JXB Advance Access originally published online on March 23, 2006
Journal of Experimental Botany 2006 57(6):1461-1469; doi:10.1093/jxb/erj127
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RESEARCH PAPER |
Protein cross-linking, peroxidase and ß-1,3-endoglucanase involved in resistance of pea against Orobanche crenata
1CSIC, Instituto de Agricultura Sostenible, E-14080 Córdoba, Apdo. 4084, Spain
2IFAPA-CICE (Junta de Andalucia), CIFA ‘Alameda del Obispo’, Área de Mejora y Biotecnología, E-14080 Cordoba, Apdo. 3092, Spain
3ETSIAM-UCO, Departamento de Genética, E-14080 Córdoba, Apdo. 3048, Spain
4CSIC, Centro de Investigaciones Biológicas, Departamento de Plant Development and Nuclear Organization, Ramiro de Maeztu 9, E-28040 Madrid, Spain
* To whom correspondence should be addressed. E-mail: bb2pelua{at}uco.es
Received 9 October 2005; Accepted 23 January 2006
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Abstract |
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Root holoparasitic angiosperms, like Orobanche spp, completely lack chlorophyll and totally depend on their host for their supply of nutrients. O. crenata is a severe constraint to the cultivation of legumes and breeding for resistance remains the most economical, feasible, and environmentally friendly method of control. Due to the lack of resistance in commercial pea cultivars, the use of wild relatives for breeding is necessary, and an understanding of the mechanisms underlying host resistance is needed in order to improve screening for resistance in breeding programmes. Compatible and incompatible interactions between O. crenata and pea have been studied using cytochemical procedures. The parasite was stopped in the host cortex before reaching the central cylinder, and accumulation of H2O2, peroxidases, and callose were detected in neighbouring cells. Protein cross-linking in the host cell walls appears as the mechanism of defence, halting penetration of the parasite. In situ hybridization studies have also shown that a peroxidase and a ß-glucanase are differently expressed in cells of the resistant host (Pf651) near the penetration point. The role of these proteins in the resistance to O. crenata is discussed.
Key words: Confocal microscopy, cytochemistry, glucanase, in situ hybridization, Orobanche crenata, parasitic plants, peroxidase, Pisum sativum, protein cross-linking, resistance
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Orobanche spp (broomrapes) are root parasitic angiosperms lacking in chlorophyll and totally dependent on their host for their supply of nutrients. O. crenata has been known to threaten pulse crops since antiquity and it is a severe constraint to the cultivation of grain legumes (Cubero et al., 1994
). Genetic resistance remains as one of the most desirable components in an integrated control strategy. However, resistance to O. crenata in legume crops such as faba bean (Vicia faba L.) and pea (Pisum sativum L.) has proved to be a polygenic character with very low heritability, making breeding for resistance a difficult task (Cubero, 1994; Rubiales, 2003). In addition, little resistance is available in commercial cultivars of crops such as pea, so turning to the wild relatives for breeding is necessary (Rubiales, 2003; Rubiales et al., 2003a; 2005).
Studies of plant–parasitic plants interactions are limited compared with other plant–pathogen systems. However, understanding the mechanisms underlying host resistance is needed in order to improve the screening methods in breeding programmes. Several defence responses activated in plants against micro-organisms have also been identified as induced in response to the parasitization process of parasitic plants. These responses include increased levels of phenolics and peroxidase activity (Goldwasser et al., 1999; Pérez-de-Luque et al., 2005a), induction of phytoalexins (Serghini et al., 2001), lignification (Goldwasser et al., 1999; Pérez-de-Luque et al., 2005b), and expression of Pathogenesis-Related proteins (PR-proteins) (Joel and Portnoy, 1998). In addition, constitutive overexpression of a 3-hydroxy-3-methylglutaryl CoA reductase encoding gene increased the resistance in the susceptible host (Westwood et al., 1998). Recent work using suppression subtractive hybridization approaches (Vieira Dos Santos et al., 2003) and proteomic techniques (Castillejo et al., 2004) have led to the identification of several genes expressed during the process of resistance to Orobanche spp. However, there is a long way to go in order to know which genes are really implicated in resistance to Orobanche and which of them are induced as part of a chain process: for example, chitinases could be induced by Orobanche infection, but these enzymes have proved to be effective only against fungi and not to plant cell walls, since chitin is not a constituent of plant cell walls.
In the present work, some of the factors involved in resistance to penetration of O. crenata in pea have been studied. Cytochemical assays allowed it to be determined whether parasite intrusive cells were stopped within the host cortex, before reaching the central cylinder. Based on these data and previous publications (Chang et al., 1992; Joel and Portnoy, 1998; Goldwasser et al., 1999; Vieira Dos Santos et al., 2003; Castillejo et al., 2004; Pérez-de-Luque et al., 2005a) a peroxidase and glucanase/PR-2 genes, previously isolated from P. sativum, were used for in situ hybridization studies.
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Materials and methods |
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Plant material and growth conditions
Orobanche crenata was grown on susceptible and resistant genotypes of pea (Pisum sativum L. cv. Messire and Pisum fulvum L. accession Pf651; respectively). The Petri dish system described by Rubiales et al. (2003b)
was used. Pea seeds were germinated in Petri dishes on wet glass fibre filter papers and kept in the dark at 20 °C for 5 d. When the radicle reached 4–5 cm in length, seedlings were transferred to new dishes (15 cm diameter) with perlite and glass fibre papers (Whatmann GF/A). O. crenata seeds (8 mg) were previously spread on the paper, after being disinfected with ethanol (70%, for 30 s) followed by sodium hypochlorite (1%, for 20 min) and placed in darkness at 20 °C for 10 d. The dishes were sealed with parafilm, covered with aluminium foil to exclude the light, and were placed vertically, the germinating host plant upwards, in trays with Hoagland nutrient solution (Hoagland and Arnon, 1950). Test plants were grown in a controlled environment chamber with a day/night temperature of 20±0.5 °C, 14 h photoperiod, and an irradiance of 200 µmol m–2 s–1.
Collection and fixation of samples for conventional microscopy
Observations were taken using a binocular microscope (Nikon SMZ1000; Nikon Europe BV, Badhoevedorp, The Netherlands). At 25 d after inoculation, seedlings of O. crenata were sampled at random with the corresponding attached parts of host roots.
For cytochemical assays with bright field and epifluorescence observations using a light microscope, the sampled material was fixed in FAA (ethanol 50%+formaldehyde 5%+glacial acetic acid 10%, in water) for 48 h. Fixed samples were then dehydrated in an ethanol series (50, 80, 95, 100, 100%: 12 h each) and transferred to an embedding solvent (Xylene; Panreac Quimica SA, Montcada i Reixac, Spain) through a xylene-ethanol series (30, 50, 80, 100, 100%: 12 h each) and finally saturated with paraffin (Paraplast Xtra; Sigma, St Louis, USA). Sections, 7 µm thick, were cut with a rotary microtome (Nahita 534; Auxilab SA, Beriain, Spain) and attached to adhesive-treated microscope slides (polysine slides; Menzel GmbH & Co KG, Braunschweig, Germany).
Some samples were kept after fixation in FAA and used for protein cross-linking determination, and others were used fresh for H2O2 and peroxidase activity determination.
Cytochemical methods for light microscopy
After removal of paraffin, the sections were stained with different dyes. (i) Alcian green–safranin (AGS) (Joel, 1983). The slides were dried and mounted with DePeX (BDH). With this staining method, carbohydrates (including cell walls and mucilage) appeared green, yellow or blue, while lignified, cutinized, and suberized walls, as well as tannin and lipid material inside cells appeared red (Joel, 1983). (ii) Pectins were detected using ruthenium red. The samples were immersed for 5 min in a solution of 0.05% ruthenium red in water. Non-methyl esterified pectins take a red/pink colour with this dye (Vallet et al., 1996). (iii) Aniline blue fluorochrome was used for callose detection under UV fluorescence (340–380 nm). The samples were stained for 15–30 min in a solution of 0.1% aniline blue fluorochrome in water (Bordallo et al., 2002).
Protein cross-linking in cell walls were determined following the procedure described by Mellersh et al. (2002). Fixed samples were submerged in 1% sodium dodecyl sulphate (SDS) for 24 h at 80 °C. They were hand cut, stained for 3–5 min in 0.1% Coomassie blue in 40% ethanol/10% acetic acid, rinsed in a solution of 40% ethanol/10% acetic acid, and mounted in distilled water. Cell walls with protein cross-linking take a deep blue colour.
For the determination of H2O2 and peroxidase activity, fresh samples were stained with 3,3-diaminobenzidine (DAB) using a modification of the procedure described by Thordal-Christensen et al. (1997). Fresh samples were submerged in DAB solution (1 mg ml–1, pH 3.8) in distilled water for 2–3 h. After that, the samples were washed with lactic acid, glycerol, and water (1:1:1 by vol.) for 1 h, hand cut with a razor blade and mounted on slides with lactic acid, glycerol, and water (1:1:1 by vol.).
The sections were observed using a light microscope (Leica DM-LB; Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) and photographed at a magnification x100 to x400 using a digital camera (Nikon DXM1200F; Nikon Europe B.V., Badhoevedorp, The Netherlands). The samples were also observed by epifluorescence under excitation at 450–490 nm.
Preparation of probes
Genomic DNA was extracted from Pisum sativum according to the protocol described by Lassner et al. (1989) and modified by Torres et al. (1993), and used for PCR amplification on an Applied Biosystems PCR System 9700. Specific primers olipx1 (5'-AAACCACACTTATTGAAATG-3')/olipx2 (5'-AACAAAGAAATACTCAATCAT-3') and oliglu1 (5'-TAGCCACAAACCAAGACAG-3')/oliglu2 (5'-CTCATCAAACATAGCAAAAAG-3') were derived from P. sativum peroxidase (GenBank accession number AF396465) and P. sativum glucanase (Chang et al., 1992) nucleotide sequences, respectively. The following PCR conditions were used: 40 cycles with denaturation at 94 °C for 35 s, annealing at 48 °C for 35 s and extension at 72 °C for 1 min. An initial denaturation step of 5 min at 94 °C and a final elongation step at 72 °C for 7 min were performed. The amplified 286 pb peroxidase and 678 pb glucanase DNA fragments were isolated from an agarose gel, cloned into pGEM-T-easu cloning system (Promega) and sequenced. Vector sequences were removed and remaining sequences were used as template for BLAST searches (BLASTN and BLASTX) in GenBank (http://www.ncbi.nlm.nih.gov/BLAST). Once verified that the cloned sequences corresponded to the target genes, isolated plasmids containing the P. sativum peroxidase or glucanase PCR fragments were used as a template to synthesize specific probes.
Specific probes of single-stranded RNA were generated by in vitro transcription using a DIG RNA labelling kit according to the manufacturer protocols (Roche). The insert was oriented such that SP6 polymerase produced the sense RNA probe (complementary to the transcribed strand of the DNA). Probe size was reduced to approximately 150 bases to improve the penetration across the vibratome sections, by a mild carbonate hydrolysis (Cox et al., 1984). Probe labelling efficiency was determined by a two step dot blot assay. 1 µl spots of the probes were applied on a nylon membrane (Hybond-N, Amershan), detected with a primary mouse anti-digoxigenin antibody (Sigma) and a secondary anti-mouse antibody conjugated to alkaline phosphatase (Sigma) and revealed using as substrate a solution of 0.015% (w/v) 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 0.03% (w/v) P-nitro blue tetrazolium chloride (NBT) in detection buffer. Positive and negative controls were taken from the DIG RNA labelling kit.
In situ hybridization in tissue sections
Samples collected in the same way as indicated previously were fixed in 4% formaldehyde in PBS buffer (PBS: 140 mM NaCl, 3 mM KCl, 4 mM Na2HPO4, 2 mM KH2PO4 pH 7.4) overnight at 4 °C. After washing in PBS, 40 µm vibratome sections (Vibratome series 1000, Intracel Electrophysiology and Biochemistry Equipment, Herts, UK) were cut under water and dried down on APTES-coated multi-well slides. The sections were dehydrated in a series of 30, 50, 70, and 100% methanol/water, then rehydrated in a series of 70, 50, and 30% methanol/water, and finally in PBS, for 5 min at each step. To facilitate penetration of the labelling reagents, the sections were treated with 2% (w/v) cellulase (Onozuka R-10) in PBS for 2 h at room temperature in a humid chamber and 0.1% Tween 20 in PBS for 10 min at room temperature. After each treatment, the sections were washed with PBS for 5 min. Then the sections were dehydrated in a series of 70, 95, and 100% ethanol/water, allowed to dry, and the hybridization mixture was added. The hybridization mixture contained: 
10 ng µl–1 digoxigenin-labelled probe; 1000 ng µl–1 unlabelled RNA transcribed from a plasmid containing an unrelated insert; 50% de-ionized formamide, 10% dextran sulphate, 300 mM NaCl, 10 mM PIPES pH 8.0, and 1 mM EDTA. Hybridization was carried out overnight at 37 °C in a humid chamber. Then the sections were washed twice in PBS for 15 min at room temperature.
Fluorescent labelling and confocal microscopy
Vibratome sections were hybridized with probes as described above. Then the hybridization signal was detected by mouse anti-digoxigenin antibodies (Sigma), applied 1/5000 in 3% BSA in PBS for 60 min at room temperature, followed by a fluorescent anti-mouse ALEXA 546 (Molecular Probes Inc., Eugene, OR, USA) antibody, applied 1/500 in 3% BSA in PBS for 60 min at room temperature. After washing in PBS, the nuclei were stained with 4'-6-diamidino-2-phenylindole (DAPI). Confocal optical section stacks were collected using a Leica TCS-SP2-AOBS-UV confocal laser scanning microscope (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany). Analysis of confocal images was performed with the LEICA software LCS, version 2.5 Build 1227.
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Results |
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Cytochemical procedures revealed that in incompatible interactions in resistant pea the parasite intrusive cells were stopped in the host cortex, before reaching the endodermis or central cylinder (Fig. 1A). By contrast, infections developed normally in compatible interactions on susceptible pea (data not shown). A preliminary study did not reveal the presence of physical barriers such as lignified or suberized host cell walls by using polarized light or fluorescence (Fig. 1C, D). Only certain red fluorescence was observed in the apoplast, at the interface between host cortical and parasite intrusive cells, and on the external surface of parasite cells. This fluorescence corresponded with the strong red staining obtained with AGS at the same points (Fig. 1B, C).
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More detailed studies showed a low presence of non-methyl esterified pectins in the apoplast at the point where the parasite intrusive cells were stopped during incompatible interactions (Fig. 2A). However, an intense staining was present on other areas previously pierced by the parasite, indicating a strong presence of non-methyl esterified pectins.
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Callose accumulation was detected in host cells at different points through the penetration pathway of O. crenata in resistant peas (Fig. 2B), but was lacking in susceptible ones (data not shown). H2O2 and peroxidase activity were observed in host cell walls and in the apoplast next to parasite intrusive cells (Fig. 2D), but these were absent in compatible interactions (Fig. 2C). Protein cross-linking was also detected in host cell walls in contact with parasite tissues (Fig. 3). Soluble proteins were previously extracted from tissues with the SDS treatment, and only those proteins strongly linked to cell walls remained and were stained by the dye. An intense blue staining of host cell walls next to parasite tissues indicated the presence of proteins (Fig. 3B) contrary to the pale, almost colourless, staining of host cell walls away from the intrusive cells (Fig. 3C).
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Finally, expression of the peroxidase and glucanase genes was observed in situ by confocal microscopy. The hybridization signal was not very intense in both cases, but clearly enough and easily differentiated from the controls incubated with the sense probe (Figs 4, 5). The expression pattern of both genes is very similar: it is restricted to cortical cells of the resistant pea and next to the penetration point of O. crenata (Figs 4B, 5B). Inside the cells, the signal was detected in small cytoplasmic areas near the cell walls and around the nucleus due to the presence of great vacuoles (Figs 4F, 5F). No hybridization signal from both genes was observed in the resistant genotype using the sense probe (Figs 4D, 5D). Only with the peroxidase anti-sense probe was a weak signal detected in the susceptible pea genotype infected with O. crenata and far away from the penetration point (Fig. 4C), but no signal was observed in the case of the ß-glucanase (Fig. 5C).
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Discussion |
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Studying resistance against parasitic plants presents some handicaps compared with other pathogens like fungi, as parasite and host are relatively close organisms that share many morphological, physiological, and biochemical traits. For example, many enzymes released by bacteria or fungi are specific and can be differentiated from plant enzymes. Furthermore, due to differences in cell wall constitution, bacterial colonies and fungal mycelia are easily distinguishable inside plant tissues. However, the question becomes more complicated if the interaction takes place between two plants which merge their tissues. For that reason, cytochemical and in situ hybridization studies are powerful tools in order to unveil the mechanisms underlying the plant–parasitic plants interaction. This is the first report of protein cross-linking as a defensive mechanism against parasitic plants, and this is the first time also that in situ hybridization techniques are used in order to identify the expression of two genes (peroxidase and ß-glucanase) involved in resistance to parasitic plants.
Protein cross-linking has been shown as a rapid and effective defensive response against intruding pathogens like bacteria or fungi (Bradley et al., 1992; Showalter, 1993; Brisson et al., 1994; Hammond-Kosack and Jones, 1996; Brown et al., 1998). Extensins and other hydroxyproline-rich glycoproteins (HRGPs), proline-rich proteins (PRPs), and glycine-rich proteins (GRPs) are structural proteins present in the cell walls. They can be rapidly insolubilized after wounding, pathogen penetration or elicitor treatment (Bradley et al., 1992; Brisson et al., 1994; Brown et al., 1998) and it is a very fast response which enhances cell wall resistance within just a few minutes after pathogen attack (Bradley et al., 1992). This process implies the formation of covalent cross-links and is mediated by H2O2 and peroxidases (Bradley et al., 1992; Brisson et al., 1994; Brady and Fry, 1997; Brown et al., 1998; Otte and Barz, 2000).
Peroxidases also appear to be implicated in the formation of papillae (Brown et al., 1998). This is another type of cell wall fortification rapidly developed under pathogen invasion (Hammond-Kosack and Jones, 1996; Brown et al., 1998). Papillae are composed mainly of callose, a ß-1,3-glucan polymer, but their construction requires cross-linking of HRGPs and phenolic residues such as ferulic acid in the primary wall. Both processes are accomplished by peroxidases in the presence of H2O2 (Brown et al., 1998).
These results showed that the parasite was stopped in the host cortex. However, other mechanisms of resistance against parasitic plants that had previously been reported, such as suberization (Zaitoun et al., 1991; Dörr et al., 1994) or lignification (Pérez-de-Luque et al., 2005b), were not identified. The deposition of callose implies that reinforcement of host cell walls is underway, but it seems that it is not enough or well located enough to stop Orobanche penetration. Nevertheless, the weak presence of non-methyl esterified pectins at the stopping point suggests that the parasitic enzymes (i.e. pectin methyl esterases) are not working properly, perhaps due to the presence of inhibitors (Giovane et al., 2004) and/or reinforcement of host cell walls by insolubilization of cell wall structural proteins (Brisson et al., 1994). This hypothesis is supported by the observation of the intense staining for H2O2 and peroxidase activity in incompatible interactions and the protein cross-linking detected in host cell walls adjacent to the parasite intrusive cells. The in situ hybridization studies showed the expression of the peroxidase gene being restricted to cortical cells in the resistant pea and only to those near the parasite intrusive cells, with a parallel pattern to that of the peroxidase activity and H2O2 distribution obtained through the cytochemical studies. It also fits with the distribution of protein cross-linking in resistant host cell walls. So it can be stated that this peroxidase is very likely implicated in the resistance of pea to O. crenata, probably mediating the reinforcement of the cell wall through oxidative cross-linking of structural proteins.
On the other hand, PR proteins are extracellular proteins that accumulate in response to pathogen infection. There are several families of PR proteins; chitinases (PR-3, -4, -8 and -11), ß-glucanases (PR-2), peroxidases (PR-9), and proteinase inhibitors (PR-6), being some of the most important (Van Loon, 1997; Van Loon and Van Strien, 1999). Chitinases and ß-glucanases play an important role in defence either by degrading cell walls of the pathogens or releasing oligosaccharide elicitors (Leubner-Metzger and Meins Jr, 1999). Chitinases play no role in resistance against parasitic plants because of the absence of chitin in their cell walls. However, ß-glucanases degrade ß-glucans, which are part of the plant cell walls, and the released oligosaccharides can play an important role as elicitors (Esquerré-Tugayé et al., 2000). This hypothesis is reinforced by the fact that expression of a ß-glucanase was observed in host cells next to the penetration point and the intrusive parasite cells. Taking into consideration that increased levels of ß-glucanases have been detected in peas resistant to O. crenata (Castillejo et al., 2004) and that transgenic pea lines expressing constitutively a tobacco ß-glucanase significantly reduced O. crenata parasitization (D Rubiales, unpublished results), it seems clear that this enzyme plays an important role in defence against this parasite. Therefore, it has been also suggested that callose accumulated in plants in response to biotic and abiotic stresses may serve as a reservoir of ß-glucans elicitors (Esquerré-Tugayé et al., 2000), which could be another possible role for the callose depositions detected in host cells.
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Acknowledgements |
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We thank Ana Moral for her help in the realisation of this work and the Confocal Microscopy Service of the CIB-CSIC (Madrid) where observations were made.
A P-d-L was a visiting researcher at the Plant Development group at the CIB-CSIC (Madrid) funded by the Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía. P G-M is a researcher at the CSIC funded by the programme ‘Ramón y Cajal’ of the Spanish Ministry of Education and Science.
This research was supported by the projects AGL2005-01781, Food-CT2004-50622, and BOS2002-03550.
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