Current Genetics

, Volume 48, Issue 6, pp 366–379 | Cite as

Diversity of the exoproteome of Fusarium graminearum grown on plant cell wall

  • Vincent Phalip
  • François Delalande
  • Christine Carapito
  • Florence Goubet
  • Didier Hatsch
  • Emmanuelle Leize-Wagner
  • Paul Dupree
  • Alain Van Dorsselaer
  • Jean-Marc Jeltsch
Research Article

Abstract

The exoproteome of the fungus Fusarium graminearum grown on glucose and on hop (Humulus lupulus, L.) cell wall has been investigated. The culture medium was found to contain a higher quantity of proteins and the proteins are more diverse when the fungus is grown on cell wall. Using both 1D and 2D electrophoresis followed by mass spectrometry analysis and protein identification based on similarity searches, 84 unique proteins were identified in the cell wall-grown fungal exoproteome. Many are putatively implicated in carbohydrate metabolism, mainly in cell wall polysaccharide degradation. The predicted carbohydrate-active enzymes fell into 24 different enzymes classes, and up to eight different proteins within a same class are secreted. This indicates that fungal metabolism becomes oriented towards synthesis and secretion of a whole arsenal of enzymes able to digest almost the complete plant cell wall. Cellobiohydrolase is one of the only four proteins found both after growth on glucose and on plant cell wall and we propose that this enzyme could act as a sensor of the extracellular environment. Extensive knowledge of this very diverse F. graminearum exoproteome is an important step towards the full understanding of Fusarium/plants interactions.

Keywords

Fungi Fusarium graminearum Hydrolases Plant cell wall Polysaccharide degradation CWDE regulation 

Introduction

Filamentous fungi constitute a very large community of organisms (Hawksworth 2001). This kingdom seems to be largely understudied although its importance is considerable in many ecosystems. One of the roles of filamentous fungi on earth is nutriment recycling through vegetal tissue degradation (Evans and Hedger 2001). On one hand, this activity could be regarded as beneficial, since many filamentous fungi are saprophytes and decay biological materials. But, on the other hand the same activity is deleterious since many filamentous fungi are pathogens mostly for plant, causing huge losses in agriculture all over the world (Agrios 1997). The raw plant material is composed of diverse polysaccharides and is insoluble. In any cases, and in order to fulfil its carbon, nitrogen, sulphur and phosphorus requirements, fungi have to secrete many enzymes from several families. Polysaccharides, proteins and lignin are degraded and the resulting oligomers or even monomers can then be internalized and used to satisfy basal metabolic requirements.

The complete genome of the saprophyte Neurospora crassa has been released (Galagan et al. 2003) and these crude data re-investigated by classification of the predicted proteins (Borkovich et al. 2004). However, clues for fungal reaction to the environment are better deciphered by global analysis of transcriptomes. The expression of the biomass-degrading enzymes was analysed for Trichoderma reesei (Foreman et al. 2003). Many of the corresponding genes, but also other genes with unknown function were shown to be regulated by cellulose. Post-transcriptional mechanisms and protein degradation may, however, result in a lack of correspondence between mRNA and protein abundances. Hence proteomic studies emerge as the most powerful tool to study the protein expression pattern in a defined condition. Nevertheless, a few studies have been performed to characterize fungal extracellular proteins. One of these, based on an exoproteome approach, was helpful for the flavonoid rutin metabolism degradation system elucidation in Aspergillus flavus (Medina et al. 2004).

Fusarium graminearum is a devastating pathogen of wheat, maize and other cereals. Direct (deficit in production) and indirect (for instance as consequences on employment) economic losses in years 1998–2000 was estimated at $2.7 billion in the United States (Fusarium Focus, Fall 2001, US Wheat and Barley Scab Initiative). In May 2003, more than 90% of the genome sequence of this fungus was released (F. graminearum Sequencing Project, Whitehead Institute/MIT Center for Genome Research). The sequencing project was initiated and funded because of the huge losses provoked in the cereals market, but the impact of F. graminearum towards plants other than cereals is probably underestimated and understudied. This fungus is indeed a potent pathogen for several dicotyledones like Arabidopsis, tobacco, tomato and soybean (Urban et al. 2002). This sheds a new light on the host spectrum of this fungus. In our laboratory, F. graminearum was repeatedly isolated over years from hop (Humulus lupulus) plants in decay. The present study provides for the first time a global insight of the F. graminearum exoproteome after growth on plant cell wall. Compared to the low quantity and poor diversity of the proteins secreted after growth on glucose, this exoproteome displays a great richness in very diverse cell wall degrading enzymes and reveals the probable high fungus reactivity towards environmental variations. This study is a prerequisite and constitutes an important step for the elucidation of F. graminearum/dicotyledonous plant interactions.

Materials and methods

Specialist reagents

Citrus polygalacturonic acid, birchwood xylan, arabinose, xylose, endo-polygalacturonase (endo-PG; EC 3.2.1.15) from Aspergillus japonicus and pectin with 30% of esterification were purchased from Sigma (UK). Oligoarabinans (Degrees of Polymerization; DP 2–4 and 8), oligoxylans (DP 2–6), konjac glucomannan, ivory nut β-1,4-mannan and lupin β-1,4-galactan were obtained from Megazyme (Ireland).

K. Johansen (Novozymes, Denmark) and H. Gilbert (University of Newcastle, UK) kindly provided us with xyloglucan-specific endoglucanase (Aspergillus aculeatus) and α-1,5-arabinanase (Pseudonomas fluorescens), β-1,4-galactanase (P. fluorescens), xylanase (P. fluorescens) and β-1,4-mannanase (Man5A; Cellvibrio japonicus), respectively. All enzymes used in this analysis were checked for the purity and/or specificity using polysaccharide analysis using carbohydrate gel electrophoresis (PACE) method as described (Goubet et al. 2002). The fluorescent probes 2-aminoacridone (AMAC) and 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) were purchased from Molecular Probes (The Netherlands). Acrylamide for PACE gels (40%; 29v acrylamide/1v bisacrylamide) was obtained from Severn Biotech Ltd (UK).

Fungal strain, media and culture condition

Fusarium graminearum (Gibberella zeae) was isolated from hop plants in decay and maintained as frozen macroconidia in the laboratory. Plant cell wall (from hop collected after flowers harvest) was prepared essentially as described for maize (Sposato et al. 1995). The fungus (103 macroconidia) was inoculated and grown in M3 minimal medium (Mitchell et al. 1997) with either 1% (w/v) glucose or 0.8% (w/v) complete hop cell wall preparation as the sole carbon source. Cultures were performed into 75 cm2 Falcon flasks filled with 100 ml in static conditions at 25°C.

Protein preparation

After 6 and 9 days of fungal growth on M3 glucose or M3 cell wall, respectively, culture was collected. Culture supernatants were obtained by centrifugation at 3,000g for 15 min at 4°C and then concentrated by using Vivacell 70 concentrator, molecular weight cutoff 10 kDa (Vivascience, Germany). Concentrated supernatants were washed with 10 volumes of 50 mM phosphate buffer, pH 6.0. Protein concentration was determined by using BioRad Protein assay reagent with bovine serum albumin as standard (Bradford 1976). The required protein amounts were cleaned-up immediately before gel loading (ReadyPrepTM 2-D Cleanup Kit, BioRad, CA, USA).

1D SDS-PAGE and western blot analyses

SDS-PAGE were performed in triplicate with 12% (w/v) acrylamide/bisacrylamide. For comparison of cell wall and glucose exoproteomes and for cellobiohydrolase content analysis, gels were loaded with the equivalent of 6.5 ml of culture supernatants. For protein identification, the volume necessary to obtain 500 μg proteins in cell wall supernatant was loaded (corresponding to 20 ml of supernatant). For accurate comparison, the same volume was loaded for glucose supernatant also. The gels were then stained by Coomassie Brilliant Blue R-250 and slices excised from the gels. For western blot analysis, proteins were transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA) and polyclonal antibodies directed against F. graminearum cellobiohydrolase were used (Hatsch et al. 2002).

2D PAGE

For 2D PAGE (in triplicate), 600 μg of proteins were dissolved in 400 μl of rehydration buffer [8 M urea, 50 mM DTT, 2% (w/v) CHAPS, 0.2% (v/v) 100x Bio-Lyte 3/10 ampholites and 0.002% (w/v) Bromophenol Blue]. For the first dimension, 17 cm pH3-10 non-linear (NL) immobilized pH gradient (IPG) strips (BioRad) hydrated with the protein solutions were submitted to electrofocusing. IPG strips were then treated according to the manufacturer and proteins were separated onto 12% (w/v) acrylamide gels in a Bio-Rad Protean II XL device. Gels were stained with colloidal Coomassie Brilliant Blue. Protein spots were visualized and excised with a Proteineer SP automated spot picker (Bruker Daltonics, Germany) according to the manufacturer’s instructions.

Mass spectrometry

In-gel protein digestion was performed with an automated protein digestion system (MassPREP Station, Waters, MA, USA) as described (Richert et al. 2004; Ventelon-Debout et al. 2004). After the overnight digestion, a double extraction was performed: firstly with 60% (v/v) acetonitrile in 5% (v/v) formic acid and then with 100% acetonitrile. The resulting peptide extracts were directly analysed by nanoLC–MS/MS either on a CapLC capillary LC system (Waters) coupled to a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer Q-TOF II (Waters) or on an Agilent 1100 Series capillary LC system coupled to a HCT Plus ion trap (Bruker Daltonics). Both instruments were equipped with a nanospray ion source and the chromatographic separations were conducted on reverse-phase (RP) capillary columns (C18, 75 μm i.d., 15 cm length, LC Packings or Agilent Technologies) with a 200 nl/min flow rate. For the CapLC-Q-Tof II system, mass data acquisitions were piloted by MassLynx software (Waters) using automatic switching between MS and MS/MS modes. We empirically derived energy curves according to the m/z value of the selected precursor ion to improve the MS/MS spectra quality. For the Agilent 1100 capillary LC/HCT Plus system, the voltage applied to the capillary cap was optimized to −2000 V. The MS/MS scanning mode was performed in the Ultra Scan resolution mode at a scan rate of 26.000 m/z per second. A total of eight scans were averaged to obtain an MS/MS mass spectrum. The complete system was fully controlled by Agilent ChemStation and EsquireControl (Bruker Daltonics) software.

Data interpretation and protein identification

A F. graminearum translated ORFs (http://biotec.u-strasbg.fr/) and public databases (NCBI, http://www.ncbi.nlm.nih.gov/ and FGBD, http://mips.gsf.de/genre/proj/fusarium) databases were used to perform the similarity searches via a local Mascot (MatrixScience, UK) server. Searches were performed with a mass tolerance of 100 ppm in MS mode and 0.25 Da in MS/MS mode. One missed cleavage per peptide was allowed and variable modifications were taken into account such as carbamidomethylation of cysteine and oxidation of methionine. Searches were performed without constraining protein molecular weight or isoelectric point and without any taxonomic restriction. Only highly significant matches were considered (Table S1). The hits with a minimum of five matching peptides or 10% of sequence coverage were unambiguously accepted. For the hits presenting less matching peptides or less coverage, the peptides sequences were manually verified by de novo sequencing and submitted to individual BLAST searches.

Functional attributions of the predicted proteins were carried out by several successive steps. First, all proteins were subjected to BlastP analysis (Altschul et al. 1997). For some proteins, the nearest homologues suggested possible functions. However, for many of them, the nearest homologues corresponded to in silico translations of fungal genome ORFs, for whom no functions were assigned. Therefore, for each protein, any homologue was identified in N. crassa (http://mips.gsf.de); Ustilago maydis and Magnaporthe grisea (http://www.broad.mit.edu). All the proteins and their homologues were analysed by BlastP and their functions predicted by the FunCat program (http://mips.gsf.de/projects/funcat). Each protein and each homologue with supposed activity on carbohydrates was searched and analysed through the carbohydrate-active enzymes classification (Coutinho and Henrissat 1999). Finally, each protein identification was individually checked.

Analysis of hop cell wall polysaccharides

ß-Glucosyl Yariv reagent (1,3,5-tris[4-ß-d-glucopyranosyloxyphenylazo]-2,4,6-trihydroxybenzene) was used to precipitate arabinogalactan protein (AGP) (Gao et al. 1999). The cell wall (8 mg except for endo-PG analysis—0.8 mg) was treated with different enzymes. Xyloglucan-specific endoglucanase and endo-PG treatments were performed in 0.1 M ammonium acetate at pH 5. Arabinanase, galactanase, xylanase and Man5A hydrolyses were performed in 0.1 M ammonium acetate at pH6. Before treatment with Man5A and xylanase, the cell wall was pre-treated with ammonium solution (Handford et al. 2003) or 4 M NaOH (Fry 2000) for 30 min, respectively. The former samples were then dried whereas the latter were neutralized with HCl solution to pH∼6. For endo-PG treatments, two conditions were performed, directly or with an NaOH pre-treatment to de-esterify all homogalacturonan (Goubet et al. 2003). Prior to PACE analysis, enzyme-produced oligosaccharides and standard oligosaccharides and monosaccharides (20 μmol) were vacuum air-dried. Derivatization using the ANTS or AMAC fluorophores and polyacrylamide gel electrophoresis were carried out as previously described (Goubet et al. 2002, 2003).

Results

Growth on hop cell wall induces high levels of protein secretion

Fusarium graminearum was grown either on glucose or on hop cell wall in order to compare the secreted proteins in each growth condition. Pilot experiments indicated that the fungus has reached its maximum of colonization possibilities after 6 days on glucose and after 9 days on cell wall. The proteins in the same volume of supernatants were collected and separated by SDS-PAGE (Fig. 1). The glucose supernatant contained proteins in much lower concentration (∼1 μg/ml) than the supernatant of fungus grown on cell wall (∼25 μg/ml).
Fig. 1

Exoproteome of F. graminearum grown on M3 glucose (1) and M3 hop cell wall (2). The equivalent of the same volume of supernatants (6.5 ml) was loaded onto 12% (w/v) polyacrylamide gels, the proteins were separated and stained with Coomassie blue. Molecular weight of marker proteins is indicated on the left

Identification of polysaccharides present in the hop cell wall

The induction of protein secretion by growth on hop cell wall suggested that the fungus secretes proteins for degradation of the cell wall. Dicotyledonous plants have a cell wall that contains high proportions of pectin: the most abundant is homogalacturonan (HGA) which can be esterified, rhamnogalacturonan-I (RG-I) and RG-II, cellulose and the hemicelluloses (AGP, xylan, xyloglucan and mannan). Hop cell wall has, however, been very little characterized. To determine if it contains the polysaccharide components usually found in a dicotyledonous cell wall, it was studied by PACE. Polysaccharides that are more accessible to hydrolases present in the F. graminearum exoproteome are likely to be present in a solubilized fraction. The complete hop cell wall medium containing polysaccharides solubilized by autoclaving and the supernatant without fibres were treated with endo-PG to reveal the presence of HGA (Fig. 2). As pectins are water soluble, HGA was detected at a high concentration in the solubilized fraction (supernatant), but was more abundant in the complete cell wall sample. Interestingly, most of the HGA was non-esterified. Some partially esterified oligogalacturonides were released from the HGA in both samples, but in very low amounts. Both β-1,4-galactan and α-1,5-arabinan, probably side chains of RG-I, were present (Fig. 3a), and mostly in the complete cell wall sample suggesting that the RG-I was less solubilized than HGA. Using Yariv precipitation, AGP was found in both complete and solubilized fractions (data not shown). We were unable to detect xyloglucan using xyloglucanase. This could indicate an absence of xyloglucan, or that xyloglucan is highly substituted, possibly preventing the access of the xyloglucanase to the xyloglucan backbone. Xylan, which is present in secondary cell wall, was detected in the complete cell wall preparation (Fig. 3b). A low level of xylan was also present in the solubilized fraction. As expected in dicotyledonous plants (Fry 2000), the xylan structure was glucuronoxylan. Glucomannan, which is often a minor component, was detected as such in both fractions (data not shown). So the hop cell wall contains all the polysaccharides normally observed in dicotyledonous plants (Fry 2000).
Fig. 2

Homogalacturonan content of hop cell wall used to grow F. graminearum. The hop cell wall (complete cell wall) was autoclaved and centrifugated to separate the solubilized fraction from the unsolubilized fraction containing the microfibrils of cellulose associated with hemicellulose. Oligosaccharides released after polygalacturonase digestion from the equivalent of 8 μg of cell wall were loaded on the gel. The analysis was performed using polysaccharide analysis using carbohydrate gel electrophoresis (PACE) as described in the Material and methods. Markers are indicated

Fig. 3

Galactan, arabinan and xylan content of hop cell wall used to grow F. graminearum. a β-1,4-galactan (digested by β-1,4-galactanase) and α-1,5-arabinan (α-1,5-arabinanase) detection and b β-1,4-glucuronoxylan (β-1,4-xylanase) detection. The analysis was performed using polysaccharide analysis using carbohydrate gel electrophoresis (PACE) as described in the Material and methods. Asterisks unspecific band from a compound present in the cell wall sample. Oligosaccharides released from the equivalent of 200 μg of cell wall (prepared as described in Fig. 2.) were loaded on the gel

Functional classification and identification of the secreted proteins after growth on glucose and plant cell wall

To investigate the exoproteome of F. graminearum grown on glucose medium, the proteins from 20 ml of the culture supernatant were first separated by SDS-PAGE. Proteins in gel slices were identified by mass spectroscopy. Twenty-three unique proteins were identified (Table 1). Several are implicated in fungal cell wall biogenesis or morphogenesis (Fig. S1). Two glucanosyltransferases, homologues of the Saccharomyces cerevisiae Gas1p, were identified (FG02022 and FG09980). Some proteins, such as the two cellulases FG00571 and FG05851, are probable polysaccharide degrading proteins (Table S2). Several other cell wall-related proteins and some unknown proteins were also found (Table 1).
Table 1

Characterization of the F. graminearum proteins secreted during growth on glucose

Fusarium graminarum protein

Molecular mass (Da)a

pIa

Predicted function

Predicted EC numberb

Predicted CAZy classc

GPI-modification (position)d

Nearest informative homologue given by BlastP

Accession [organism]

E value

FG00415

29124

5.51

Unknown

  

Yes (255)

  

FG00571

54653

6.00

Cellulose 1,4-β-cellobiosidase

3.2.1.91

GH7

 

EAA69232.1 [Gibberella zeae]

0.0

FG02022

58627

4.77

β-1,3-glucanosyltransferase

2.4.1.-

GH72

Yes (518)

AAP91684.1 [Paracoccidioides brasiliensis]

e-126

FG02560

14571

5.43

Unknown

     

FG02720

21381

4.54

Endo-1,3(4)-β-glucanase

3.2.1.6

GH16

Yes (455)

AAG53947.1 [Bacillus circulans]

8e-14

FG03017

41375

4.38

Endo-1,4-β-xylanase

3.2.1.8

GH16

Yes (363)

AAM63080.1 [Arabidopsis thaliana]

1e-14

FG03628

49029

5.65

Endo-1,4-β-cellulase

3.2.1.4

GH6

 

EAA73192.1 [Gibberella zeae]

0.0

FG03662

21113

9.01

Cell wall protein

   

CAA09585.1 [Emericella nidulans]

3e-25

FG03875

70548

4.73

Lysophospholipase

3.1.1.5

  

CAE76554.1 [Neurospora crassa]

0.0

FG04074

20535

7.57

Cell wall protein

   

CAA09585.1 [Emericella nidulans]

3e-22

FG04213

20591

8.69

Unknown

     

FG04735

16128

8.88

Unknown

     

FG04741

15045

6.71

Unknown

     

FG05757

29036

4.73

Cell wall protein

 

GH16

 

AAQ14297.1 [Gibberella acuminata]

3e-84

FG05851

26894

9.27

Endo-1,4-β-cellulase

3.2.1.4

GH12

 

AAM77705.1 [Nectria ipomoeae]

1e-89

FG06130

26135

8.74

Unknown

     

FG06278

62034

5.42

Glucan 1,4-α-glucosidase

3.2.1.3

GH15

 

XP_327956.1 [Neurospora crassa]

0.0

FG08122

20782

9.20

Cell wall protein

   

CAA09585.1 [Emericella nidulans]

9e-27

FG09366

47616

4.63

Glucan 1,3-β-glucosidase

3.2.1.58

GH17

Yes (430)

AAO34674.1 [Gibberella zeae]

2e-28

FG09980

48580

4.95

Glucanosyltransferase

2.4.1.-

GH72

Yes (422)

AAC35942.1 [Aspergillus fumigatus]

e-119

FG10089

42301

4.66

Cell wall biogenesis protein

  

Yes (373)

T38309 [Schizosaccharomyces pombe]

1e-24

FG10212

14489

9.10

SnodProt

   

XP_328493.1 [Neurospora crassa]

1e-36

FG10316

25670

8.26

Unknown

     

a Molecular masses and pI have been calculated for predicted precursor proteins

bhttp://www.expasy.org/enzyme/

chttp://afmb.cnrs-mrs.fr/CAZY/;GH glycoside hydrolase family

dhttp://mendel.imp.univie.ac.at/gpi/fungi_server.html; GPI fungal prediction server

To characterize the secreted proteins of F. graminearum grown in hop cell wall-containing medium, the proteins from 20 ml of the culture supernatant were separated by SDS-PAGE. In contrast to the sample for glucose containing medium, the protein mixture was very complex (Fig. 1). Consequently, proteins were also separated by 2D gels. Proteins in excised plugs (192 spots: from 1 to 96 and 101 to 196, Fig. 4) and gel slices were analysed by MS. Eighty-four proteins were recovered (Table 2).
Fig. 4

2D PAGE mapping of the secreted F. graminearum proteins. The fungus was grown on hop cell wall and 20 ml of the supernatant collected was concentrated. Proteins were separated by using a pH 3–10NL strip followed by 12% SDS-PAGE. The gel was stained by Coomassie blue. Excised protein spots are circled and the numbers (1–196) correspond to the spots list (Table 2)

Table 2

Characterization of the F. graminearum proteins secreted during growth on hop cell wall preparation

Fusarium graminarum protein

Spot number (Fig. 4)

Molecular mass (kDa)a

pIa

Predicted function

Predicted EC numberb

Predicted CAZy classc

Nearest informative homologue given by BlastP

Accession [organism]

E value

FG00571

43–48/multipled

54653

6.00

Cellulose 1,4-β-cellobiosidase

3.2.1.91

GH7

EAA69232.1 [Gibberella zeae]

0.0

FG00806

126/127

45100

8.36

Serine protease

3.4.21.-

 

AAL75578.1 [Tolypocladium inflatum]

e-130

FG01283

16–17/multiple

75708

5.49

Chitinase

3.2.1.14

GH20

AAB47060.1 [Trichoderma harzianum]

0.0

FG01603

NAe

63074

5.38

Lipase

3.2.1.3

 

AAU87359.1 [Botryotinia fuckeliana]

0.0

FG01671f

166

33827

9.28

Protein phosphatase

  

NP_171993.1 [Arabidopsis thaliana]

9e-26

FG01748

NA

48012

6.92

Xylan 1,4-β-xylosidase

3.2.1.37

GH39

NP_149280.1 [Clostridium acetobutylicum]

1e-12

FG01818

179

39645

6.01

Leucine aminopeptidase

3.4.11.10

 

AAN31395.1 [Aspergillus sojae]

e-113

FG02059

143/145

44719

8.33

Alpha-galactosidase

3.2.1.22

GH27

CAB46229.1 [Aspergillus niger]

1e-74

FG02202

NA

34273

5.56

Endo-1,4-β-cellulase

3.2.1.4

GH61

CAA71999.1 [Hypocrea jecorina]

1e-68

FG02204

13/14/15g

59619

5.16

Serine peptidase

3.4.14.2

 

NP_922290.1 [Oryza sativa]

5e-24

FG02356

121/167/194

86540

5.71

Aldehyde dehydrogenase

1.2.1.3

 

ZP_00094192.1 [Novosphingobium aromaticivorans]

6e-93

FG02977

128/171

25010

8.21

Pectate lyase

4.2.2.2

GH8

AAC64368.1 [Fusarium oxysporum]

e-126

FG03002

120

33966

7.72

Endo-1,5-α-arabinanase

3.2.1.99

GH43

NP_626671.1 [Streptomyces coelicolor]

2e-85

FG03003

149

36906

8.48

Xylan 1,4-β-xylosidase

3.2.1.37

GH43

NP_827557.1 [Streptomyces avermitilis]

3e-71

FG03131

147

42889

4.80

Pectate lyase

4.2.2.2

GH8

NP_827558.1 [Streptomyces avermitilis]

2e-62

FG03143

66

44231

4.80

Transferase?h

2.5.1.31

GH88

NP_346999.1 [Bacilus subtilis]

6e-64

FG03406

NA

34531

9.10

Pectin esterase

3.1.1.11

GH8

AAD51853.1 [Vitis riparia]

4e-99

FG03467

177g

68643

5.52

Elastinolytic metalloprotease

3.4.24.-

 

CAA83015.1 [Aspergillus fumigatus]

0.0

FG03530

NA

25888

9.39

Rhamnogalacturonan acetylesterase

3.1.1.-

CE12

BAC66097.1 [Gibberella zeae]

e-130

FG03591

144/187

45136

5.35

Chitinase

3.2.1.14

GH18

JQ1975 [Aphanocladium album]

e-158

FG03598

114

42270

8.58

Alpha-N-arabinofuranosidase

3.2.1.55

GH93

BAC76689.1 [Penicillium chrysogenum]

2e-97

FG03609

NA

58446

8.70

Xylan 1,4-β-xylosidase

3.2.1.37

GH43

ZP_00310780.1 [Cytophaga hutchinsonii]

3e-64

FG03624

138/139/182

24529

9.16

Endo-1,4-β-xylanase

3.2.1.8

GH11

CAB52417.1 [Setosphaeria turcica]

4e-73

FG03628

49/51/multiple

49029

5.65

Endo-1,4-β-cellulase

3.2.1.4

GH6

EAA73192.1 [Gibberella zeae]

0.0

FG03695

131/132/multiple

25450

7.12

Endo-1,4-β-cellulase

3.2.1.4

GH61

CAF31975.1 [Aspergillus fumigatus]

1e-57

FG03795

69–72/multiple

41892

5.65

Endo-1,4-β-cellulase

3.2.1.4

GH5

CAE81955.1 [Neurospora crassa]

e-142

FG03813

91–93/188

51946

6.14

Alpha-N-arabinofuranosidase

3.2.1.55

GH54

CAD90582.2 [Fusarium oxysporum]

0.0

FG03842

50/52/multiple

50752

5.48

Alpha-amylase

3.2.1.1

GH13

JN0588 [Aspergillus oryzae]

1e-99

FG03865

55

45420

5.20

l-sorbosone dehydrogenase

  

AAO55227.1 [Pseudomonas syringae]

7e-21

FG03883

NA

120556

4.97

Unknown

    

FG03905

112/178/multiple

39692

7.05

Endo-1,4-β-xylanase

3.2.1.8

GH43

NP_827557.1 [Streptomyces avermitilis]

4e-91

FG03908

114/119

34324

8.62

Pectate lyase

4.2.2.2

GH8

EAA65383.1 [Aspergillus nidulans]

e-131

FG03909

62/192

33949

4.99

Pectate lyase

4.2.2.2

GH8

AAA87382.1 [Fusarium solani]

1e-39

FG04213

NA

20591

8.69

Unknown

    

FG04678

68

41169

5.18

Mannan endo-1,4-β-mannosidase

3.2.1.78

GH5

AAA67426.1 [Aspergillus aculeatus]

1e-85

FG04681

76

28820

5.88

Endo-1,4-β-cellulase

3.2.1.4

GH61

CAA71999.1 [Hypocrea jecorina]

6e-55

FG04738

11–12

81161

6.16

Ceramidase

  

NP_200706.1 [Arabidopsis thaliana]

e-118

FG04768

135g

31659

6.15

Glucan endo-1,3-β-glucosidase

3.2.1.39

GH16

AAC38290.1 [Oerskovia xanthineolytica]

8e-34

FG04848

NA

27446

9.08

Rhamnogalacturonan acetylesterase

3.1.1.-

CE12

CAA61858.1 [Aspergillus aculeatus]

5e-59

FG04936

64

52033

5.93

Aminopeptidase

3.4.11.-

 

NP_009845.1 [Saccharomyces cerevisiae]

1e-67

FG05906

123

37037

8.05

Lipase

3.1.1.3

 

AAQ23181.1 [Gibberella zeae]

0.0

FG06278

20–23/multiple

62034

5.42

Glucan 1,4-α-glucosidase

3.2.1.3

GH15

XP_327956.1 [Neurospora crassa]

0.0

FG06445

185

40463

8.90

Endo-1,4-β-xylanase

3.2.1.8

GH1

JC5861 [Humicola grisea]

1e-95

FG06452

172

27587

8.59

Chitin deacetylase

3.5.1.41

GH4

AAO34677.1 [Gibberella zeae]

2e-29

FG06466

75/77/154

25997

6.05

Cip1 (Hemicellulase?)

  

AAP57751.1 [Hypocrea jecorina]

3e-80

FG06545

NA

53608

8.63

Aminopeptidase

3.4.11.-

 

NP_251629.1 [Pseudomonas aeruginosa]

5e-58

FG06549

31/176g

50191

6.87

Chitin deacetylase

3.5.1.41

GH4

AAK84438.1 [Blumeria graminis]

3e-80

FG06605

8–10

96037

5.11

Beta-glucosidase

3.2.1.21

GH3

AAP57755.1 [Hypocrea jecorina]

0.0

FG06616

8–10g

86465

5.17

Glucan endo-1,3-β-glucosidase

3.2.1.39

GH55

AAF80600.1 [Trichoderma atroviride]

0.0

FG07551

27g

51505

8.31

Polygalacturonase

3.2.1.15

GH28

AAF12737.2 [Fusarium oxysporum]

0.0

FG07608

159g

29685

6.30

Acid phosphatase

3.1.3.2

 

CAA46331.1 [Yarrowia lipolytica]

7e-49

FG07625

141/173g

47747

6.53

Alpha-N-arabinofuranosidase

3.2.1.55

GH62

BAA85252.1 [Aspergillus sojae]

e-121

FG07694

Multipleg

100185

4.95

Xylan 1,4-β-xylosidase

3.2.1.37

GH43

ZP_00311746.1 [Clostridium thermocellum]

e-167

FG07695

157g

57304

8.91

Xylan 1,4-β-xylosidase

3.2.1.37

GH8

ZP_00117292.1 [Cytophaga hutchinsonii]

2e-66

FG07794

148/175

36761

6.66

Pectate lyase

4.2.2.2

 

AAD43564.1 [Colletotrichum gloeosporioides]

5e-47

FG07822f

73

31968

5.04

Serine protease

3.4.21.-

 

CAA44828.1 [Coccidioides posadasii]

0.001

FG07912

32g

82007

5.52

Endo-1,4-β-cellulase

3.2.1.4

GH61

CAA71999.1 [Hypocrea jecorin]

2e-73

FG08825

NA

28423

8.72

Dienelactone hydrolase

3.1.1.45

 

AAR23260.1 [Chaetomium globosum]

6e-30

FG08907

NA

35015

8.30

Unknown

    

FG09085

1–7/multiple

85846

6.65

Cellobiose dehydrogenase

1.1.3.25

CBM1

AAC26221.1 [Thielavia heterothallica]

0.0

FG09142

NA

36851

4.73

Cell polarity protein

  

NP_639451.1 [Xanthomonas campestris]

2e-32

FG10595

180

40660

6.02

Alkaline proteinase

  

BAA00951.1 [Aspergillus oryzae]

7e-80

FG10656

30

61708

5.36

Unknown

    

FG10782

NA

42652

4.80

Aspartyl proteinase

3.4.23.-

 

AAM81358.1 [Leptosphaeria maculans]

e-162

FG11036

184

29305

9.03

Feruloyl esterase

3.1.1.73

CE1

CAC85738.1 [Penicillium funiculosum]

6e-60

FG11037

133/137

26182

6.81

Endo-1,4-β-cellulase

3.2.1.4

GH12

AAM77703.1 [Fusarium equiseti]

2e-92

FG11048

NA

38268

9.00

Endo-1,4-β-arabinogalactosidase

3.2.1.89

GH53

CAE76342.1 [Neurospora crassa]

e-131

FG11049

173

32994

8.92

Acetylxylan esterase

3.1.1.72

CE1

BAD12626.1 [Aspergillus oryzae]

2e-93

FG11066

56–59/multiple

48529

5.11

Mannan endo-1,4-β-mannosidase

3.2.1.78

GH5

AAA67426.1 [Aspergillus aculeatus]

3e-65

FG11097

24/195g

96796

4.74

Glyoxal oxidase

1.1.3.9

 

CAE76318.1 [Neurospora crassa]

0.0

FG11163

116–118/174

33682

8.63

Pectate lyase

4.2.2.2

GH8

AAD43566.1 [Colletotrichum gloeosporioides]

e-123

FG11164

NA

24976

9.05

Trypsin

3.4.21.4

 

AAB27568.1 [Fusarium oxysporum]

3e-99

FG11169

18/19g

59928

5.17

Alpha-galactosidase

3.2.1.22

GH27

AAM45068.1 [Arabidopsis thaliana]

1e-69

FG11184

102/104/162

47439

6.35

Endo-1,4-β-cellulase

3.2.1.4

GH5

NP_189244.1 [Arabidopsis thaliana]

4e-38

FG11208

86–88

77891

6.05

Xyloglucanase

3.2.1.-

GH74

AAP57752.1 [Hypocrea jecorina]

0.0

FG11249

124/125

44269

7.01

Carboxypeptidase

3.4.17.1

GH29

AAB68600.1 [Metarhizium anisopliae]

e-102

FG11254

27g

67961

4.95

Alpha-l-fucosidase

3.2.1.51

 

NP_866909.1 [Rhodopirellula baltica]

9e-87

FG11255

103g

86930

5.63

Unknown

    

FG11258f

155

25228

5.55

Endo-1,4-β-xylanase

3.2.1.8

GH10

NP_869272.1 [Rhodopirellula baltica]

2e-38

FG11280

74

26579

5.30

Rhamnogalacturonan acetylesterase

3.1.1.-

CE12

AAO34675.1 [Gibberella zeae]

4e-61

FG11304

107–110/multiple

41301

6.71

Endo-1,4-β-xylanase

3.2.1.8

GH10

AAC06239.1 [Fusarium oxysporum]

e-144

FG11348

NA

41936

8.75

3-Carboxymuconate cyclase

  

ZP_00267186.1 [Pseudomonas fluorescens]

6e-18

FG11366

53/multiple

49144

5.46

Xylan 1,4-β-xylosidase

3.2.1.37

GH43

ZP_00312459.1 [Clostridium thermocellum]

6e-46

FG11428

126/multipleg

32028

8.63

Feruloyl esterase

3.1.1.73

CE1

CAC14144.1 [Penicillium funiculosum]

2e-74

a Molecular masses and pI have been calculated for predicted precursor proteins

bhttp://www.expasy.org/enzyme/

chttp://afmb.cnrs-mrs.fr/CAZY/, (GH glycoside hydrolase family; CE carbohydrate esterase family and CBM carbohydrate binding module)

d This means that the protein was found in many spots but predominantly in the mentioned ones

eNA Not applicable: protein found only in the 1D gel. Among the 17 proteins that were evidenced only in the 1D experiment, 13 have pI below 5.0 or above 8.4 (for the mature proteins)

f Protein which is predicted to be devoid of signal peptide for secretion

g Protein found only in these spots but not necessarily as the most abundant or proteins for whose physico-chemical parameters do not correspond well with prediction

h To be confirmed by experimental data

The majority of the proteins identified on cell wall are involved in cell wall polysaccharide degradation (45% of the total proteins, Fig. 5a). Fungal cell wall biogenesis proteins, proteases and some stress-related proteins are recovered (8, 9 and 5%, respectively). The carbohydrate-active enzymes were re-analysed to determine the nature of the polymer they could use as substrate. During the growth on plant cell wall, the fungus secretes many proteins likely to degrade cellulose, pectin and hemicellulose (11, 19 and 25 proteins, respectively, Fig. 5b and Table 3). In contrast, during growth on glucose, the fungus secretes much less carbohydrate-active enzymes (Fig. S1).
Fig. 5

Functional class analysis of the identified proteins from exoproteomes of F. graminearum grown on hop cell wall. a Each unique protein was first submitted to FunCat version 2.0 analysis (http://mips.gsf.de/proj/funcatDB). Unclassified proteins were then compared to the genome annotations available for other fungi. Finally, carbohydrate active enzymes were analysed through the CAZy classification (http://afmb.cnrs-mrs.fr/CAZY/). Percentage of each category is given into brackets. b Sub-classification of carbohydrate active enzymes secreted by F. graminearum. The nature of the polymer which could serve as substrate is given, followed by the number of protein concerned in brackets

Table 3

Classification of F. graminearum proteins active on polysaccharides recovered after growth on hop cell wall according to the enzyme nomenclature and references

EC number

Putative function

Fusarium graminearum protein

Reference

Miscellaneous

1.1.3.25

Cellobiose dehydrogenasea

FG09085

Bao and Renganathan (1992)

Enhances crystalline cellulose hydrolysis (Phanerochaete)

3.1.1.-

Rhamnogalacturonan acetylesteraseb

FG03530; FG04848; FG11280

  

3.1.1.11

Pectine esteraseb

FG03406

Valette-Collet et al. (2003)

Required for full virulence of Botrytis

3.1.1.72

Acetylxylane esterasec

FG11049

  

3.1.1.73

Feruloyl esteraseb, c

FG11036; FG11428

  

3.2.1.-

Xyloglucanasec

FG11208

Grishutin et al. (2004)

Exo-action on oligoxyloglucans

3.2.1.1

Alpha-amylased

FG03842

Skadsen and Hohn (2004)

Not found in F. graminearum during barley infection

3.2.1.3

Glucan 1,4-α-glucosidased

FG06278

Martel et al. (2002)

Secreted by Sclerotinia

3.2.1.4

Endo-1,4-β-cellulasea

FG02202; FG03628; FG03695; FG03795; FG04681; FG07912; FG11037; FG11184

Jahr et al. (2000)

Homologue of FG11184: implicated in pathogenesis in Clavibacter

3.2.1.8

Endo-1,4-β-xylanasec

FG03624; FG03905; FG06445; FG11258; FG11304

Apel et al. (1993)

Isolated form the pathogen Cochliobolus

3.2.1.14

Chitinase

FG01283; FG03591

  

3.2.1.15

Polygalacturonaseb

FG07551

Shieh et al. (1997), Ten Have et al. (1998), Isshiki et al. (2001), Oeser et al. (2002)

Required for: cotton invasion by Aspergillus full virulence of Botrytis, citrus infection by Alternaria, Claviceps pathology on rye

3.2.1.21

Beta-glucosidasea, c

FG06605

  

3.2.1.22

Alpha-galactosidasec

FG02059; FG11169

Civas et al. (1984)

Aspergillus enzyme produced on galactomannan

3.2.1.37

Xylan 1,4-β-xylosidasec

FG01748; FG03003; FG03609; FG07694; FG07695; FG11366

Wegener et al. (1999)

Secreted by Cochliobolus on maize cell walls

3.2.1.39

Glucan endo-1,3-β-glucosidasec

FG04768; FG06616

  

3.2.1.51

Alpha-l-fucosidaseb, c

FG11254

Tsuji et al. (1990)

Two forms in F. oxysporum

3.2.1.55

Alpha-N-arabinofuranosidaseb, c

FG03598; FG03813; FG07625

Chacon-Martinez et al. (2004)

Upregulated during infection by F. oxysporum (FG03813 homologue)

3.2.1.78

Mannan endo-1,4-β-mannosidasec

FG04678; FG11066

Civas et al. (1984)

Aspergillus enzyme produced on galactomannan

3.2.1.89

Arabinogalactane endo-1,4-β-galactosidaseb

FG11048

Kimura et al. (1998)

Aspergillus enzyme produced on beet pulp

3.2.1.91

Cellulose 1,4-β-cellobiosidasea

FG00571

Medve et al. (1994), Gusakov et al. (2005), Müller et al. (1997)

Two genes in Trichoderma Two forms in Chrysosporium Produced during infection

3.2.1.99

Endo-1,5-α-arabinanaseb,c

FG03002

Van de Veen et al. (1991)

Aspergillus enzyme produced on beet pulp

3.5.1.41

Chitin deacetylase

FG06452; FG06549

  

4.2.2.2

Pectate lyaseb

FG02977; FG03131; FG03908; FG03909; FG07794; FG11163

Rogers et al. (2000), Yakoby et al. (2001)

Required for infection by Nectria, virulence of Colletotrichum

aCellulose degradation

bPectin degradation

cHemicellulose degradation

dStarch degradation

Among the unique proteins secreted on cell wall, several cellulose degrading enzymes were present (Tables 2, 3), including eight endo-β-1,4 cellulases (EC 3.2.1.4), a cellobiohydrolase (β-1,4 cellobiosidase, FG00571), and a β-glucosidase (FG06605; EC 3.2.1.21). A cellobiose dehydrogenase (FG09085, EC 1.1.3.25) was also secreted. Additionally, two 1,3-β-glucosidase (EC 3.2.1.39) were found in this exoproteome.

Pectins are important polysaccharides in the hop cell wall. Pectin esterase (EC 3.1.1.11; FG03406) which deesterifies pectin is essential for pectin main chain degradation since it allows the subsequent action of polygalacturonases (EC 3.2.1.15, one is detected, FG07551, Table 3) and pectate lyases (EC 4.2.2.2, six detected) to split the pectin backbone. Another pectin molecule is rhamnogalacturonan-I (RG I). Degradation of this polymer requires rhamnogalacturonase that was apparently not found in this study. However, the exoproteome has three putative rhamnogalacturonan acetylesterases (EC 3.1.1.-) able to initiate RG I degradation. The hairy region of the RG I contains α-1,5-arabinan and β-1,4-galactan chains which could be substrates for two endo-acting enzymes FG03002 (EC 3.2.1.99) and FG11048 (EC 3.2.1.89), respectively.

Five endoxylanases (EC 3.2.1.8), crucial for xylan backbone depolymerization, were found (Table 3). β-Xylosidase, as other exo-acting enzymes (xylan acetylesterase, EC 3.1.1.72, FG11049) may facilitate the access for endo-acting enzymes. FG11208 was classified by similarities as a xyloglucanase (EC 3.2.1.-). Two putative endo-β-1,4-mannanases (EC 3.2.1.78) and two α-galactosidases (EC 3.2.1.22) detected in our study have the mannan layer as substrate.

Both arabinogalactan side chains of RG I and xylan could be substituted by ferulic acid residues which are substrates of ferulic acid esterase. Two representatives of such enzyme (EC 3.1.1.73) were found (Table 3). Alpha-l-fucosidase (EC 3.2.1.51; FG11254) liberates the l-fucose residue from non-reducing ends in various glycoproteins, glycolipids and oligosaccharides including pectin and xyloglucan. α-Arabinofuranosidase (EC 3.2.1.55, three representatives) also acts towards arabinoxylan and pectin to eliminate l-arabinofuranoside ends thus completing the potential endo-degradation of arabinan by EC 3.2.1.99 (FG03002).

Some enzymes secreted by F. graminearum could act on other polymers (Table 3). The fungus would possess the ability to hydrolyse starch in both endo- (FG03842, EC 3.2.1.1) and exo- (FG06278, EC 3.2.1.3) manner. F. graminearum secretes also two chitinases (EC 3.2.1.14) responsible for 1,4-β linkages breakdown and two chitin deacetylases (EC 3.5.1.41) which transform chitin present in fungal cell wall to the water soluble compound chitosan. Finally, other enzymes are not polysaccharide-active enzymes but their putative function is still very interesting: two lipases (EC 3.1.1.3), a carboxypeptidase (FG11249) and other proteases (FG00806 and FG10595, Table 2).

Notably, only four proteins secreted during growth on hop cell wall were recovered after growth on glucose also: the cellobiohydrolase, FG00571; a cellulase (FG03628); an α-glucosidase (FG06278) and FG04213 an unknown protein (Tables 1, 2).

Cellobiohydrolase is the major protein and appears as multiple forms

Cellobiohydrolase (FG00571) is identified in both culture conditions (glucose and cell wall). This protein is the major protein found in four strings of spots, each with different pIs and PMs (spots numbers 24/195; 13-15/19; 32-37/9/176 and 43-48/60/90, Fig. 4). The apparent PMs of some of these spots are higher than the mature cellobiohydrolase predicted one (54.6 kDa). This protein displays four potential acceptor sites for N-glycosylation and heterogeneity in actual glycosylation could explain differences. The fact that cellobiohydrolase peptides were actually recovered from 68 spots (data not shown) is in accordance with a high amount produced. To evaluate the difference between the cellobiohydrolase quantities secreted in the presence of glucose versus cell wall, western blot experiments using anti-cellobiohydrolase antibody were carried out. It is clear that cellobiohydrolase is expressed in much larger quantity in the presence of hop cell wall than for the glucose condition using the same volume of culture (Fig. 6). Furthermore, in accordance with 2D gels, it shows a multiform pattern.
Fig. 6

Cellobiohydrolase visualization and relative quantification in the exoproteome of the fungus grown on glucose (1) or hop cell wall (2). Concentrated supernatants (6.5 ml equivalent) were loaded onto 12% (w/v) polyacrylamide gels. Western analysis was performed using anti-cellobiohydrolase antibodies. Molecular weight of marker proteins is indicated on the left

Discussion

This study compares the exoproteome of F. graminearum when grown on glucose to that when grown on hop cell wall. We used high confidence validation criteria for protein identification (Table S1) and when the peptides coverage was low, peptides were verified by manual sequencing. Furthermore, both 1D and 2D gels were analysed, and many proteins were recovered from both experiments. Coupled with the fact that the sequence of all F. graminearum putative proteins is known, this approach led to unambiguous protein identifications. Although classification by similarity is only a first guess for the actual activity of a given protein, the great wealth of highly similar fungal proteins in the databases render the activity predictions highly likely. Only three proteins found in the exoproteome did not seem to have secretion signal peptides (FG01671, FG07822 and FG11258; using the Sigcleave program from the EMBOSS package). This indicates that almost all the putative proteins described here are actually secreted. As secreted proteins they are likely to be highly modified which could hamper their analysis using gel-based systems. Therefore, some secreted proteins were probably not identified.

Functional assignment of the differentially secreted proteins indicates clearly that Fusarium grown on cell wall produces an extraordinarily rich arsenal of glycosyl hydrolases, proteases and lipases in accordance with the fact that polysaccharides, proteins and lipids are carbon sources for the fungus. The hop cell wall composition was analysed and correlates well with the fungal proteome. Indeed, the presence of glucomannan (a substrate for mannanase and glucosidase), RG-I (β-1,4-galactanase, α-1,5-arabinanase, rhamnogalacturonan esterase and ferulic acid esterase), xylan (xylosidase, xylanase and ferulic acid esterase), AGP (arabinanase and galactosidase) and HGA (endo-PG, pectate lyases) was confirmed by PACE and the corresponding degrading enzymes were identified in this study. Xyloglucan was not detected by PACE probably because it could be highly substituted but a putative xyloglucanase was detected.

When F. graminearum was grown on medium containing plant cell wall, a massive production of secreted proteins was observed (Fig. 1). This exoproteome includes 84 proteins of which more than 40 are classified as polysaccharide degradation or carbohydrate catabolism enzymes (FunCat classification). It is clear that the F. graminearum plant cell wall exoproteome is targeted towards carbohydrate degradation, perhaps more than other microorganisms. In their review, Borkovich et al. (2004) suggested only 48 putative cell wall degrading enzymes (CWDE) are encoded in the N. crassa genome, and fewer than 5% of N. crassa putative proteins are involved in carbohydrate metabolism (Mannhaupt et al. 2003). In the phytopathogen bacterium Erwinia chrysanthemi exoproteome, 14 CWDE including as many as eight pectate lyases were found (Kazemi-Pour et al. 2004). Our study shows that in presence of plant cell wall, not only does the fungus secrete many more proteins but also that these build a much more diverse arsenal of carbohydrate-active enzymes (24 different EC numbers) than it does when glucose is the carbon source. Indirect evidence for secreted CWDE activity was earlier suggested by the observation that wheat cellulose, xylan and pectin layers are reduced in size after contact with F. graminearum (Wanjiru et al. 2002).

Fusarium grown on hop cell wall can secrete up to eight carbohydrate-active proteins with the same EC number. It may be that their enzymatic properties differ slightly so that the fungus has the maximum potential to degrade substrates even if the conditions vary (for example as a result of plant defence mechanisms). The redundancy could explain the large number of disruption experiments that have forced their authors to conclude an absence of a role of a given CWDE on the pathology process as reviewed by Di Pietro et al. (2003). For future studies it would be advisable to consider the disruption of regulatory proteins or of whole gene families rather than single or double disruptions of CWDE.

Trace amounts of starch are present in the cell wall preparation (data not shown). The identification of two starch degrading enzymes could be interpreted as a way to gain energy through plant starch mobilization. The fungus also produces proteases and lipases likely to disrupt the cell membrane to access this internal substrate. Fusarium-infected kernels of wheat show present partial cell wall digestion, protein matrix reduction and damage to starch granules (Jackowiak et al. 2005). Our study provides the evidence that the fungus secretes all the degrading enzymes needed to perform the whole process (CWDE, proteases and two kinds of starch degrading enzymes).

Only a few secreted enzymes were detected when the fungus is grown on glucose (Table 1). All of the seven exoproteome proteins predicted to be glycosylphosphatidylinositol (GPI) modified (according to Eisenhaber et al. 2004) were found in the glucose exoproteome. Four of these (FG02022, FG02720, FG03017 and FG09980) are involved in cell wall biogenesis and belong to GH families 16 or 72. Other proteins identified after growth on glucose are involved in cell morphogenesis or differentiation (FG04735 and FG10089). One quite surprising trait shown by this work was the secretion of enzymes responsible for polysaccharide degradation during growth on glucose (FG00571 and FG05851). One hypothesis is that these proteins play an essential role in fungal cell wall remodelling to support mycelia growth. Cellobiohydrolase (FG00571) is furthermore abundantly secreted and in multiple forms when the fungus is grown on hop cell wall. Thus, another hypothesis is that these enzymes act as extracellular guards. On glucose, the fungus constitutively secretes a few CWDE in small quantities. Early after contact with plant, these enzymes start to degrade cell wall, thus liberating oligosaccharides and disaccharides, especially cellobiose. These molecules may serve as signals, probably through sensor molecules, to indicate to the fungus the presence of plant cell wall materials. This model is quite similar to the behaviour of plants in the presence of fungi. Oligosaccharide elicitors, resulting from fungi enzyme activities and detected by plant receptors, are hence the start point of the plant defence strategy against pathogens (Bergey et al. 1996). This is in accordance with the two recently proposed models for CWDE regulation (De Vries and Visser 2001; Aro et al. 2005). In these models, the system is controlled by XlnR, a transcriptional activator of the xylanolytic system, which regulates xylanases but also other classes of CWDE. Our research on Fusarium genome shows that there is no XlnR homologue in this fungus (within the slight doubt linked to the small part of the genome not sequenced to date) in contrast to Neurospora (NCU06971) and Magnaporthe (MG01414). In Fusarium, the candidate elicitor could be the cellobiose released by the action of the cellulases secreted constitutively at low levels. The glucose/cellobiose ratio (which is also dependent on β-glucosidase activity) could direct the metabolism on catabolic repression or in contrast on massive CWDE secretion. This is consistent with the observation that such a regulation is not specific for a particular polysaccharide (Margolles-Clark et al. 1997; De Vries and Visser 2001). The regulation by cellobiose could likely affect production of other CWDEs as well.

Exoproteome analysis emerges as a powerful tool for studying interactions between a fungi and a host. Of course, biochemical studies have to be performed to validate the enzyme classification predictions. Furthermore, histological studies interpreted through the presence and the abundance of pertinent markers (cellobiohydrolase and others revealed in this study) could be interesting to test the regulation model. Transcription studies of the whole arsenal can now be designed more rationally by using microarrays and Q-RT-PCR. Gaining a complete insight into fungal behaviour when in contact with raw biological materials is crucial for our understanding of plant cell wall degradation and plant pathology.

Notes

Acknowledgements

This work was supported by the Cophoudal (Brumath, France) and by the French agency for agriculture development (ADAR). Didier Hatsch was funded by a Ph.D. fellowship from the Region Alsace. We thank the Bruker Daltonics society and the CNRS for Christine Carapito’s Ph.D. fellowship and Aventis for François Delalande’s post-doc fellowship. Anne Forster and Daniéle Thierse are greatly acknowledged for their technical competence. We are grateful to Prof. Pierre Oudet and Jan DeMey for helpful and interesting discussions and materials. The work performed at Cambridge was supported by grants from the BBSRC.

Supplementary material

294_2005_40_MOESM1_ESM.pdf (154 kb)
Supplementary material

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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Vincent Phalip
    • 1
    • 4
  • François Delalande
    • 2
  • Christine Carapito
    • 2
  • Florence Goubet
    • 3
  • Didier Hatsch
    • 1
  • Emmanuelle Leize-Wagner
    • 2
  • Paul Dupree
    • 3
  • Alain Van Dorsselaer
    • 2
  • Jean-Marc Jeltsch
    • 1
  1. 1.UMR 7175—Laboratoire de PhytopathologieUniversité Louis PasteurIllkirchFrance
  2. 2.UMR 7512—Science Analytique et Interactions Ioniques, Moléculaires et BiomoléculairesUniversité Louis PasteurStrasbourgFrance
  3. 3.Department of BiochemistryUniversity of CambridgeCambridgeUK
  4. 4.Ecole Supérieure de Biotechnologie de StrasbourgBoulevard Sébastien BrandtIllkirch-Graffenstaden CedexFrance

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