and strong hypersensitive response (HR) in tomato, tobacco and Arabidopsis leaves, and is responsible for pathogenicity.53 In previous proteomic studies on secretome of B. cinerea, BcSpl1 was identified under various culture conditions.3,12,14 The protein has also been reported to be induced by ethylene in B. cinerea.54 Here, we first reported that BcSpl1 was regulated by ambient pH signal.

Up and Specifically Accumulated Proteins at pH 6

As compared with protein profile at pH 4, 24 spots (corresponding to 12 unique proteins) had differential accumulation under pH 6 condition, including 12 common spots and 12 specifically accumulated spots. Putative functions of most spots were classified as carbohydrate metabolism or proteolysis, except U19, a pathogenicity factor, and U14, an oxidoreductase. With the exception of spots S27 and 28, all spots were predicted as extracellular location by software of SingalP or SecretomeP. A close-up view of the gels and changes in abundance of these spots are shown in Figure 2B and D. Different with that in pH 4, function of most differential protein spots (18 out of 24 spots) at pH 6 was related to carbohydrate metabolism, and most of these proteins were CWDEs. Among them, 9 spots (4 up-accumulated and 5 specifically accumulated spots) were matched to exocellulase (gene ID: BC1G_06035). The protein was also identified from secretome of B. cinerea in cultures3,12 and interaction with tomato fruit15 in previous studies. Cellulose, as a straight-chain polymer of glucose (β-1,4-glucan), is a major component of plant cell wall, especially in leaves and stems.45 For complete hydrolysis of cellulose, synergistic action of three different cellulases is required, including endo-β-1,4-glucanases (endocellulase, EC 3.2.1.4), which cleave internal glycosidic bonds leaving shorter polysaccharide chains; cellobiohydrolases (cellobiosidase, EC 3.2.1.91), which release the disaccharide cellobiose from the nonreducing ends of cellulose; and exo-β1,4-glucanases (exocellulase, EC 3.2.1.74), which liberate successive glucose units from the polymer ends.46 In Clavibacter michiganensis subsp. michiganensis, a gram-positive bacterium, the endo-β-1,4-glucanase CelA is required during its infection on tomato,47 whereas knocking out of an endo-β-1,4-glucanase coding gene, cel5A, from B. cinerea strain B05.10, had no effect on pathogenicity of B. cinerea.46 In this study, compared with that at pH 4, the 4 common spots (U8−11) matched to exocellulase were up-accumulated for 2.4, 2.6, 2.9, 2.8 folds, respectively (Figure 2D). In addition, 5 specifically accumulated spots also show high abundance at pH 6 compared with other spots in the gels (Figures 1 and 2). These results provided evidence that accumulation of exocellulase was dramatically induced at pH 6. So far, little information is available about the relationship between the exocellulase and pathogenicity in B. cinerea.

Changes in mRNA Expression of Related Genes at pH 4 and 6

It is well-known that extracellular proteins related to carbohydrate metabolism or proteolysis are important for pathogenicity and life cycle of plant pathogens. Here, 15 proteins of the all of 21 identified proteins were predicted as secreted and related to carbohydrate metabolism or proteolysis. A time-coursed gene expression analysis of these proteins was performed by qRT-PCR. BcpacC, a widely reported upregulated gene in fungi under neural or alkaline condition, was used as a positive control. As expected, gene expression of BcpacC showed a higher level at pH 6 than that at pH 4 at each sampling time point, in particular at 72 hpi (Figure 3). Notably, BC1G_03070 (encoding aspartic protease BcAP8) and BC1G_06035 (encoding exocellulase) presented the highest relative expressions among all the tested genes at the first 12 h under pH 4 or 6 condition, respectively. Further, the expression pattern of 16 tested genes was clustered using PermutMatrix software version 1.9.3 (Pearson’s algorithm) on the basis of their relative expression at different pH treatments. There were two main clusters of genes with different expression patterns (Figure 4). Cluster I included 10 genes that generally showed a higher expression at pH 6, and 7 of them were corresponded to the up or specifically accumulated proteins at pH 6, except BcpacC (control), BC1G_04994, and BC1G_09180. Six genes in cluster II presented a higher expression at pH 4, and all the genes were corresponded to the up or specifically accumulated proteins at pH 4, except BC1G_06836. These results demonstrate the expression of differential proteins under different ambient pH values was mainly regulated at transcription level. Transcription factor plays a very important role when organisms respond to the change of environmental conditions. In A. nidulans, transcription factor PacC can activate neural-toalkaline expressed genes, including itself, after the fungus apperceives the changes of ambient pH from acidic to neural or 4257



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PR proteins for defense, so inducing secretion of proteins with the function of proteolysis is more important than that of CWDEs; in contrast, for leaves and stem, which have higher tissue pH and stiffer cell walls, CWDEs are more urgently required, as all pathogenicity factors should be secreted where they are expected to be most needed in B. cinerea.22 Our results provided some new evidence that can explain why B. cinerea causes gray mold rot in a wide range of plant hosts, which is beneficial not only for understanding the complicated infecting mechanisms, but also for developing new targets for fungicides.

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ASSOCIATED CONTENT

* Supporting Information S

Table S1 shows primer sets used for quantitative real-time PCR. Table S2 shows MS/MS peptide sequence of the identified proteins. Figure S1 shows dark pellets secreted by B. cinerea under pH 4 and 6 conditions. Figure S2 shows annotated spectra for identifications based on assignment of less than two peptides with ion score above the threshold. Figure S3 shows manual correction of initiation codons of BC1G_03991. Figure S4 shows changes in aspartic protease activity of B. cinerea culture medium under pH 4 and 6 conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 4. Hierarchical clustering analysis of changes in expression of genes encoding predicted secreted proteins related to carbohydrate metabolism and proteolysis under pH 4 and 6 conditions. Genes corresponding to those in Figure 3 were clustered according to their relative expression using the Pearson clustering algorithm.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-10-62836559. Fax: +86-10-82594675. E-mail: tsp@ ibcas.ac.cn. Notes

alkaline.17 Similar regulating mechanism has been reported in C. gloeosporioides,18 F. oxysporum,19 S. sclerotiorum,20 A. alternata.21 Differently, the regulating mechanism and involved transcription factors under acid condition are still unknown, though a few specific acid-expressed genes, such as Bcap8,7 Bcpg35 in B. cinerea, and pepg1 in P. expansum,55,56 have been reported. In this study, we found that 9 and 6 genes encoding secreted proteins in B. cinerea were up-regulated under pH 6 or 4 condition, respectively. These results will be helpful to elucidate the regulating mechanisms of secreted proteins in B. cinerea under different ambient pH, especially under acidic condition.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Tudzynski for supplying Botrytis cinerea strain B05.10 and Dr. Tongfei Lai for his help in analysis of 2D gels. This study was supported by National Natural Science Foundation of China Grant (31030051, 31171765), by National High Technology Research (863) Program of China (2012AA101607), and by the Chinese Academy of Sciences (KSCX2-EW-G-6).

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CONCLUSIONS In the present study, we studied the effect of ambient pH on secretome of B. cinerea strain B05.10 with a comparative proteomic method based on 2-DE. An improved protein isolation method was used in the study, which resulted in a better separating performance of protein spots in the gels. We ascertained that secretome of B. cinerea had distinct differences between pH 4 and 6 treatments, and 47 differential spots (corresponding to 21 unique proteins) were identified by the technique of MALDI-TOF/TOF. Most of these proteins are CWDEs or proteases, which is consistent with previous studies on secretome of B. cinerea,3,12−15 and indicates the importance of the two categories of secreted proteins for B. cinerea. Results from analysis of gene expression indicate that expressions of differential proteins under different ambient pH values were mainly regulated at transcription level. At pH 4, more proteins related to proteolysis were induced, whereas more CWDEs were induced at pH 6. Ripe fruits generally have lower tissue pH and weakened cell walls and accumulate a large number of

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Exploring Pathogenic Mechanisms of Botrytis cinerea Secretome

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