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First published online August 10, 2007; 10.1104/pp.107.097089 Plant Physiology 145:437-449 (2007) © 2007 American Society of Plant Biologists Medicago truncatula as a Model for Nonhost Resistance in Legume-Parasitic Plant Interactions1,[C]Consejo Superior de Investigaciones Científicas, Institute for Sustainable Agriculture, 14080 Cordoba, Spain (M.D.L.-B., E.P., D.R.); and Instituto de Investigación y Formación Agraria y Pesquera de Andalucía (Junta de Andalucía), Centro Alameda del Obispo, Área de Mejora y Biotecnología, 14080 Cordoba, Spain (M.T.M., A.P.-d.-L.)
Crenate broomrape (Orobanche crenata) is a root parasitic weed that represents a major constraint for grain legume production in Mediterranean and West Asian countries. Medicago truncatula has emerged as an important model plant species for structural and functional genomics. The close phylogenic relationship of M. truncatula with crop legumes increases its value as a resource for understanding resistance against Orobanche spp. Different cytological methods were used to study the mechanisms of resistance against crenate broomrape of two accessions of M. truncatula, showing early and late acting resistance. In the early resistance accession (SA27774) we found that the parasite died before a tubercle had formed. In the late resistance accession (SA4327) the parasite became attached without apparent problems to the host roots but most of the established tubercles turned dark and died before emergence. The results suggest that there are defensive mechanisms acting in both accessions but with a time gap that is crucial for a higher success avoiding parasite infection.
Crenate broomrape (Orobanche crenata) is one of the most important parasitic plants attacking legume crops in Mediterranean area, devastating crops and making unusable infested land (Rubiales, 2001  , 2003; Rubiales et al., 2002). Being a broomrape (Orobanche sp.), crenate broomrape is an obligate root holoparasite lacking in chlorophyll and depending entirely on the host for its supply of nutrients (Joel et al., 2007). The knowledge of the mechanisms of resistance against the parasite is crucial to develop strategies of control, like breeding for resistance. With this purpose, we have chosen Medicago truncatula as a crenate broomrape host model plant due to its characteristics.
M. truncatula is an annual forage legume in the Mediterranean area. Contrary to other legume crops, M. truncatula is an autogamous self-fertile plant with a small and diploid genome, a short life cycle, and a prolific seed production (Blondon et al., 1994
Nowadays, the most numerous and important works about parasitic plants were focused on the development in susceptible host, as Orobanche spp. (Joel and Losner-Goshen, 1994 This lack of knowledge is due to the complexity of this interaction. The study of this host-parasite interaction presents important limitations because they both are plants, which implies sharing similar morphological, physiological, and biochemical traits. To solve it, we have chosen cytological and cytochemical techniques as powerful tools to reveal the mechanisms underlying the host-parasitic plant interaction. These studies, complemented with omic studies using M. truncatula as a host model, will be a valuable addition to our knowledge about the plant-parasitic plant interactions.
In this work we have used two M. truncatula accessions previously evaluated against crenate broomrape infection (Rodríguez-Conde et al., 2004
Minirhizotron Studies This system allowed the study of the infection process in both accessions of M. truncatula and the collection of samples at the right time for cytochemical analysis. It also permitted a quantitative study of the expression of the resistance, allowing characterization of both accessions as early and late resistant plants. Addition of GR24 to the minirhizotrons assured a high and homogeneous germination of crenate broomrape seeds (40%). A similar number of attachments was formed on both accessions during the first 3 weeks (Table I ). However, both accessions differed already by this time on percentage of crenate broomrape germinated seeds in contact with M. truncatula roots that successfully formed a tubercle, being the success on tubercle formation much lower in SA27774 (7.7%). This reaction was characterized by stoppage of parasite seedlings penetration into the host root, usually accompanied by darkening of host and/or parasite tissues around the point of attachment. On the contrary, at the same observation date, most of the attachments in SA4327 had evolved to small tubercles. As a consequence, 30 d after GR24 application there were almost no tubercles (4.3) in accession SA27774 contrasting with the number of them established on SA4327 (23.6). But a new incompatible reaction was found at this point: More than half of the tubercles (67.9%) on SA4327 had become dark and stopped their development.
Samples of both kinds of incompatible interactions and compatible interactions were collected and used for cytochemical studies.
To characterize the mechanisms of resistance setting in play by the host, different histochemical procedures were employed. For comparison, tissues of the M. truncatula accession SA4087 were included as controls. This accession is the most susceptible one to crenate broomrape attack described to date (Rodríguez-Conde et al., 2004 Sections of both successful and unsuccessful penetration attempts are presented in Figure 1 , corresponding to accessions SA4087 (Fig. 1, A and B), SA4327 (Fig. 1, C and D), and SA27774 (Fig. 1, E and F), respectively. The toluidine blue O (TBO) staining was used as a general dye to get preliminary information about the incompatible interaction. In accessions SA4087 and SA4327 the parasite was able to pierce through the cortex and penetrated into the host central cylinder, beginning the development of the haustorium. Parasite intrusive cells in SA27774 also reached the host central cylinder but some abnormalities were observed. A dark stained deposit accumulated at the interface between host and parasite and a wall thickening inside host xylem vessels next to parasite tissues was developed (Fig. 1F).
Staining of sections from the early resistance accession with alcian green safranin (AGS) confirmed that parasite intrusive cells reached the host central cylinder, but an intense red coloration of the parasite tissues (corresponding to noncarbohydrate substances) was observed (Fig. 2, A and D ), contrary to the common green staining (carbohydrates) expected for normal and healthy tissues in SA4087 (Fig. 2G). The parasite tissues also presented a disrupted and disorganized aspect. Accumulation of a red-stained material in the apoplast within and around the penetration pathway of the parasite was observed (Fig. 2, A and D) corresponding with the dark-stained material (unspecific substances) found using the TBO procedure. Host cells in contact and near the parasite tissues were impregnated with this red substance and it was also present inside some host xylem vessels (Fig. 2D). Observation of samples under UV excitation (340–380 nm) revealed a strong blue-white fluorescence from the walls and middle lamellae of cells surrounding the parasite intrusive tissues, including some xylem vessels (Fig. 2, B, C, and E). This fluorescence was shown only in host vessels near or in contact with the parasite cells (Fig. 2, C and E), but not in those located away from the infection point. The thickening of the cell walls found inside the xylem vessels also presented this fluorescence (Fig. 2E). Observation of samples under polarized light proved that the secondary thickening of xylem walls was not due to birefringence (O'Brien and McCully, 1981 ), as it is common in the case of lignified and suberized walls (Fig. 2F). Sections of tubercles becoming dark on the late resistant accession and stained with the same technique (AGS) showed a normal aspect at the first sight (Fig. 2H) compared with those corresponding to healthy tubercles (Fig. 2G). However, a few details were observed: First, the presence of a dark brown deposit in the apoplast and some vessels from the haustorium (Fig. 2J), which did not stain; and second, the presence of a slight blue-white fluorescence in host xylem walls in contact with the haustorium (Fig. 2K). The dark deposit did not present fluorescence and quenched that of the impregnated vessels (Fig. 2K). It also eliminated the birefringence of the affected vessels (Fig. 2I) under polarized light. The blue-white fluorescence was very similar to that found in the case of the early resistant accession (Fig. 2, B, C, and E).
To check the possible role of lignins and suberins in this resistance, phloroglucinol-HCl staining was used (Fig. 3 ). In compatible interactions on SA4087, xylem walls appeared with a light pink stain (Fig. 3, E and F), indicating their normal lignification. However, an intense red coloration was observed in walls of xylem vessels near the parasite intrusive cells in sections of incompatible interactions on SA27774 (Fig. 3, A and C). These vessels presented also the thickened walls observed with TBO and AGS staining. Some xylem vessels were also filled with a material that was strongly stained with this method. When observed under UV excitation (340–380 nm; Fig. 3, B and D), lignin autofluorescence was quenched by the staining and only suberin fluorescence remained. As can be seen in Figure 3, B and D, only the suberized walls corresponding to the endodermal cells showed fluorescence; lignified cell walls did not fluoresce.
To identify other mechanisms implicated in reinforcement of host cell walls against Orobanche penetration, aniline blue fluorochrome was used for identification of callose under UV excitation (340–380 nm; Fig. 4 ). Our results showed that host cell walls from the cortex (Fig. 4C) in contact with parasite intrusive tissues and some cells in the central cylinder (Fig. 4D) presented a slight accumulation of callose. No presence of callose was detected in compatible interactions (Fig. 4F).
Observations of fresh hand cut sections were taken under UV excitation (340–380 nm; Fig. 5 ) to detect the presence of phenolic compounds. No fluorescence in tissues was detected neither in uninfected roots (Fig. 5, A and B) or compatible interactions (Fig. 5, C and D). On the contrary, a strong fluorescence was found in host tissues adjacent to parasite intrusive cells (Fig. 5, E and F) and haustoria (Fig. 5, G and H) in sections of incompatible interactions.
Confocal Laser Scanning Microscopy Confocal microscopy studies were developed to get a more secure localization of phenolic compounds in tissues (Fig. 6 ). The emission spectra were collected using two channels (green and red) for the same excitation, to check differences in the fluorescence of the accumulated compounds. The fluorescence was detected within the host central cylinder (Fig. 6, A–D) and cortical cells (Fig. 6, E–H) in incompatible interactions on the early resistant accession (SA27774). Also, accumulation of phenolics was observed in the attachment organ of some parasites in contact with the host root (Fig. 6, A–D). Regarding incompatible interactions on the late resistant accession (SA4327), accumulation of phenolics was observed in the haustoria and tubercles of the parasite (Fig. 6, I–L) and within host xylem vessels connected with the parasite haustorium (Fig. 6, M–O). No presence of phenolics compounds was detected in compatible interactions on both accessions SA4327 (Fig. 6P) and SA4087 (data not shown).
Cell Viability Assay Trypan blue staining was used in fresh hand cut sections to check the viability of the cells in compatible and incompatible interactions (Fig. 7 ). In compatible interactions (Fig. 7, A and B) and uninfected roots (Fig. 7E) all the cells exclude the dye, confirming they were alive. However, the parasite intrusive cells and those corresponding to the attachment organ (Fig. 7, C and D) in incompatible interactions were clearly stained by the dye, indicating no viable cells. Moreover, parasite cells located in the distal part of the tubercle in incompatible interactions on the late resistant accession were also stained (Fig. 7F) and consequently were not viable.
Identification of Phytoalexins Thin-layer chromatography (TLC) plates showed the presence of known phytoalexins in the methanolic extracts of inoculated plants from both soluble and cell wall-bound phenolics fractions (Fig. 8 ). Medicarpin and maackiain were found in the soluble phenolic fraction of inoculated plants of both M. truncatula accessions. Scopoletin was identified in the cell wall-bound phenolic fraction of inoculated plants of M. truncatula accession SA4327. Pisatin was not detected in any of the extracts. Several other compounds appeared on the TLC plate corresponding to soluble phenolics from inoculated plants but they did not correlated with any of the phytoalexins used as standards. The retention factor of the known phytoalexins was 0.158 for scopoletin, 0.526 for pisatin, 0.595 for maackiain, and 0.632 for medicarpin.
Mechanisms of resistance against crenate broomrape were characterized in two genotypes of M. truncatula. One of them, SA27774, shows an early expression of the resistance to this parasitic plant and does not allow the establishment of the parasite. On the contrary, SA4327 is infected with crenate broomrape and allows the establishment and development of parasite tubercles. However, it is not completely susceptible to the pathogen attack and a late resistance is expressed after tubercles establishment. This does not prevent completely the development of the parasitic plant, but limits the amount of individuals of the pathogen growing on it. Both kind of incompatible interactions, appearing before or after the development of the parasite haustorium, have been previously reported in resistant host to parasitic plants (Labrousse et al., 2001 ; Serghini et al., 2001; Rubiales et al., 2003; Zehhar et al., 2003; Pérez-de-Luque et al., 2005a, 2005b, 2006a, 2006b; Echevarría-Zomeño et al., 2006). They are indicative of resistance, but the exact nature of the mechanisms underlying such resistance is not well understood. For example, stoppage of seedling penetration has been usually associated to a hypersensitive response (HR; Dörr et al., 1994; Goldwasser et al., 1997), but there is no conclusive evidence that a HR really occurs in these interactions (Rubiales et al., 2003; Pérez-de-Luque et al., 2005b) in a manner similar to that described for fungal attack (Heath, 1999; Richael and Gilchrist, 1999). For that reason, histological and histochemical studies are of great value to know what is really happening inside host tissues.
The cytological data show that the penetration of crenate broomrape in the early resistant M. truncatula accession (SA27774) is stopped once the parasite intrusive cells have reached the host central cylinder. To our knowledge, this is the first time that a prehaustorial mechanism of resistance against parasitic root plants is located inside the central cylinder of the host. Usually, the prehaustorial defensive mechanisms against parasitic plants have been located in the host cortex (Echevarría-Zomeño et al., 2006
The only physical barrier detected against the parasite intrusion is the thickening of host xylem walls. This was reported some time ago (Dörr et al., 1994
So the lack of strong physical barriers preventing the parasite penetration into the host must be complemented and reinforced by another type of defensive mechanism: The fluorescence points toward the presence of phenolic compounds (phytoalexins) as the mechanism responsible to stop parasite intrusion, as has been recently reported in the sunflower (Helianthus annuus)-crenate broomrape interaction (Echevarría-Zomeño et al., 2006
In the case of the late resistance found in SA4327, the accumulation and secretion of phenolic compounds seems to be operating but at a later stage. Once the parasite has formed a haustorium and established vascular connections with the host, the last one produces phenolic compounds that are translocated through the vascular system and reach the parasite. The first evidence for this is the presence of a dark deposit in parasite vessels of the haustorium. This deposit probably corresponds with the oxidation of phenolic compounds, which usually originates dark brown components (Takahama, 2004
All these results are in accordance with those obtained by Dita et al. (2007)
Recent studies have revealed that multiple factors are involved in resistance to parasitic plants. Behind the observation of incompatible interactions (unsuccessful attachment penetration and darkening of established tubercles) underlies a complex system of multiple mechanisms of resistance. To date, most of those described were based mainly in physical barriers preventing parasite penetration into the host central cylinder (prehaustorial mechanisms) and blocking of host vessels disrupting the nutrient fluxes between host and parasite (posthaustorial mechanisms). In this work we presented one mechanism of resistance, accumulation of phytoalexins (phenolic compounds), that does not rely on physically stopping parasite penetration into the host: The parasite penetrates reaching the central cylinder, but it seems to be poisoned and killed before developing a haustorium. Despite other mechanisms of resistance that could be involved, a crucial difference between both accessions is the moment at which the host detects and reacts against the parasite. It determines a more effective resistance against the pathogen: The earliest the parasite is detected, the most effective are the defensive mechanisms activated, and the infection is lower.
Plant Material and Growth Conditions Crenate broomrape (Orobanche crenata) was grown on accessions of Medicago truncatula showing early or late resistance to this pathogen (accessions SA27774 and SA4327, respectively).
The petri dish system described by Pérez-de-Luque et al. (2005a)
M. truncatula seeds were supplied from the South Australian Research and Development Institute Genetic Resource Centre in Australia (origin: Yugoslavia for SA4327 and Morocco for SA27774). Seeds were scarified with a metal sheet and sterilized in commercial bleach (20% in sterile water) for 10 min. For synchronize germination, seeds were place at 4°C for 36 h in sterile water. During this period, we replaced sterile water two or three times to help germination. After that, seeds were rinsed with sterile water at room temperature for 3 or 4 h, changing it six or eight times. Finally, seeds were placed in petri dishes on wet glass fiber filter papers (Whatmann GF/A) and kept in darkness at 20°C for 1 to 2 d. When the radicle reached 2 cm length, seedlings were transferred to new dishes (15 cm diameter) with perlite and new glass fiber papers (Pérez-de-Luque et al., 2005a
Crenate broomrape seeds were collected from infected faba bean (Vicia faba) plants at Córdoba during 2000. They were disinfected with formaldehyde according to González-Verdejo et al. (2005)
At the same time plants were growing, crenate broomrape seeds were conditioned. For conditioning, parasite seeds need to be in darkness at 20°C for 10 d (Pérez-de-Luque et al., 2005a
After conditioning period, we applied 5 mL of the synthetic stimulant GR24 (1 mg/mL; Magnus et al., 1992
The infection process was followed using a binocular microscope (Nikon SMZ1000; Nikon Europe B.V.). Fifteen days after GR24 application, the percentage of crenate broomrape attach seedlings on M. truncatula roots was calculated. The total of 200 crenate broomrape seedlings close (<3 mm) to the M. truncatula roots were visualized in each petri dish and the number of attached seedlings was referred to the total number of seedlings. At 22 d after GR24 application, the percentage of compatible and incompatible attachments against total attachments was scored. An attachment was considered compatible when it resulted in tubercle formation. Finally, 30 d after GR24 application established broomrapes were quantified and expressed as absolute value per plant. In addition, the number of darkened tubercles was recorded and expressed as a percentage respect to the total number of established tubercles per plant.
Observations were taken using a binocular microscope. At 15 d after GR24 application, seedlings of crenate broomrape were sampled at random with the corresponding attached parts of host roots. For staining methods and confocal laser scanning microscopy, the samples were fixed in 4% formaldehyde in phosphate-buffered saline (PBS), pH 7.3 at 4°C overnight. After washing in PBS (3 x 15 min), they were stored in 0.1% formaldehyde in PBS at 4°C. Fixed samples were then dehydrated in ethanol series (50%, 80%, 95%, 100%, and 100%: 12 h each) and transferred to an embedding solvent (Xylene; Panreac Quimica S.A.) through a xylene-ethanol series (30%, 50%, 80%, 100%, 100%: 12 h each) and finally saturated with paraffin (Paraplast Xtra; Sigma). Seven micrometer-thick sections were cut with a rotary microtome (Nahita 534; Auxilab S.A.) and attached to adhesive-treated microscope slides (polysine slides; Menzel GmbH & Co. KG).
After removal of paraffin, sections were stained with different dyes: (1) Staining with 0.05% TBO in PO4 buffer (pH 5.5) during 5 to 10 min was used. In this case the dye was applied before removal of paraffin (Ruzin, 1999 Transverse sections were observed using a light microscope (Leica DM-LB, magnification x100–x400; Leica Microsystems Wetzlar GmbH) and photographed using a digital camera (Nikon DXM1200F; Nikon Europe B.V.). The samples were also observed by epifluorescence under excitation at 340 to 380 nm (UV).
Hand cut sections (with a razor blade) were obtained from fresh root samples to observe accumulation of phenolic compounds by epifluorescence under excitation at 340 to 380 nm (UV).
Ten fresh samples of crenate broomrape seedlings with the corresponding attached parts of host roots were immersed in a solution of 0.1% (w/v) diphenyl boric acid 2-aminoethylester (Naturstoffreagenz A: NA) in buffer (100 mM KPi pH 6.8, 1% NaCl [w/v]) according to Hutzler et al. (1998)
Trypan blue dye exclusion was used to assess cell viability. Samples were immersed in a commercial Trypan Blue solution of 50% (Sigma) and observed using a light microscope. Viable (alive) and nonviable (dead) cells were identified microscopically under bright field optics, as those that had excluded and taken up the trypan blue stain, respectively.
The petri dish system was used for plant material collection from M. truncatula accessions showing early or late resistance (SA27774 and SA4327, respectively) to crenate broomrape as described before. M. truncatula roots from noninfected and infected plants were sampled 30 d after GR24 application, the date corresponding to establishment of the parasite. For noninfected plants, small root pieces (approximately 1 cm) were sampled at random. For infected plants, parts of the root with a crenate broomrape attachment were sampled, removing the parasite tissues. Then, samples were washed with tap and then distilled water, blotted dry with filter paper, frozen in liquid nitrogen, and stored at –80°C until biochemical analysis (Pérez-de-Luque et al., 2005a Frozen root tissue (0.04 g fresh weight) was homogenized in 1 mL methanol by using a pestle and mortar. After filtering off the solvent extract, the residue was further sequentially extracted twice with a similar volume of methanol and centrifuged twice at 15,000g for 15 min. The combined solvent extracts were dried and phenolic compounds were resuspended in 0.24 mL of methanol. The pellet residue was resuspended in 0.08 mL of 2 M NaOH and incubated at 70°C for 16 h. The suspension was cooled down, neutralized with 0.08 mL of 2 M HCl, and centrifuged (15,000g for 15 min). Then, the suspension was dried and resuspended in the same volume of methanol.
TLC analysis of the methanolic extract was performed according to Prats et al. (2003)
Minirhizotron assays were performed with two plants per petri dish and 10 petri dishes per M. truncatula accession (SA27774 and SA4327). Data were recorded from three randomized areas in each plant. To study possible interactions due to cultivation of plants in different dishes, each petri dish was treated as a block and an ANOVA was performed (Statistix v1.1 for Windows). No significant differences were found between blocks so each petri dish was considered as a replicate. For cytochemical and confocal studies, at least 10 samples were selected at random from several petri dishes for each study. Percentages were transformed according to the formula Y = arcsin(
We thank the microscopy service of the University of Córdoba-Servicio Centralizado de Apoyo a la Investigación where confocal laser microscopy observations were made. Received February 1, 2007; accepted August 6, 2007; published August 10, 2007.
1 This work was supported by the project FP6–2002–FOOD–1–5062232004–2008. A.P.-d.-L. is a researcher at the Instituto de Investigación y Formación Agraria y Pesquera de Andalucía funded by the program Juan de la Cierva of the Spanish Ministry of Education and Science.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Alejandro Pérez-de-Luque (bb2pelua{at}uco.es).
[C] Some figures in this article are displayed in color online but in black and white in the print edition. www.plantphysiol.org/cgi/doi/10.1104/pp.107.097089 * Corresponding author; e-mail bb2pelua{at}uco.es.
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-glucans elicitors (Esquerré-Tugayé et al., 2000
[X%/100]) prior to statistical treatment. 
