Journal of Experimental Botany, Vol. 52, No. 354, pp. 85-90,
January 2001
© 2001 Oxford University Press
Original Papers |
Rapid deposition of wheat cell wall structural proteins in response to Fusarium-derived elicitors
1 University of Westminster, School of Biosciences, 115 New Cavendish St., London W1M 8JS, UK
2 Department of Nutrition, High Institute of Public Health, University of Alexandria, 165 El-Horye Avenue, El-Hadrah, Alexandria, Egypt
3 Agriculture and Agri-Food, Cereal Research Centre, 195 Dafoe Rd., Winnipeg, Manitoba R3T 2M9, Canada
4 Milling, 19 Elsawah Square, Alameria, Cairo, Egypt
5 John Innes Centre, Protein Sequencing and Peptide Synthesis Facility, Norwich Research Park, Colney, Norwich NR4 7UH, UK
6 Instituto de Tecnologia Química e Biológica, Apartado 127, Oeiras 2781-901, Portugal
Received 2 May 2000; Accepted 4 August 2000
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Abstract |
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Two novel cell wall structural proteins of spring wheat (Triticum aestivum L. em Thell.). undergo rapid deposition in the cell wall matrix in a H2O2-dependent reaction after the elicitation of cultures with Fusarium graminearum (L.)-derived elicitor. The amino acid compositions of these proteins were remarkably similar and indicated that they were highly acidic (pI 3.8). These proteins contained 13–17% each of Gly, Glx and Ser with lesser amounts (6–8%) of Ala, Asx and Thr, and it has been suggested that they are known as glycine- and serine-rich proteins (GSRPs). SELDI-MS ionization spectra demonstrated that these proteins have low molecular masses of 8590 and 4292 Da. These results are discussed in relation to the possible role of these novel proteins in rapid, cell wall defensive reactions to pathogenic attack.
Key words: Structural proteins, elicitation, cell wall, Triticum aestivum L.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The role of cell wall structural proteins in defence and response to wounding has been investigated extensively in dicots (see reviews by Showalter, 1993
; Cassab, 1998). Specific cell wall structural proteins have been shown to be oxidatively cross-linked in French bean cells within 15 min after application of an elicitor preparation from cell walls of Colletotrichum lindemuthianum (Wojtaszek et al., 1995). A similar situation was found in suspension-cultured cells of soybean (Bradley et al., 1992) and tomato (Brownleader et al., 1995).
Monocot cell walls differ significantly from those of dicots. However, reasonable homology exists between certain structural proteins of non-graminaceous plants and grasses (Carpita, 1996). Hydroxyproline-rich glycoproteins (HRGPs) rich in threonine (THRGP) or histidine (HHRGP) have been purified from maize suspension cultures (Kieliszewski and Lamport, 1987) and pericarps (Hood et al., 1988). Gene sequences obtained for HRGPs from maize, teosinte, sorghum (Raz et al., 1992), and rice (Caelles et al., 1992) revealed amino-acid sequences common to HRGP proteins of both monocots and dicots. Similar homologies have been found in proline-rich proteins (Raines et al., 1991) and glycine-rich proteins (GRPs) (Goddemeier et al., 1998).
The function of the cell walls of cereal species in disease resistance is not well understood. Cell wall thickening has been associated with fungal attack in several cereals (Sherwood and Vance, 1980) and correlated with resistance to Pseudocercosporella sp. in winter wheat (Murray and Bruehl, 1983). A highly localized deposition of THRGPs was observed in the extrahaustorial matrix in both compatible and incompatible host–pathogen combinations (Hippe-Sanwald et al., 1994). Gene expression for HRGP has been associated with the wound response in maize (Ludevid et al., 1990).
Wheat Head Blight or Scab, caused by Fusarium graminearum, is a disease of significant economic importance in the temperate wheat growing regions of the world. Scab of wheat has a multigenic background and is highly affected by ambient conditions found in warmer, drier locations (Bai and Shaner, 1994). Recently, one of our laboratories has produced doubled haploid lines of spring wheat (Triticum aestivum, L. em Thell.) from the cross of RL4761 and 93FHB37, and tested the lines for resistance to F. graminearum (J Gilbert, unpublished results). In this work, callus was produced from a resistant line (DH1015), and used as a model system to study cell wall proteins in wheat–Fusarium interactions. The purification and characterization of novel cell wall proteins from these callus tissues is described. A putative role for these proteins in plant defence, as suggested by their deposition in situ by Fusarium-derived elicitors and by a wheat spike-derived peroxidase, is discussed.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Material
The doubled haploid wheat line DH1015, previously characterized as resistant to Fusarium head blight was selected for callus production. The doubled haploid line were derived from the cross FHB37/RL4761 (FHB37=HY611/Ning 8331; RL4761=Roblin BSR). Fusarium graminearum (L) Schwabe (Isolate 192132) was obtained from Dr J Gilbert, Agriculture and Agri-Food, Cereal Research Center, Canada). Wheat (Ning 8331) spike-derived peroxidase was provided by Phil Clarke from the University of Westminster, London and the Cereal Research Centre, Canada. All consumables, unless otherwise stated, were purchased from Sigma (Poole, UK).
Growth conditions of wheat callus
Callus tissues derived from wheat seed embryos were grown on a solidified Murashige and Skoog-based tissue culture medium (1.0 l contained 30 g sucrose, 4.4 g Sigma basal salt mixture (Sigma, Poole, UK), 0.4 mg thiamine/HCl, 100 mg glutamine, 1 mg 2,4-dichloro-phenoxyacetic acid, and 2.5 g PhytagelTM) (Murashige and Skoog, 1962). Callus was transferred every 3–4 weeks to fresh medium and grown at 25 °C in the dark.
Preparation of Fusarium culture filtrate
Fusarium graminearum (L) Schwabe was grown in liquid medium (1.0 l contained 15 g carboxymethylcellulose, 1 g NH4NO3, 1 g KH2PO4, 0.5 g MgSO4.7H2O, and 1 g yeast extract, and was adjusted to pH 7.0) and incubated with aeration for 5–7 d under cool fluorescent light at 24 °C. The resulting fungus/media solution was filter-sterilized by 0.22 µm ultrafiltration.
Isolation and purification of GSRP1 and GSRP2
Wheat callus (3.0 g) was washed with 20 ml of dH2O and the washed cells collected by vacuum-assisted Buchner-filtration. The callus was then soaked in 6 ml of 1 M KCl for 5 min. to elute ionically-bound cell surface proteins. The KCl eluate was then collected by the application of vacuum, clarified by centrifugation at 10000 g for 10 min and concentrated by 10 kDa ultrafiltration (4 ml VivaspinTM, Vivascience, Gloucestershire, UK). The concentrated eluate was then fractionated by Superose-12 gel-filtration FPLC column chromatography (HR 10/30, Amersham Pharmacia Biotech, St. Albans, UK). The column was equilibrated with degassed 0.1 M sodium acetate buffer pH 5.0 and 0.1 M NaCl at a flow rate of 0.3 ml min-1. The eluate was monitored at 280 nm. Peak fractions corresponding to cell surface proteins, GSRP1 (eluting at 27 min.) and GSRP2 (eluting at 60 min.) were purified further by reverse phase C8-HPLC (15 cm length, 4.6 mm i.d., DiscoveryTM; Supelco, Poole, UK). GSRP1 and GSRP2 were eluted from the column using a linear 15–75% acetonitrile (+0.1% TFA) gradient over 30 min and the eluate was monitored at 220 nm. Fractions containing GSRP1 and GSRP2 were pooled and desalted using a PD-10 column (Amersham Pharmacia Biotech, UK) equilibrated with 25 ml dH2O. The fractions were freeze-dried then dissolved in 1 ml dH2O. Aliquots (20 µl) of each sample were re-injected on to the reverse phase C8-HPLC column to establish sample homogeneity.
Assay of insolubilization of GSRP1 and GSRP2
Wheat callus (0.4 g) were inoculated with 1 ml of filter-sterilized Fusarium culture filtrate for 0–30 min or incubated with 1 ml of 75 µM H2O2 or 15 µg ml-1 of wheat-spike-derived peroxidase for 10 min. The reaction was stopped by washing the callus with 20 ml of dH2O. Cell surface protein was eluted with 1 ml of 1 M KCl as described above, and centrifuged at 10 000 g for 10 min. The clarified eluate was concentrated to a final volume of 400 µl by 0.5 ml VivaspinTM centrifugal devices (10 kDa molecular weight cut off, Vivascience) and 100 µl concentrate was injected on to a Superose-12 column. The eluate was monitored at 280 nm. The Superose-12 column was calibrated using markers ranging in size from catalase (Mr 240 000) to a tri-peptide Tyr-Gly-Gly (Mr 300).
Amino acid analysis of GSRP1 and GSRP2
GSRP1 and GSRP2 were hydrolysed in 6 M HCl at 110 °C for 16 h (Brownleader and Dey, 1993). The acid hydrolysates were analysed on an LKB Alpha-Plus Analyser (Pharmacia LKB Biotechnology, St Albans, UK) using sodium buffers and ninhydrin determination.
Effect of H2O2 upon covalently-bound cell wall protein
Wheat callus (1 g) was incubated with 1 ml of 75 µM H2O2, or 1 ml dH2O (as control) for 30 min at 30 °C and then washed with 50 ml of dH2O. After ionically bound cell wall protein were eluted with 2 ml 1 M KCl, the callus was washed again with 50 ml of dH2O, then ground in a pestle and mortar with liquid nitrogen. dH2O (1 ml) was added to the ground callus and the resultant suspension was centrifuged at 3000 g for 15 min, 4 °C, to pellet the cell wall fraction. After the cell wall was freeze-dried, its amino acid composition was determined under identical conditions used for amino acid composition analyses of soluble GSRP1 and GSRP2.
Surface Enhanced Laser Desorption/Ionization-Mass Spectrometry
Mass spectra were obtained using a SELDI-mass spectrometer. GSRP1 and GSRP2 eluted from the C8-reverse phase HPLC column were spotted on to a reverse-phase (H4) protein array chip surface (C16 carbon backbone) and allowed to air dry. After drying, samples were washed twice with 4 µl 10% (v/v) acetonitrile. Sinapinic acid matrix (0.7 µl) was added just before samples were allowed to dry. The matrix was a saturated solution in 50% (v/v) acetonitrile: water containing 0.5% TFA. Data from 50 laser shots were acquired and averaged to produce the spectra. Insulin (Mr 5.733) was used as the external calibration standard.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Wheat callus provides a convenient model system to investigate controlled elicitation of early wheat defence responses in the cell wall. Double haploid wheat lines which are resistant (var. DH1015) to Fusarium graminearum (see Materials and methods) were selected for callus production.
Prior to the exposure of wheat calluses with sterilized Fusarium culture filtrate, KCl eluates of wheat callus contained only 2 main protein peaks which eluted at 27 min and 60 min on a Superose-12 gel filtration column (Fig. 1, trace 0 min). Based upon their rates of elution, the two protein peaks were provisionally designated P27 and P60. Insolubilization in situ of P27 and P60 was observed after challenging wheat callus with sterilized Fusarium culture filtrate prior to elution of cell surface proteins with 1 M KCl (Fig. 1, traces 2–30 min). This time-course analysis showed that almost complete insolubilization of P27 occurred after 2 min. The insolubilization of P60 clearly required more time, and neared completion only after 10 min incubation with Fusarium derived-elicitors (Fig. 1A, trace 10 min).
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The high levels of soluble P27 and P60 (Fig. 1
trace 0 min.) were also rapidly insolubilized after exposing wheat callus to H2O2 (Fig. 2, trace H2O2). In addition, no insolubilization was observed in the absence of H2O2, suggesting that the insolubilization process is mediated by an endogenous wheat peroxidase. Similar results were obtained when wheat cells were incubated with a pathogen-induced wheat spike-derived peroxidase for 10 min without the addition of exogenous H2O2 (trace WPox). This work follows previous findings (Bolwell et al., 1995) that plant peroxidases are able to generate H2O2 in the presence of a suitable reductant. The fact that the insolubilization in situ of these two proteins occurred only in the presence of H2O2 or peroxidase suggests that cross-linking after elicitation by Fusarium-derived elicitors could be driven by the oxidative burst when H2O2 is formed.
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The rapid and complete insolubilization of both proteins into the cell wall suggested that P27 and P60 each consisted of a single structural protein species. These proteins were isolated from KCl eluates by Superose-12 chromatography and then further purified by C8-RP HPLC (see Materials and methods). Both P27 and P60 were eluted from the C8-RP column as single symmetrical A220-absorbing peaks at 40% acetonitrile (Fig. 3A
, B), suggesting that they had been purified to homogeneity. P27 and P60 therefore appear to have identical polarities.
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Superose-12 gel filtration chromatography of these proteins suggested that they have substantially different molecular weights. By comparing the Ve values of the two proteins, the apparent molecular mass of P27 was estimated to be in excess of 240 kDa, whereas that for P60 was estimated to be 3.2 kDa. However, estimation of molecular weight by gel-filtration has proven to be unreliable for some structural proteins (Brownleader and Dey, 1993
). Therefore, the molecular masses of these proteins were determined by SELDI-mass spectrometry. Using this method, the preparations of both P27 and P60 presented single molecular masses, confirming their purity. P27 was shown to have a molecular weight of 8590 Da (Fig. 4A), substantially lower than that determined by gel-filtration. The pseudo-molecular ion for P60 appears at 4292 Da (Fig. 4B), in close agreement with data obtained with Superose-12 chromatography. These two proteins therefore appear to have low molecular weights relative to the majority of known cell wall structural proteins. Only a few examples of similarly small structural proteins have been identified, including a 7138 Da tyrosine and lysine-rich protein from tomato which is deposited in xylem and sclerenchyma cells walls (Domingo et al., 1994).
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Amino acid composition analysis of these proteins demonstrated that they have a remarkably similar composition (Table 1
). Five amino acids make up 60% of each of the two proteins; Gly, Glu/Gln, Ser, Ala, and Asp/Asn. The estimated pI for both P27 and P60 is about pH 3.8 (Patrickios and Yamasaki, 1995).
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The complete absence of hydroxyproline in these proteins precludes their similarity to threonine and hydroxyproline-rich glycoproteins, which have been extensively characterized in the monocot system (Caelles et al., 1992
; Ruiz-Avila et al., 1992; Smallwood et al., 1995). P27 and P60 demonstrate some similarity with GRPs and nodulin GRPs, which also have high levels of Gly, Ser, Ala, and Glu/Gln (Cassab, 1998). However, P27 and P60 have a substantially lower glycine content, are considerably smaller than proteins of this type, and therefore appear to be novel proteins. In order to distinguish these structural proteins from GRPs, it is proposed that they be referred to as glycine- and serine-rich proteins (GSRPs). Hence, P27 and P60 can be referred to as wheat GSRP1 and GSRP2, respectively.
It was important to demonstrate that the loss of soluble GSRPs correlated with their deposition in the cell wall matrix. Therefore, the amino acid compositions of cell walls from control cells and those incubated for 10 min with H2O2 were compared (Table 1). The largest increases were seen in Gly, Ser, Glu/Gln, Ala, and Asp/Asn, the major amino acids of GSRP1 and GSRP2. This confirms that the rapid reduction in the levels of these soluble GSRPs occurs with their deposition in the cell wall. The large changes in cell wall amino acid composition after the deposition of these proteins indicates that they can effect significant changes to the covalently bound cell wall structure during an early response to elicitation. This suggests that the deposition of wheat GSRPs, like other structural proteins, might increase cell wall resistance to fungal lytic enzymes (Brisson et al., 1994) and plant resistance to pathogens (Esquerré-Tugayé et al., 1979).
The available evidence suggests that GSRP1 and GSRP2 are closely related proteins. Both are rapidly cross-linked in the cell wall matrix in a peroxide-dependent reaction, and have nearly identical amino acid compositions. That they are both eluted from C8-RP columns at 40% acetonitrile suggests further similarities in physico-chemical properties. Attempts were made to obtain the N-terminal sequences of GSRP1 and GSRP2. Unfortunately, both proteins proved to be blocked at their N-terminus. However, SELDI-mass spectroscopy demonstrated that GSRP1 has a molecular mass (8590 Da) almost exactly twice that of GSRP2 (4292 Da). It is therefore tempting to suggest that GSRP1 represents the dimeric form of GSRP2. Dimerization of GSRP2 would conceivably increase the number of structural motifs available for intermolecular cross-linking. This might explain why the deposition of GSRP1 occurs at a faster rate than GSRP2.
The deposition of GSRP1 and GSRP2 at the cell wall has been associated with an early and rapid wheat defensive response to Fusarium. Further characterization of these novel low molecular weight proteins and the enzymatic basis for their deposition will undoubtedly enhance the understanding of rapid defence mechanisms in wheat.
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Acknowledgments |
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Wael El-Gendy is grateful to The British Council Chevening Section for financial support. Dr Philip Jackson acknowledges a research fellowship awarded by PRAXIS XXI (ref. BPD/ 16316/98). We thank Dr Lee Lomas (Ciphergen, UK) for performing SELDI-mass spectrometric analyses.
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Notes |
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7 To whom correspondence should be addressed. Fax: +351 21 4433644. E-mail: Phil{at}itqb.unl.pt
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