A method was developed for isolating and sequencing proteins present in the extracellular matrix (ECM) of germlings and hyphae of filamentous fungi. Surface proteins of the cereal pathogen Bipolaris sorokiniana were labelled with a membrane impermeable biotinylating agent and extracted using a glycine–HCl buffer. Extracted proteins were purified by affinity binding to streptavidin-conjugated magnetic beads or by two-dimensional gel electrophoresis. Four of the biotinylated proteins from the ECM of B. sorokiniana were isolated, in gel digested with trypsin and partly sequenced by tandem mass spectrometry. No significant sequence similarities to proteins in databases were obtained.
Extracellular matrix protein
Two-dimensional gel electrophoresis
Plant pathogenic fungus
Many plant pathogenic fungi secrete an extracellular matrix (ECM) that can be associated with spores, germ tubes and hyphae . A number of investigations have suggested that components in the ECM, particularly glycoproteins, can function as adhesives enabling a firm binding of the fungus to the host surface. Others have proposed that fungal ECM is important for concentrating and localizing enzymes important for the infection of the host. Another possible function of the ECM is that the matrix can protect the fungus from desiccation and from toxic metabolites of the host plant . Despite many studies indicating the importance of the ECM in fungal pathogenesis, the molecular structure of such a matrix is not well known. Several investigations have indicated that fungal adhesives consist of glycoproteins. Such proteins can be insoluble in water and resistant to chemicals, making them difficult to dissolve and isolate for further molecular characterizations .
In this paper, we have developed a method for isolating and sequencing proteins present in the ECM layer of the cereal pathogen Bipolaris sorokiniana. This fungus causes root rot and leaf spot diseases, mainly on wheat and barley. Microscopic studies have shown that conidia, germlings and hyphae of B. sorokiniana are surrounded by an ECM . These studies indicated that the germling ECM is mainly composed of proteins and its composition changes during the development of the fungus. Further experiments have provided evidence that glycoproteins in the ECM are important for the fungus to adhere to solid surfaces .
2 Materials and methods
B. sorokiniana (syn. Helminthosporium sativum; teleomorph Cochliobolus sativus) isolate Tellus was maintained and conidia were collected as previously described .
2.2 Biotinylation and extraction
To tag surface proteins of B. sorokiniana, conidia (approximately 5×107), germlings and hyphae were biotinylated. Germlings and mycelium were obtained by inoculating conidia (1×105 ml−1) in 50 ml of 2% (w/v) potato dextrose broth (PDB) on a rotary shaker (150 rpm, 21°C) for 4 h (germlings) or 24 h (hyphae). In addition, a so-called attachment ring was collected consisting of mycelium and extracellular material attached to the wall of a culture flask (incubated with orbital shaking for 24 h). For biotinylation, the fungal material was transferred to 50 ml Falcon tubes, and washed with 3×50 ml ice-cold PBS (10 mM sodium phosphate buffer pH 7.4 and saline 0.15 M NaCl). The pellet was suspended in PBS containing 2 mM PMSF. Sulfo-NHS-LC-biotin (Pierce, USA) was added to a final concentration of 0.5–0.7 mg ml−1 and the samples were incubated on a bottom-up-bottom mixer for 1 h at 4°C. The biotinylation reaction was stopped by adding 1 mg ml−1 glycine and the material was washed with 4×45 ml PBS. The fungus was also biotinylated when attached to a solid surface. Polystyrene Petri dishes were incubated with 10 ml of a conidial suspension (2×105 conidia ml−1, in 2% PDB) for 24 h at room temperature. Attached germlings were washed with 4×30 ml of PBS on a rotary shaker at 100 rpm (4°C, 10 min). During the biotinylation reaction, the Petri dishes were incubated on a rotary shaker at 150 rpm (4°C, 1 h). For fluorescence microscopy, the fungus was biotinylated when attached to glass slides .
The proteins from fungal pellets were extracted in 0.2 M glycine–HCl, pH 2.2 for 15 min at room temperature. The extracted proteins were concentrated either by TCA (20% w/v) precipitation or by binding to streptavidin-conjugated magnetic beads (Dynal, Norway).
2.3 Fluorescence microscopy
To block unspecific binding, biotinylated fungal material was incubated for 1 h in PBS-B (containing 1% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) Tween 20 in PBS). After three washes with PBS-B, the fungus was incubated with anti-biotin-FITC labelled antibody (Sigma) (1:20, in PBS-B) for 1 h at room temperature and in darkness. The samples were washed three times with PBS containing 0.05% Tween, then they were mounted in glycerol–PBS (1:2).
2.4 Affinity binding
The glycine–HCl extracts were dialyzed against PBS in Slide-A-Lyze 10 MWCO dialysis cassettes (Pierce, USA) overnight at 4°C. Streptavidin-conjugated magnetic beads (40 μl) were pre-washed with 3×400 μl PBS containing 0.1% (w/v) BSA and incubated in 1.5 ml of the extracts for 2 h at room temperature. The beads were washed in 3×400 μl of 0.05% Tween 20 in PBS.
2.5 Gel electrophoresis
For sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the TCA precipitated proteins or proteins bound to streptavidin beads were dissolved in SDS sample buffer (4% (w/v) SDS, 100 mM Tris–HCl (pH 8.8), 200 mM dithiothreitol (DTT), 20% (w/v) glycerol) by heating at 95°C for 7 min. For analysis by MS/MS (tandem mass spectrometry) the concentration of DTT in the sample buffer was 20 mM and after denaturation/reduction, the proteins were alkylated with 1/10 volume of 0.5 M freshly prepared iodoacetamide (10–20 min in darkness). SDS–PAGE was performed in a Mini Protean II Ready Gel Cell (Bio-Rad) using pre-cast linear gradient gels (4–20% Tris–HCl).
For two-dimensional (2-D) PAGE, the TCA precipitated proteins were dissolved in rehydration medium (2 M thiourea, 8 M urea, 20 mM Tris, 4% CHAPS, 65 mM DTT, 2% IPG sample buffer pH 3–10 NL, Pharmacia Biotech, Sweden), sonicated 2×20 s, and incubated for 1 h at room temperature or overnight at 4°C. The samples were centrifuged (13 000 rpm, 15°C, 30 min) and the protein content in the supernatant was determined . Dry non-linear pre-cast immobilized pH gradient gel strips (IPG, 13 cm, pH 3–10 NL, Pharmacia Biotech) were rehydrated with 250 μl proteins (40–180 μg) in the rehydration medium. After focusing in a Multiphor II apparatus (Pharmacia Biotech), the IPG strips were incubated (20 min) in a SDS equilibration solution (50 mM Tris–HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 1% (w/v) SDS) supplemented with 2% (v/v) DTT and trace of bromophenol blue, followed for another 20 min in the SDS equilibration solution supplemented with 4.5% (v/v) iodoacetamide. The equilibrated strips were transferred to a 10% (Tris–HCl) acrylamide slab gel (160×160×1.5 mm), and separated (Protean II xi cell, Bio-Rad). Proteins were silver-stained  or transferred to PVDF membranes.
2.6 Western blots
The proteins were transferred to an Immobilon PVDF membrane (pore size 0.45 μm, Millipore) using a semi-dry electroblotter equipment (JKA, Biotech, Denmark) (80 min, 0.8 mA cm−2). The membrane was blocked in PBS-B for 1 h at room temperature, washed 3×5 min in 0.05% Tween 20 in PBS, incubated with 0.15 U ml−1 streptavidin–POD conjugate (Boehringer Mannheim Scandinavia AB, Sweden) in PBS-B for 1 h at room temperature and washed 3×10 min in 0.05% Tween 20 in PBS. The membrane was developed using ImmunoPure Methal Enhanced DAB Substrate kit (Pierce, Rockford, IL, USA).
2.7 Peptide sequencing
Proteins were excised from 1-D or 2-D gels and subjected to in gel tryptic cleavage . Resulting peptides were desalted  or separated on a SMART HPLC system (Pharmacia, Uppsala, Sweden) equipped with a 1×150 mm 300SB-C8 column (Hewlett Packard), at a flow rate of 30 μl min−1, and using a gradient of 0–50% B in 75 min (A=0.1% TFA and B=95% acetonitrile containing 0.09% TFA). Detection was at 214 (main), 280 and 254 nm. Before MS analysis, peptide fractions were pooled, dried in a Speedvac, re-dissolved in 80% formic acid and diluted (10-fold) with 50% acetonitrile in water. Mass spectrometric analysis was performed in a Micromass Q-TOF electrospray instrument (Manchester, UK). After the initial peptide scanning, individual peptides were selected for further fragmentation. The final analysis of the fragmentation pattern was made with the aid of Biolynx Peptide sequencing software.
3 Results and discussion
Fluorescence microscopy of biotinylated samples of B. sorokiniana demonstrated an extensive labelling of the ECM of germlings and hyphae (Fig. 1). No intracellular labelling was detected. Conidia showed weak or no labelling. Fluorescence of non-biotinylated hyphae was negligible.
Visualization of biotinylated proteins in the ECM of B. sorokiniana. (A) Germlines incubated on glass surface for 3.5 h; (B) corresponding light micrograph; (C) hyphae from 1-day-old mycelium incubated in a liquid medium. The fungus was biotinylated, probed with anti-biotin-FITC and examined with fluorescence microscopy. Bar=25 μm.
Attempts were made to extract biotinylated ECM proteins using buffers of alkaline and acid pH values, since it has been demonstrated that strong bases and acids can dissolve the ECM layer of B. sorokiniana . All extracts, except the one using a glycine–HCl buffer (pH 2.2), had complex patterns of fuzzy protein bands (Fig. 2). Extracts made using the glycine buffer, previously used for extracting cell surface proteins of Helicobacter pylori , showed at least seven distinct protein bands (molecular masses corresponding to ca. 39, 42, 52, 82, 95, 125, 140 kDa) and were repeatedly observed (Fig. 2, lane 6). Several of these bands were also present in the SDS/Tris extracts.
Isolation of biotinylated ECM proteins from the hyphae of B. sorokiniana using different extraction buffers. Lane 1: SDS sample buffer (95°C, 7 min); lane 2: 2% SDS, 25 mM Tris–HCl, pH 6.8 (22°C, 15 min); lane 3: 2% SDS, 25 mM Tris–HCl, pH 8.8 (22°C, 15 min); lane 4: 2% SDS, 40 mM Tris, pH 11 (22°C, 15 min); lane 5: 40 mM Tris, pH 11 (22°C, 15 min); lane 6: 0.2 M glycine–HCl, pH 2.2 (22°C, 15 min). The proteins were separated using SDS–PAGE, blotted to PVDF membrane and probed for biotin using a streptavidin–POD conjugate.
The glycine–HCl buffer was used to extract proteins from biotinylated conidia and germlings, and from an attachment ring consisting of fungal material adhering to the wall of the culture vessel. No protein bands were detected in the extracts of conidia (Fig. 3, lane 1). The extracts of germlings had a higher background of non-specific labelling than the hyphal extracts (Fig. 3, lanes 2 and 3). Some of the protein bands in the germling extracts were not present in the hyphal extracts, including several low molecular mass proteins (<35 kDa). Other protein bands including those of 82 and 95 kDa appeared to be present in both germlings and hyphae. The attachment ring, consisting of mycelium and extracellular material attached on the wall of the culture flask, showed a similar pattern of biotinylated proteins as the hyphae (Fig. 3, lanes 3 and 4). The similarity in protein patterns between the attachment ring and the hyphal ECM was confirmed by comparing the profile of biotinylated proteins concentrated on streptavidin-conjugated magnetic beads (Fig. 4, lanes 1 and 2). The profile of ECM proteins of hyphae attached to a solid surface was similar to that of hyphae grown in a bulk medium (Fig. 4, lanes 1 and 3). Bands at 39, 42, 52, 82, 95 and 125 kDa were present in both samples.
Extraction of biotinylated ECM proteins from different stages of B. sorokiniana using the glycine–HCl buffer. Lane 1: conidia; lane 2: germlings (4 h of incubation); lane 3: hyphae (24 h); lane 4: attachment ring, i.e. fungal material attached to the glass wall of the culture vessel collected after 24 h of incubation. The proteins were separated using SDS–PAGE, blotted to PVDF membrane and probed for biotin using a streptavidin–POD conjugate.
Isolation of biotinylated ECM proteins using streptavidin-conjugated magnetic beads. Lane 1: hyphae (incubated for 24 h); lane 2: attachment ring (recovered after 24 h of incubation); lane 3: hyphae attached (for 24 h) to polystyrene surface. The proteins were extracted using the glycine–HCl buffer. Proteins bound to streptavidin were separated using SDS–PAGE, blotted to PVDF membrane and probed for biotin using a streptavidin–POD conjugate. The band eluting close to 35 kDa (X) in lane 3 corresponds to the streptavidin dimer that appears in blots, if the removal of unbound biotin has not been complete.
Attempts were made to purify the biotinylated ECM proteins for subsequent peptide sequencing using affinity binding to streptavidin-conjugated magnetic beads. A comparison of ‘crude’ and affinity-purified extracts of hyphal proteins showed that the biotinylated proteins bound selectively to the streptavidin-conjugated magnetic beads (Fig. 2, lane 6 and Fig. 4, lane 1). The binding of several of the weaker bands was inconsistent from preparation to preparation. Furthermore, several of the protein bands were fuzzy after the affinity binding, which indicates that the bands contained several unresolved proteins.
Analyses using 2-D gel electrophoresis confirmed that some of the biotinylated ECM protein bands (including those at 39 kDa) contained multiple proteins (Fig. 5). For example, the bands at 39 and 82 kDa were resolved in the IEF dimension into several spots. Silver-stained gels contained protein spots that could not be labelled or were labelled only weakly for biotin in Western blots. In contrast, few proteins detected on Western blots were not found on silver-stained gels.
2-D gel electrophoresis of biotinylated proteins of hyphae (incubated for 24 h) of B. sorokiniana. Proteins were extracted using the glycine–HCl buffer. (A) Silver-stained gel (total amount of applied protein ca. 175 μg). Encircled area indicates proteins weakly stained or not detected when probed for biotin. Arrows indicate proteins recovered for MS/MS sequencing. (B) Western blot (total amount of protein ca. 120 μg). Encircled area indicates proteins detected only when probed for biotin and which did not appear on silver-stained gels.
Four proteins isolated by 2-D PAGE (designated ECM39, ECM82, ECM125 and ECM140) and one isolated using SDS–PAGE and streptavidin-conjugated magnetic beads (ECM82) were digested with trypsin and sequenced by MS/MS (Table 1). No significant matches to sequence information in the NCBI databases were obtained . Notably, two of the peptides isolated from the ECM140 protein contained a stretch of acidic amino acid residues (EDLE). Repeats of acidic residues have recently been found in several fungal surface proteins [9,10].
Peptide sequences of ECM proteins isolated from the hyphae of B. sorokiniana using biotinylation followed by 2-D gel electrophoresis and MS/MS analysisa
aProteins were extracted with glycine–HCl. Internal peptides were obtained by in gel tryptic digestion. Isolated peptides were sequenced using MS/MS.
bECM39 designates a protein with a molecular mass of 39 kDa (as estimated from SDS–PAGE).
cL designates L or I, since these amino acids cannot be distinguished by MS/MS analysis. (CS)(SC) designates CS or SC.
dNumber of analyzed amino acid (AA) residues.
ePeptide mass fingerprinting showed that ECM82 is identical to a 82-kDa protein isolated using streptavidin-conjugated magnetic beads (cf. Fig. 4). The given sequences are from the MS/MS analysis of the protein purified using the streptavidin beads.
Biotinylation has previously been used for characterizing cell wall proteins of Candida albicans and Saccharomyces cerevisiae [11,12]. We have demonstrated that biotinylation can be used to label proteins present in the ECM layer of filamentous fungi. Furthermore, after extraction with a glycine–HCl buffer, and separation with 2-D gel electrophoresis, the ECM proteins can be sequenced with MS/MS. Such sequence information should make it possible to clone and identify genes encoding ECM proteins in fungal plant pathogens. The method could be used to follow compositional changes in the ECM layer during growth and development of the fungus, and it has the sensitivity to characterize proteins expressed in the ECM layer of a fungus attached to a solid surface.
This work was supported by a grant from the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Science Research Council. We thank Drs. Tomas Johansson and Stefan Rosén for valuable comments on the manuscript.