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Immunocytochemical localization of β-1,3-glucanase and chitinase in Fusarium culmorum -infected wheat spikes
Physiological and Molecular Plant Pathology (2002) 60, 141±153 doi:10.1006/pmpp.2002.0386, available online at http://www.idealibrary.com on
Immunocytochemical localization of b-1,3-glucanase and chitinase in Fusarium culmorum-infected wheat spikes Z . K A N G * and H . B U C H E N A U E R Institute of Phytomedicine (360), University Hohenheim, D-70593, Stuttgart, Germany (Accepted for publication 11 February 2002) Two antisera raised against acidic b-1,3-glucanase and acidic chitinase from tobacco were used to investigate the subcellular localization of the two enzymes in Fusarium culmorum-infected wheat spike by means of the immunogold labelling technique. The studies demonstrated that the distribution of b-1, 3-glucanase and chitinase were very similar in the uninoculated healthy and infected wheat spikes. The enzymes were localized mainly in the cell walls of dierent tissues including the lemma, ovary and rachis of the wheat spike, while the cytoplasm and organelles of cells in these tissues showed almost no labelling. However, the accumulation of b-1,3-glucanase and chitinase in the infected wheat spikes diered distinctly between resistant and susceptible wheat cultivars. The labelling densities for the two enzymes in the infected lemma, ovary and rachis of the susceptible cultivar Agent increased only slightly as compared to the corresponding uninoculated healthy tissues, whereas higher labelling densities of b-1,3-glucanase and chitinase were found in the infected tissues of wheat spikes from the resistant cultivar Arina compared to the corresponding uninoculated healthy tissues. Furthermore, the labelling of b-1,3-glucanase and chitinase also occurred over the cell walls of the hyphae in the infected wheat spike, but not over the hyphal cytoplasm. In addition, labelling for the two enzymes was often detected over the cell wall appositions and the electron-dense material located between the host cell and the hyphal cell in the infected tissues of the resistant wheat cultivar. The ®ndings reported in the present study indicate that b-1,3-glucanase and chitinase accumulation in the F. culmorum-infected wheat spike may be involved in c 2002 Elsevier Science Ltd. * resistance to pathogen spread in the host tissue. Keywords: b-1,3-glucanase; chitinase; immunocytochemistry; resistance; Triticum aestivum; Fusarium culmorum.
INTRODUCTION Plant responses to fungal attack involve rapid induction of defence mechanisms including morphological, structural and biochemical changes, such as deposition of lignin, callose and phenolic compounds, formation of papillae or cell wall appositions, and synthesis of pathogenesis related (PR)-proteins [1, 3, 4, 27]. Among PR-proteins, two plant hydrolases, b-1,3-glucanase and chitinase, have been intensively studied for their accumulation in the infected plant tissues and their function in plant defence reactions in dierent fungal pathogen±plant systems [3±5, 9, 10, 16, 17, 29, 34, 35]. These two enzymes are of particular interest because many pathogenic fungi contain b-1,3-glucans and chitin as major structural cell wall components [32]. It had been demonstrated in vitro * To whom correspondence should be addressed. Biotech Center and Plant Protection College, Northwestern Sci-Tech University of Agriculture & Forestry, Yangling, Shaanxi 712100, People's Republic of China. Abbreviations used in text: DON, deoxynivalenol; FHB, Fusarium head blight; PR, pathogenesis related.
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that the two enzymes b-1,3-glucanase and chitinase are able to degrade fungal wall components, resulting in growth inhibition of fungi [2, 18, 36]. Furthermore, breakdown products of fungal wall components, released by the activity of the two enzymes, have been shown to act as elicitors of plant defence responses [8, 15, 30]. Studies on the subcellular localization of b-1,3-glucanase and chitinase in planta showed that these hydrolases accumulated at higher concentrations in infected host plant tissues and on fungal cell walls, especially, at sites where host cells were in close contact with fungal hyphae. In incompatible plant±pathogenic fungal interactions higher activities of both hydrolytic enzymes were detected compared with the compatible interactions [3, 4, 9, 29, 34]. This indicates that b-1,3-glucanse and chitinase could play an important role in the active defence of plants against the fungal pathogens. The infection process and route of F. culmorum colonization of wheat spikes has been elucidated using light and electron microscopy [12]. Our cytochemical analyses con®rmed that F. culmorum also produced cell wall degrading enzymes at early stages of infection [13]. c 2002 Elsevier Science Ltd. *
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During infection of wheat spikes by Fusarium graminearum and F. culmorum, the pathogens can produce trichothecene mycotoxins, such as deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) [19, 21, 28]. Our immunogold labelling studies revealed a relationship between the accumulation of Fusarium toxins in the infected wheat spike and the pathogenic changes in the host cells, symptom appearance and the colonization by the pathogen in the host tissues [11]. Studies on the toxicity of Fusarium trichothecenes in eukaryotic cells indicated that the toxin inhibited protein synthesis [25, 33]. Fusarium trichothecene toxins are phytotoxic [7, 25, 31] and may be regarded as virulence factors involved in development of Fusarium head blight (FHB) [23]. More recently, Miller and Ewen [20] reported that DON inhibited protein synthesis in vitro through binding to ribosomes isolated from wheat leaves. It is well known that plant defence responses to pathogen attack depend on rapid and intensive transcription and translation reactions of host tissues. Thus, it seems likely that DON may interfere in post-infectional defence reactions. Furthermore, cytological examinations revealed that F. culmorum grew more slowly in resistant wheat cultivars than in the susceptible cultivar, and plant structural defence reactions such as the formation of thick layered appositions and large papillae were essentially more pronounced in the infected host tissues of resistant cultivars than in the susceptible one [14], indicating that the FHB resistant cultivars are able to develop active structural defence reactions during infection and spreading of the pathogen in the host tissue. In order to elucidate further mechanisms involved in defence responses of wheat to FHB, it was therefore of interest to study the accumulation of b-1,3-glucanase and chitinase in wheat spikes infected by F. graminearum or F. culmorum. The localization of both hydrolytic enzymes in the wheat spikes after infection by Fusarium spp. has not been studied so far. However, the accumulation and
distribution of b-1,3-glucanase in wheat leaves infected by rust fungi has been reported [9, 29]. In the present study, we describe the subcellular localization of b-1,3glucanase and chitinase in wheat spikes of two wheat cvs. diering in their susceptibility to F. culmorum by means of immunogold labelling techniques. MATERIALS AND METHODS
Plant material, fungal strain and inoculation Two winter wheat cultivars, Agent (susceptible) and Arina (resistant), were used throughout the present experiments. The wheat plants were grown as described previously [11]. When the plants were at the middle stage of ¯owering (GS 65) [37], the wheat spikes were inoculated with macroconidia of F. culmorum (isolate No. 46; Institute of Phytomedicine, University Hohenheim, Germany) [11]. For inoculation, the macroconidia suspension was adjusted to 1 105 spores ml 1. The wheat spikes were inoculated by pipetting 10 ml of a macroconidia suspension into the ¯oral cavity between the lemma and palea of the ®rst ¯oret of spikelets. Every third ¯oret at one side of the spike was inoculated. Control plants were inoculated with distilled water instead of the macroconidial suspension. Inoculated spikes were covered with plastic bags and plants were transferred into a growth chamber with 16 h ¯uorescent light at 238C and 8 h darkness at 188C. Two days later the plastic bags were removed and the plants were returned to outdoor conditions.
Tissue processing for electron microscopy The pathogen inoculated and control inoculated ¯orets were sampled after 3, 5 and 8 days covering speci®c stages of the infection and colonization process [12]. At the same times samples of healthy, uninoculated tissues were taken. The lemmas, ovaries and rachis were excised from the
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F I G S . 1±6. Immunogold localization of b-1,3-glucanase in the uninoculated healthy and F. culmorum-infected wheat spike of the susceptible cv. Agent. F I G . 1. A parenchyma cell in the lemma from the uninoculated wheat spike, 3 days after treatment (dat) with water. A low density of gold particles was localized over the cell wall while the cytoplasm and organelles were almost free of gold particles. F I G . 2. Parenchyma cells in the ovary from the uninoculated wheat spike, 3 dat. The cell walls showed a few gold particles. F I G . 3. A cortical cell in the rachis from the uninoculated wheat spike, 5 dat. Gold particles were deposited over the cell wall, but not over the cytoplasm. F I G . 4. Xylem vessel in the rachis from the uninoculated wheat spike, 5 dat. Dense gold particles were observed over the secondary thickening. F I G . 5. Infected lemma, 3 dai by F. culmorum. A low density of gold particles was found over the plant cell walls. The hyphal cell wall also showed a low density of gold particles. F I G . 6. Infected ovary, 3 dai. Few gold particles were observed over the parenchyma cell walls and the hyphal cell wall. All bars 0.5 mm. CH: chloroplast; H: hyphal cell; ST: secondary thickening; X: xylem vessel.
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¯orets and ®xed in 3 % (v/v) glutaraldehyde in 50 mM phosphate buer ( pH 6.8) for 3±6 h at 48C. Thereafter, the samples were thoroughly rinsed with 50 mM phosphate buer ( pH 6.8) and post-®xed with 1 % (w/v) osmium tetroxide in the same buer for 2 h at 48C. The samples were then dehydrated in a graded ethanol series, embedded in LR White (TAAB Laboratories, Munich, Germany) and polymerized at 508C for 2 days. Ultrathin sections of the samples were cut with a diamond knife and collected on 200-mesh nickel grids for immunogold labelling. The experiments were repeated three times.
Immunogold labelling for b-1,3-glucanase and chitinase The rabbit polyclonal antisera against acidic b-1,3glucanase and acidic chitinase from tobacco were obtained from Prof. Fritig (C.N.R.S., Institut de Biologie Moleculaire des Plantes, Strasbourg, France) and showed high speci®city with b-1,3-glucanase and chitinase, respectively, from wheat leaves and shoots by western blotting as demonstrated by Kemp et al. [16] and Siefert [26]. The secondary antibodies (goat anti-rabbit IgG) coupled to 15 nm gold particles were purchased from British Biocell International Ltd (Cardi, U.K.). Immunogold labelling was carried out as follows: (i) incubation of ultrathin sections with blocking solution containing 1 % (w/v) of BSA in Tris-buered saline (TBS, 10 mM Tris±HCl, 150 mM NaCl, pH 7.4) for 20 min; (ii) incubation of the sections with the primary antibody diluted at 1 : 200 in the blocking solution for 2 h at room temperature; (iii) washing in four 10 min baths in TBS; (iv) incubation of the sections with the secondary antibody diluted at 1 : 40 in TBS; (v) rinsing with TBS followed by a distilled water rinse. After contrasting with uranyl acetate and lead citrate, the sections were examined with a ZeissEM10 electron microscope at 80 kV.
Immunocytochemical controls Speci®city of labelling was assessed by the following control tests: (i) incubation of the ultrathin sections with the rabbit pre-immune serum instead of the primary antibody; and (ii) incubation with the secondary antibody and omitting the primary antibody step.
Quanti®cation of labelling The labelling densities of gold particles detecting b-1,3glucanase and chitinase over cell walls in dierent tissues of uninoculated and infected wheat spikes were determined by counting the number of gold particles mm 2 over speci®ed cell wall areas on 10±15 microphotographs. The dierence in gold particles between uninoculated healthy and infected host tissues were statistically analysed by the t-test. RESULTS
Immunocytochemical localization of b-1,3-glucanase in uninoculated healthy and infected wheat spikes Incubation of ultrathin sections of dierent tissues from the uninoculated wheat spikes with anti-b-1,3-glucanase antiserum and the secondary antibody resulted in labelling of b-1,3-glucanase. The labelling patterns and labelling densities in the healthy tissues were very similar between the susceptible wheat cv. Agent and resistant cv. Arina, 3 days after treatment (dat) with water. A low density of gold particles was detected over the cell walls in the lemma and ovary, whereas the cytoplasm and organelles such as nuclei, Golgi bodies, mitochondria and chloroplasts showed very few gold particles (Figs 1 and 2). In the rachis of the uninoculated wheat spike of cv. Agent (5 dat with water) and cv. Arina (8 dat with water), gold particles were unevenly deposited over the walls of the cortical cells,
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F I G S . 7±11. Immunogold localization of b-1,3-glucanase in F. culmorum-infected wheat spikes of the susceptible cv. Agent and the resistant cv. Arina, 3 and 5 dai. F I G . 7. Infected lemma of the resistant cv. Arina, 3 dai. Appositions were formed between the cell wall and the plasmalemma of the host cells which were in contact with fungal cells. A high density of gold particles was found over the plant cell walls. The hyphal cell also showed more gold particles over the hyphal wall, especially at the region of contact with the host cell (arrowhead). F I G . 8. Infected ovary of the resistant cv. Arina, 3 dai. Marked wall appositions had been formed between the cell wall and the plasmalemma in the host cells (arrowheads). Many gold particles were deposited over host cell walls, wall appositions and the hyphal cell walls, while the host cytoplasm and the hyphal cytoplasm showed almost no labelling. F I G . 9. Infected lemma of the resistant cv. Arina, 3 dai. Gold particles were deposited over the host cell wall, hyphal wall, wall appositions and the electron-dense material located between the host cell and the hyphal cell (arrowhead). F I G . 10. A cortical cell in the infected rachis from the susceptible cv. Agent, 5 dai. A low density of gold particles was deposited over the host cell wall and the hyphal cell wall. F I G . 11. An infected xylem vessel in the rachis of the susceptible cv. Agent, 5 dai. Dense gold particles were observed over the secondary thickenings in the vessel. All bars 0.5 mm. H: hyphal cell; AP: wall apposition; ST: secondary thickening; X: xylem vessel.
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T A B L E 1. Labelling density of gold particles conjugated with the anti-b-1,3-glucanase antibody over cell walls in dierent tissues of non-inoculated healthy and F. culmorum-infected wheat spikes in the cvs. Agent and Arinaa Agent Healthy Parenchyma cell wall of lemma Parenchyma cell wall of ovary Cortical cell wall of rachis
7.25b 5.16 8.82
Arina Infected 7.86 5.78 9.40
Healthy nc n n
7.12 5.04 8.60
Infected 22.40 13.46 27.66
sd s s
a Densities of labellings in the cell walls of the lemma and the ovary of both cvs. were determined 3 dai and in the cortical cell walls of the rachis of cv. Agent 5 dai and in the cv. Arina 8 dai. The labelling density was expressed by the number of gold particles per mm2. b Mean values (numbers) of the gold particles determined per mm2. The average densities for uninoculated healthy and infected tissues were compared by t-test, P 0.05. c n Statistically not signi®cant dierent values in the same row. d s Statistically signi®cant dierence values in the same row.
xylem vessels and sieve tubes in the vascular bundles, while cytoplasm and organelles of these tissues were almost free of labelling (Figs 3 and 4). However, the secondary thickenings of the xylem vessels usually showed more gold particles than the walls of the other cells in the rachis. Three days after inoculation with conidia of F. culmorum, the hyphae spread inter- and intracellularly in the ovary, and only intercellularly in the lemma tissue. Pronounced formations of wall appositions and papillae in the host cells were often observed in the infected tissues of the resistant cv. Arina (Figs 7 and 8), but not in the susceptible cv. Agent (Figs 5 and 6). Labelling studies with b-1,3-glucanase revealed similar patterns of the infected host tissues of both the susceptible cv. Agent and the resistant cv. Arina compared to the uninoculated host tissues. However, the labelling densities in the infected host tissues varied markedly between the resistant and susceptible cultivars. In the susceptible cv. Agent, the labelling density over the cell walls of lemma and ovary showed only a slight increase in b-1,3-glucanase labelling as compared to the corresponding uninoculated healthy tissues (Figs 5 and 6, Table 1). On the other hand, gold
particles over the cell walls of the infected lemma and the ovary of the resistant cv. Arina increased considerably compared to the corresponding uninoculated host tissues (Figs 7±9, Table 1). Gold particles were usually detected over the wall appositions ( papillae) formed between the plant cell wall and plasmalemma and over the electrondense material located between the hyphal cell and the host cell in the resistant cv. Arina (Figs 8 and 9). In fungal cells in infected host tissues, either from the resistant or from the susceptible cultivar, the hyphal walls showed uneven labelling while the hyphal cytoplasm displayed only few gold particles. Five days after inoculation, more hyphae were found in the infected ovary and lemma. The inter- and intracellular growth of the hyphae resulted in severe damages of the cells of the susceptible cv. Agent, such as disintegration of the cytoplasm and organelles, whereas most cells of the infected tissue of the resistant cv. Arina were still intact. The pathogen reached the rachis of the susceptible cv. Agent from the infected ovary and lemma 5 days after inoculation, whereas it took 8 days for the pathogen to reach the rachis in the resistant cv. Arina. The hyphae
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F I G S . 12 and 13. Immunogold localization of b-1,3-glucanase in F. culmorum-infected wheat spikes of the resistant cv. Arina, 8 dai. F I G . 12. A cortical cell in the infected rachis. Many gold particles were observed over the plant cell wall.
F I G . 13. An infected xylem vessel in the rachis. A high density of gold particles was deposited over the secondary thickenings of the vessel and over the hyphal wall, while the electron-dense coating materials on the secondary thickenings showed only a few gold particles. F I G S . 14±17. Immunogold localization of chitinase in the uninoculated healthy wheat spikes of the susceptible cv. Agent. F I G . 14. A parenchyma cell in the lemma. The cell wall showed a low density of gold particles, whereas the cytoplasm and organelles were almost free of labelling. F I G . 15. Parenchyma cells in the ovary. A few gold particles were observed over the walls, but not over the cytoplasm. F I G . 16. A cortical cell in the rachis. The cell wall was labelled with gold particles. F I G . 17. A xylem vessel in the rachis. Many gold particles were found over the secondary thickening of the vessel. All bars 0.5 mm. CH: chloroplast; H: hyphal cell; ST: secondary thickening; X: xylem vessel.
Immunocytochemical localization of b-1,3-glucanase and chitinase extended inter- and intracellularly in the cortical tissue and vascular bundles of the rachis (Figs 10±13). The labelling patterns for b-1,3-glucanase in the infected and uninfected rachis tissues were similar, but the increase in the labelling density in the infected rachis diered markedly between the susceptible and resistant wheat cultivar. The number of gold particles over the cell walls
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of the infected rachis of the resistant cv. Arina was signi®cantly higher than on the walls of the uninoculated healthy host cells, whereas the labelling density over the cell walls of the infected rachis of the susceptible cv. Agent was only slightly increased compared to the corresponding uninoculated host tissues (Figs 10±13, Table 1). In addition, a high density of gold particles
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T A B L E 2. Labelling density of gold particles conjugated with the anti-chitinase antibody over cell walls in dierent tissues of noninoculated healthy and F. culmorum-infected wheat spikes in the cvs. Agent and Arinaa Agent Healthy Parenchyma cell wall of lemma Parenchyma cell wall of ovary Cortical cell wall of rachis
6.80b 4.94 7.92
Arina Infected 7.72 5.66 8.60
Healthy nc n n
6.68 5.20 8.38
Infected 18.00 11.64 23.42
sd s s
a
Densities of labellings in the parencyhma cell walls of the lemma and the ovary of both cvs. were determined 3 dai and in the cortical cell walls of the rachis of cv. Agent 5 dai and in the cv. Arina 8 dai. The labelling density was expressed by the number of gold particles per mm2. b Mean values (numbers) of the gold particles determined per mm2. The average densities for uninoculated healthy and infected tissues were compared by t-test, P 0.05. cn Statistically not signi®cant dierent values in the same row. d s Statistically signi®cant dierence values in the same row.
was often detected over the secondary thickenings of the xylem vessels in the infected rachis of the resistant cv. Arina, while the electron-dense coating material on the secondary thickenings showed only few gold particles or was free of labelling (Fig. 13).
Immunocytochemical localization of chitinase in uninoculated healthy and infected wheat spikes Incubation of the sections of uninoculated healthy wheat spikes with anti-chitinase antiserum and the secondary antibody resulted in irregular labelling over the cell walls of the host tissues while the cytoplasm and organelles such as chloroplasts, mitochondria and vacuoles in the host tissues showed few gold particles or were free of labelling. The labelling patterns and labelling densities for chitinase in the host tissues did not dier between the resistant cv. Arina and the susceptible cv. Agent. A low density of gold particles was observed over the cell walls in the lemma, ovary and cortical tissue of the rachis (Figs 14±16), whereas more gold particles were usually
deposited over the secondary thickenings of the xylem vessel in the rachis (Fig. 17). Three days after inoculation, gold labelling of chitinase in the infected host tissues and in the hyphal cells showed dierent labelling densities. In the fungal cells, gold particles were usually deposited over the walls while the hyphal cytoplasm showed almost no labelling. In the susceptible cv. Agent, a low density of gold particles was unevenly deposited over the host cell walls and the hyphal cell walls in the infected lemma and ovary (Figs 18 and 19). The density of the gold particles demonstrated a slight increase over the cell walls in the infected compared to uninoculated tissues, but the values were not signi®cantly dierent from those of the corresponding uninoculated healthy tissues (Table 2). On the other hand, in the infected lemma and ovary of the resistant cv. Arina a high density of gold particles was observed over the host cell wall as well as over the hyphal walls, particularly within the area of contact of the host and fungal cell (Figs 20±22). A signi®cant increase in gold particles over the cell walls in the infected tissues of the resistant cv. Arina was found, as compared to the uninoculated healthy
Y F I G S . 18±22. Immunogold localization of chitinase in the F. culmorum-infected wheat spikes of the susceptible cv. Agent and the resistant cv. Arina. F I G . 18. Infected lemma from the susceptible cv. Agent, 3 dai. The plant cells were labelled with a low density of gold particles over the cell walls, but not over the cytoplasm and organelles. The hyphal cell wall showed a few gold particles. F I G . 19. Infected ovary from the susceptible cv. Agent, 3 dai. A few gold particles were deposited over the plant cell walls and the hyphal cell wall. F I G . 20. Infected lemma of the resistant cv. Arina, 3 dai. Many gold particles were deposited over the plant cell wall and the wall apposition. The hyphal cell wall in contact with the host cell showed more gold particles (arrowhead). F I G . 21. Infected lemma of the resistant cv. Arina, 3 dai. The electron-dense materials located between the host cell and the hyphal cell showed gold labelling (arrowhead). F I G . 22. Infected ovary of the resistant cv. Arina, 3 dai. Gold particles were observed over the host cell wall, wall appositions (arrowhead) and the hyphal cell walls, whereas the host cytoplasm and the hyphal cytoplasm showed almost no labelling. All bars 0.5 mm. CH: chloroplast; H: hyphal cell; AP: wall apposition.
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tissues (Table 2). Also, wall appositions in the host cells and electron-dense material between the host cell and hyphal cell in the intercellular space of the infected tissues usually showed intense gold labelling (Fig. 21). Five days after inoculation, the labelling patterns and labelling densities for chitinase in the infected lemma and ovary from the resistant cv. Arina and the susceptible cv. Agent did not dier markedly from those of the corresponding infected host tissues three days after inoculation. Gold labelling of the infected rachis tissue was detected over the walls of the cortical cells, xylem vessels and sieve tubes in the vascular bundles, and also over the walls of the hyphal cells. More gold particles accumulated over the cell walls of the infected rachis of the resistant cv. Arina as compared to the susceptible cv. Agent (Figs 23±26, Table 2). Labelling was also often detected over the electron-dense material between host cells and hyphal cells in the intercellular space in the infected rachis of the resistant cv. Arina (Fig. 25). Although the coating material on the secondary thickenings of xylem vessels in the infected rachis of the resistant cv. Arina displayed few gold particles or were free of labelling, the secondary thickenings of xylem vessels in the same tissues were usually labelled with a high density of gold particles (Fig. 26).
Immunocytochemical controls Following incubation of the sections of uninoculated healthy and infected wheat spikes with pre-immune serum and the secondary antibody, or with the secondary antibody alone, no labelling was observed over the host plant tissues and over the hyphal cells (Fig. 27).
DISCUSSION The studies demonstrated the subcellular distribution of two plant hydrolases, b-1,3-glucanase and chitinase in
uninoculated healthy and F. culmorum-infected wheat spikes by means of the immunogold labelling technique. The antibodies used were raised against acidic b-1,3glucanase and acidic chitinase from tobacco. The two hydrolytic enzymes were localized mainly in the cell walls of dierent spike tissues including the lemma, ovary and rachis while the cytoplasm and organelles in these tissues were almost free of labelling. This indicates that the two enzymes accumulate extracellularly in the infected wheat spikes. The described labelling pattern is in accordance with an earlier report that acidic b-1,3-glucanase and acidic chitinase accumulated extracellularly in tomato leaves infected by Cladosporium fulvum, suggesting that they might play a role in the successful defence of the tomato plants [10] against pathogenic fungi. While labelling of the two enzymes in the infected tissues of wheat spikes of the susceptible cv. Agent was only slightly increased compared to the corresponding uninoculated healthy tissues, the number of gold particles in the infected lemma, ovary and rachis of the resistant cv. Arina was signi®cantly higher than in corresponding uninoculated healthy tissues. Thus, the study revealed that the labelling densities for b-1,3-glucanase and chitinase were especially pronounced over the hyphal cell walls where the hyphae were in contact with the host cells. From this it can be concluded that the two hydrolases may diuse from host cells to the hyphal cell surface and may act synergistically in hydrolysing fungal cell wall components and thus interfering with hyphal growth. This situation is similar to ®ndings in tomato and eggplant roots infected by F. oxysporum [3, 4], tomato leaves infected by C. fulvum [10] and wheat leaves infected by Puccinia recondita [9]. Mauch et al. [18] showed that the fungal growth in vitro was inhibited synergistically by combination of plant chitinase and b-1,3-glucanase compared to the single enzyme treatments. Chitin, one of the main components in the fungal walls, is considered to be embedded in a matrix of amorphous material including b-glucans [6], which makes the chitin inaccessible to
Y F I G S . 23±26. Immunogold localization of chitinase in the F. culmorum-infected wheat spikes of the susceptible cv. Agent and resistant cv. Arina. F I G . 23. A cortical cell in the infected rachis from the susceptible cv. Agent, 5 dai. The host cell wall and the hyphal cell wall were labelled with a low density of gold particles. F I G . 24. An infected xylem vessel in the rachis of the susceptible cv. Agent, 5 dai. The secondary thickenings of the vessel showed numerous gold particles. F I G . 25. A cortical cell in the infected rachis from the resistant cv. Arina, 8 dai. Many gold particles were deposited over the plant cell walls and the hyphal wall. The materials located between the plant cell and the hyphal cell were also labelled with gold particles. F I G . 26. An infected xylem vessel in the rachis of the resistant cv. Arina, 8 dai. Numerous gold particles were deposited over the secondary thickenings of the vessel, but not over the electron-dense coating materials on the secondary thickenings. The hyphal cell wall in contact with the host cell showed a dense labelling with gold particles. F I G . 27. Immunocytochemical control test. The infected lemma from the resistant cv. Arina, 3 dai, incubated with pre-immuno antiserum and the secondary antibody. No labelling is found over the plant cell and the hyphal cell. All bars 0.5 mm. H: hyphal cell; ST: secondary thickening; X: xylem vessel.
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chitinase. It has been demonstrated that preliminary treatment with b-1,3-glucanase renders chitin more susceptible to chitinase in Schizophyllum commune [32]. Benhamou et al. [3, 4] also suggested that previous alteration of fungal cell walls by the in¯uence of b-1,3glucanase would make chitin more accessible to plant chitinase in the F. oxysporum-tomato system. To what extent the synthesis of b-1,3-glucanase and chitinase in the infected plant tissues might be aected by the trichothecenes produced and secreted by the pathogens F. culmorum or F. graminearum already at early stages of infection of wheat spikes [11] needs further investigation, since it is known that the trichothecene toxins interfere in protein synthesis of wheat leaves [20]. Besides the accumulation of b-1,3-glucanase and chitinase, further mechanisms might be involved in resistance of wheat spikes to F. culmorum or F. graminearum. Recently, we found that lignin and callose depositions were higher in the F. culmorum-infected wheat spike tissues of the resistant wheat cvs. Frontana and Arina than in the susceptible cv. Agent [14]. Furthermore, Pritsch et al. [22] reported that the defence response genes enconding peroxidase, PR-1, PR-4 and PR-5 (thaumatin-like protein) were also transcribed in F. graminearum-infected wheat spikes at early stages of infection. These ®ndings suggest that b-1,3-glucanase and chitinase as well as the structural cell wall alterations (like depositions of lignin, thick layered appositions and papillae) are implicated in defence reactions and eventually contribute to resistance of wheat spike tissues against F. culmorum. The authors wish to thank Prof. Fritig (C.N.R.S., Institut de Biologie Moleculaire des Plantes, Strasboug, France) for kindly supplying antibodies, and H. Brandl for excellent technical assistance. G. Moll is gratefully acknowledged for preparing photographs. This study has been ®nancially supported by the Deutsche Forschungsgemeinschaft (DFG).
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Immunocytochemical localization of b-1,3-glucanase and chitinase in Fusarium culmorum-infected wheat spikes Z . K A N G * and H . B U C H E N A U E R Institute of Phytomedicine (360), University Hohenheim, D-70593, Stuttgart, Germany (Accepted for publication 11 February 2002) Two antisera raised against acidic b-1,3-glucanase and acidic chitinase from tobacco were used to investigate the subcellular localization of the two enzymes in Fusarium culmorum-infected wheat spike by means of the immunogold labelling technique. The studies demonstrated that the distribution of b-1, 3-glucanase and chitinase were very similar in the uninoculated healthy and infected wheat spikes. The enzymes were localized mainly in the cell walls of dierent tissues including the lemma, ovary and rachis of the wheat spike, while the cytoplasm and organelles of cells in these tissues showed almost no labelling. However, the accumulation of b-1,3-glucanase and chitinase in the infected wheat spikes diered distinctly between resistant and susceptible wheat cultivars. The labelling densities for the two enzymes in the infected lemma, ovary and rachis of the susceptible cultivar Agent increased only slightly as compared to the corresponding uninoculated healthy tissues, whereas higher labelling densities of b-1,3-glucanase and chitinase were found in the infected tissues of wheat spikes from the resistant cultivar Arina compared to the corresponding uninoculated healthy tissues. Furthermore, the labelling of b-1,3-glucanase and chitinase also occurred over the cell walls of the hyphae in the infected wheat spike, but not over the hyphal cytoplasm. In addition, labelling for the two enzymes was often detected over the cell wall appositions and the electron-dense material located between the host cell and the hyphal cell in the infected tissues of the resistant wheat cultivar. The ®ndings reported in the present study indicate that b-1,3-glucanase and chitinase accumulation in the F. culmorum-infected wheat spike may be involved in c 2002 Elsevier Science Ltd. * resistance to pathogen spread in the host tissue. Keywords: b-1,3-glucanase; chitinase; immunocytochemistry; resistance; Triticum aestivum; Fusarium culmorum.
INTRODUCTION Plant responses to fungal attack involve rapid induction of defence mechanisms including morphological, structural and biochemical changes, such as deposition of lignin, callose and phenolic compounds, formation of papillae or cell wall appositions, and synthesis of pathogenesis related (PR)-proteins [1, 3, 4, 27]. Among PR-proteins, two plant hydrolases, b-1,3-glucanase and chitinase, have been intensively studied for their accumulation in the infected plant tissues and their function in plant defence reactions in dierent fungal pathogen±plant systems [3±5, 9, 10, 16, 17, 29, 34, 35]. These two enzymes are of particular interest because many pathogenic fungi contain b-1,3-glucans and chitin as major structural cell wall components [32]. It had been demonstrated in vitro * To whom correspondence should be addressed. Biotech Center and Plant Protection College, Northwestern Sci-Tech University of Agriculture & Forestry, Yangling, Shaanxi 712100, People's Republic of China. Abbreviations used in text: DON, deoxynivalenol; FHB, Fusarium head blight; PR, pathogenesis related.
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that the two enzymes b-1,3-glucanase and chitinase are able to degrade fungal wall components, resulting in growth inhibition of fungi [2, 18, 36]. Furthermore, breakdown products of fungal wall components, released by the activity of the two enzymes, have been shown to act as elicitors of plant defence responses [8, 15, 30]. Studies on the subcellular localization of b-1,3-glucanase and chitinase in planta showed that these hydrolases accumulated at higher concentrations in infected host plant tissues and on fungal cell walls, especially, at sites where host cells were in close contact with fungal hyphae. In incompatible plant±pathogenic fungal interactions higher activities of both hydrolytic enzymes were detected compared with the compatible interactions [3, 4, 9, 29, 34]. This indicates that b-1,3-glucanse and chitinase could play an important role in the active defence of plants against the fungal pathogens. The infection process and route of F. culmorum colonization of wheat spikes has been elucidated using light and electron microscopy [12]. Our cytochemical analyses con®rmed that F. culmorum also produced cell wall degrading enzymes at early stages of infection [13]. c 2002 Elsevier Science Ltd. *
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During infection of wheat spikes by Fusarium graminearum and F. culmorum, the pathogens can produce trichothecene mycotoxins, such as deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) [19, 21, 28]. Our immunogold labelling studies revealed a relationship between the accumulation of Fusarium toxins in the infected wheat spike and the pathogenic changes in the host cells, symptom appearance and the colonization by the pathogen in the host tissues [11]. Studies on the toxicity of Fusarium trichothecenes in eukaryotic cells indicated that the toxin inhibited protein synthesis [25, 33]. Fusarium trichothecene toxins are phytotoxic [7, 25, 31] and may be regarded as virulence factors involved in development of Fusarium head blight (FHB) [23]. More recently, Miller and Ewen [20] reported that DON inhibited protein synthesis in vitro through binding to ribosomes isolated from wheat leaves. It is well known that plant defence responses to pathogen attack depend on rapid and intensive transcription and translation reactions of host tissues. Thus, it seems likely that DON may interfere in post-infectional defence reactions. Furthermore, cytological examinations revealed that F. culmorum grew more slowly in resistant wheat cultivars than in the susceptible cultivar, and plant structural defence reactions such as the formation of thick layered appositions and large papillae were essentially more pronounced in the infected host tissues of resistant cultivars than in the susceptible one [14], indicating that the FHB resistant cultivars are able to develop active structural defence reactions during infection and spreading of the pathogen in the host tissue. In order to elucidate further mechanisms involved in defence responses of wheat to FHB, it was therefore of interest to study the accumulation of b-1,3-glucanase and chitinase in wheat spikes infected by F. graminearum or F. culmorum. The localization of both hydrolytic enzymes in the wheat spikes after infection by Fusarium spp. has not been studied so far. However, the accumulation and
distribution of b-1,3-glucanase in wheat leaves infected by rust fungi has been reported [9, 29]. In the present study, we describe the subcellular localization of b-1,3glucanase and chitinase in wheat spikes of two wheat cvs. diering in their susceptibility to F. culmorum by means of immunogold labelling techniques. MATERIALS AND METHODS
Plant material, fungal strain and inoculation Two winter wheat cultivars, Agent (susceptible) and Arina (resistant), were used throughout the present experiments. The wheat plants were grown as described previously [11]. When the plants were at the middle stage of ¯owering (GS 65) [37], the wheat spikes were inoculated with macroconidia of F. culmorum (isolate No. 46; Institute of Phytomedicine, University Hohenheim, Germany) [11]. For inoculation, the macroconidia suspension was adjusted to 1 105 spores ml 1. The wheat spikes were inoculated by pipetting 10 ml of a macroconidia suspension into the ¯oral cavity between the lemma and palea of the ®rst ¯oret of spikelets. Every third ¯oret at one side of the spike was inoculated. Control plants were inoculated with distilled water instead of the macroconidial suspension. Inoculated spikes were covered with plastic bags and plants were transferred into a growth chamber with 16 h ¯uorescent light at 238C and 8 h darkness at 188C. Two days later the plastic bags were removed and the plants were returned to outdoor conditions.
Tissue processing for electron microscopy The pathogen inoculated and control inoculated ¯orets were sampled after 3, 5 and 8 days covering speci®c stages of the infection and colonization process [12]. At the same times samples of healthy, uninoculated tissues were taken. The lemmas, ovaries and rachis were excised from the
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F I G S . 1±6. Immunogold localization of b-1,3-glucanase in the uninoculated healthy and F. culmorum-infected wheat spike of the susceptible cv. Agent. F I G . 1. A parenchyma cell in the lemma from the uninoculated wheat spike, 3 days after treatment (dat) with water. A low density of gold particles was localized over the cell wall while the cytoplasm and organelles were almost free of gold particles. F I G . 2. Parenchyma cells in the ovary from the uninoculated wheat spike, 3 dat. The cell walls showed a few gold particles. F I G . 3. A cortical cell in the rachis from the uninoculated wheat spike, 5 dat. Gold particles were deposited over the cell wall, but not over the cytoplasm. F I G . 4. Xylem vessel in the rachis from the uninoculated wheat spike, 5 dat. Dense gold particles were observed over the secondary thickening. F I G . 5. Infected lemma, 3 dai by F. culmorum. A low density of gold particles was found over the plant cell walls. The hyphal cell wall also showed a low density of gold particles. F I G . 6. Infected ovary, 3 dai. Few gold particles were observed over the parenchyma cell walls and the hyphal cell wall. All bars 0.5 mm. CH: chloroplast; H: hyphal cell; ST: secondary thickening; X: xylem vessel.
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¯orets and ®xed in 3 % (v/v) glutaraldehyde in 50 mM phosphate buer ( pH 6.8) for 3±6 h at 48C. Thereafter, the samples were thoroughly rinsed with 50 mM phosphate buer ( pH 6.8) and post-®xed with 1 % (w/v) osmium tetroxide in the same buer for 2 h at 48C. The samples were then dehydrated in a graded ethanol series, embedded in LR White (TAAB Laboratories, Munich, Germany) and polymerized at 508C for 2 days. Ultrathin sections of the samples were cut with a diamond knife and collected on 200-mesh nickel grids for immunogold labelling. The experiments were repeated three times.
Immunogold labelling for b-1,3-glucanase and chitinase The rabbit polyclonal antisera against acidic b-1,3glucanase and acidic chitinase from tobacco were obtained from Prof. Fritig (C.N.R.S., Institut de Biologie Moleculaire des Plantes, Strasbourg, France) and showed high speci®city with b-1,3-glucanase and chitinase, respectively, from wheat leaves and shoots by western blotting as demonstrated by Kemp et al. [16] and Siefert [26]. The secondary antibodies (goat anti-rabbit IgG) coupled to 15 nm gold particles were purchased from British Biocell International Ltd (Cardi, U.K.). Immunogold labelling was carried out as follows: (i) incubation of ultrathin sections with blocking solution containing 1 % (w/v) of BSA in Tris-buered saline (TBS, 10 mM Tris±HCl, 150 mM NaCl, pH 7.4) for 20 min; (ii) incubation of the sections with the primary antibody diluted at 1 : 200 in the blocking solution for 2 h at room temperature; (iii) washing in four 10 min baths in TBS; (iv) incubation of the sections with the secondary antibody diluted at 1 : 40 in TBS; (v) rinsing with TBS followed by a distilled water rinse. After contrasting with uranyl acetate and lead citrate, the sections were examined with a ZeissEM10 electron microscope at 80 kV.
Immunocytochemical controls Speci®city of labelling was assessed by the following control tests: (i) incubation of the ultrathin sections with the rabbit pre-immune serum instead of the primary antibody; and (ii) incubation with the secondary antibody and omitting the primary antibody step.
Quanti®cation of labelling The labelling densities of gold particles detecting b-1,3glucanase and chitinase over cell walls in dierent tissues of uninoculated and infected wheat spikes were determined by counting the number of gold particles mm 2 over speci®ed cell wall areas on 10±15 microphotographs. The dierence in gold particles between uninoculated healthy and infected host tissues were statistically analysed by the t-test. RESULTS
Immunocytochemical localization of b-1,3-glucanase in uninoculated healthy and infected wheat spikes Incubation of ultrathin sections of dierent tissues from the uninoculated wheat spikes with anti-b-1,3-glucanase antiserum and the secondary antibody resulted in labelling of b-1,3-glucanase. The labelling patterns and labelling densities in the healthy tissues were very similar between the susceptible wheat cv. Agent and resistant cv. Arina, 3 days after treatment (dat) with water. A low density of gold particles was detected over the cell walls in the lemma and ovary, whereas the cytoplasm and organelles such as nuclei, Golgi bodies, mitochondria and chloroplasts showed very few gold particles (Figs 1 and 2). In the rachis of the uninoculated wheat spike of cv. Agent (5 dat with water) and cv. Arina (8 dat with water), gold particles were unevenly deposited over the walls of the cortical cells,
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F I G S . 7±11. Immunogold localization of b-1,3-glucanase in F. culmorum-infected wheat spikes of the susceptible cv. Agent and the resistant cv. Arina, 3 and 5 dai. F I G . 7. Infected lemma of the resistant cv. Arina, 3 dai. Appositions were formed between the cell wall and the plasmalemma of the host cells which were in contact with fungal cells. A high density of gold particles was found over the plant cell walls. The hyphal cell also showed more gold particles over the hyphal wall, especially at the region of contact with the host cell (arrowhead). F I G . 8. Infected ovary of the resistant cv. Arina, 3 dai. Marked wall appositions had been formed between the cell wall and the plasmalemma in the host cells (arrowheads). Many gold particles were deposited over host cell walls, wall appositions and the hyphal cell walls, while the host cytoplasm and the hyphal cytoplasm showed almost no labelling. F I G . 9. Infected lemma of the resistant cv. Arina, 3 dai. Gold particles were deposited over the host cell wall, hyphal wall, wall appositions and the electron-dense material located between the host cell and the hyphal cell (arrowhead). F I G . 10. A cortical cell in the infected rachis from the susceptible cv. Agent, 5 dai. A low density of gold particles was deposited over the host cell wall and the hyphal cell wall. F I G . 11. An infected xylem vessel in the rachis of the susceptible cv. Agent, 5 dai. Dense gold particles were observed over the secondary thickenings in the vessel. All bars 0.5 mm. H: hyphal cell; AP: wall apposition; ST: secondary thickening; X: xylem vessel.
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T A B L E 1. Labelling density of gold particles conjugated with the anti-b-1,3-glucanase antibody over cell walls in dierent tissues of non-inoculated healthy and F. culmorum-infected wheat spikes in the cvs. Agent and Arinaa Agent Healthy Parenchyma cell wall of lemma Parenchyma cell wall of ovary Cortical cell wall of rachis
7.25b 5.16 8.82
Arina Infected 7.86 5.78 9.40
Healthy nc n n
7.12 5.04 8.60
Infected 22.40 13.46 27.66
sd s s
a Densities of labellings in the cell walls of the lemma and the ovary of both cvs. were determined 3 dai and in the cortical cell walls of the rachis of cv. Agent 5 dai and in the cv. Arina 8 dai. The labelling density was expressed by the number of gold particles per mm2. b Mean values (numbers) of the gold particles determined per mm2. The average densities for uninoculated healthy and infected tissues were compared by t-test, P 0.05. c n Statistically not signi®cant dierent values in the same row. d s Statistically signi®cant dierence values in the same row.
xylem vessels and sieve tubes in the vascular bundles, while cytoplasm and organelles of these tissues were almost free of labelling (Figs 3 and 4). However, the secondary thickenings of the xylem vessels usually showed more gold particles than the walls of the other cells in the rachis. Three days after inoculation with conidia of F. culmorum, the hyphae spread inter- and intracellularly in the ovary, and only intercellularly in the lemma tissue. Pronounced formations of wall appositions and papillae in the host cells were often observed in the infected tissues of the resistant cv. Arina (Figs 7 and 8), but not in the susceptible cv. Agent (Figs 5 and 6). Labelling studies with b-1,3-glucanase revealed similar patterns of the infected host tissues of both the susceptible cv. Agent and the resistant cv. Arina compared to the uninoculated host tissues. However, the labelling densities in the infected host tissues varied markedly between the resistant and susceptible cultivars. In the susceptible cv. Agent, the labelling density over the cell walls of lemma and ovary showed only a slight increase in b-1,3-glucanase labelling as compared to the corresponding uninoculated healthy tissues (Figs 5 and 6, Table 1). On the other hand, gold
particles over the cell walls of the infected lemma and the ovary of the resistant cv. Arina increased considerably compared to the corresponding uninoculated host tissues (Figs 7±9, Table 1). Gold particles were usually detected over the wall appositions ( papillae) formed between the plant cell wall and plasmalemma and over the electrondense material located between the hyphal cell and the host cell in the resistant cv. Arina (Figs 8 and 9). In fungal cells in infected host tissues, either from the resistant or from the susceptible cultivar, the hyphal walls showed uneven labelling while the hyphal cytoplasm displayed only few gold particles. Five days after inoculation, more hyphae were found in the infected ovary and lemma. The inter- and intracellular growth of the hyphae resulted in severe damages of the cells of the susceptible cv. Agent, such as disintegration of the cytoplasm and organelles, whereas most cells of the infected tissue of the resistant cv. Arina were still intact. The pathogen reached the rachis of the susceptible cv. Agent from the infected ovary and lemma 5 days after inoculation, whereas it took 8 days for the pathogen to reach the rachis in the resistant cv. Arina. The hyphae
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F I G S . 12 and 13. Immunogold localization of b-1,3-glucanase in F. culmorum-infected wheat spikes of the resistant cv. Arina, 8 dai. F I G . 12. A cortical cell in the infected rachis. Many gold particles were observed over the plant cell wall.
F I G . 13. An infected xylem vessel in the rachis. A high density of gold particles was deposited over the secondary thickenings of the vessel and over the hyphal wall, while the electron-dense coating materials on the secondary thickenings showed only a few gold particles. F I G S . 14±17. Immunogold localization of chitinase in the uninoculated healthy wheat spikes of the susceptible cv. Agent. F I G . 14. A parenchyma cell in the lemma. The cell wall showed a low density of gold particles, whereas the cytoplasm and organelles were almost free of labelling. F I G . 15. Parenchyma cells in the ovary. A few gold particles were observed over the walls, but not over the cytoplasm. F I G . 16. A cortical cell in the rachis. The cell wall was labelled with gold particles. F I G . 17. A xylem vessel in the rachis. Many gold particles were found over the secondary thickening of the vessel. All bars 0.5 mm. CH: chloroplast; H: hyphal cell; ST: secondary thickening; X: xylem vessel.
Immunocytochemical localization of b-1,3-glucanase and chitinase extended inter- and intracellularly in the cortical tissue and vascular bundles of the rachis (Figs 10±13). The labelling patterns for b-1,3-glucanase in the infected and uninfected rachis tissues were similar, but the increase in the labelling density in the infected rachis diered markedly between the susceptible and resistant wheat cultivar. The number of gold particles over the cell walls
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of the infected rachis of the resistant cv. Arina was signi®cantly higher than on the walls of the uninoculated healthy host cells, whereas the labelling density over the cell walls of the infected rachis of the susceptible cv. Agent was only slightly increased compared to the corresponding uninoculated host tissues (Figs 10±13, Table 1). In addition, a high density of gold particles
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T A B L E 2. Labelling density of gold particles conjugated with the anti-chitinase antibody over cell walls in dierent tissues of noninoculated healthy and F. culmorum-infected wheat spikes in the cvs. Agent and Arinaa Agent Healthy Parenchyma cell wall of lemma Parenchyma cell wall of ovary Cortical cell wall of rachis
6.80b 4.94 7.92
Arina Infected 7.72 5.66 8.60
Healthy nc n n
6.68 5.20 8.38
Infected 18.00 11.64 23.42
sd s s
a
Densities of labellings in the parencyhma cell walls of the lemma and the ovary of both cvs. were determined 3 dai and in the cortical cell walls of the rachis of cv. Agent 5 dai and in the cv. Arina 8 dai. The labelling density was expressed by the number of gold particles per mm2. b Mean values (numbers) of the gold particles determined per mm2. The average densities for uninoculated healthy and infected tissues were compared by t-test, P 0.05. cn Statistically not signi®cant dierent values in the same row. d s Statistically signi®cant dierence values in the same row.
was often detected over the secondary thickenings of the xylem vessels in the infected rachis of the resistant cv. Arina, while the electron-dense coating material on the secondary thickenings showed only few gold particles or was free of labelling (Fig. 13).
Immunocytochemical localization of chitinase in uninoculated healthy and infected wheat spikes Incubation of the sections of uninoculated healthy wheat spikes with anti-chitinase antiserum and the secondary antibody resulted in irregular labelling over the cell walls of the host tissues while the cytoplasm and organelles such as chloroplasts, mitochondria and vacuoles in the host tissues showed few gold particles or were free of labelling. The labelling patterns and labelling densities for chitinase in the host tissues did not dier between the resistant cv. Arina and the susceptible cv. Agent. A low density of gold particles was observed over the cell walls in the lemma, ovary and cortical tissue of the rachis (Figs 14±16), whereas more gold particles were usually
deposited over the secondary thickenings of the xylem vessel in the rachis (Fig. 17). Three days after inoculation, gold labelling of chitinase in the infected host tissues and in the hyphal cells showed dierent labelling densities. In the fungal cells, gold particles were usually deposited over the walls while the hyphal cytoplasm showed almost no labelling. In the susceptible cv. Agent, a low density of gold particles was unevenly deposited over the host cell walls and the hyphal cell walls in the infected lemma and ovary (Figs 18 and 19). The density of the gold particles demonstrated a slight increase over the cell walls in the infected compared to uninoculated tissues, but the values were not signi®cantly dierent from those of the corresponding uninoculated healthy tissues (Table 2). On the other hand, in the infected lemma and ovary of the resistant cv. Arina a high density of gold particles was observed over the host cell wall as well as over the hyphal walls, particularly within the area of contact of the host and fungal cell (Figs 20±22). A signi®cant increase in gold particles over the cell walls in the infected tissues of the resistant cv. Arina was found, as compared to the uninoculated healthy
Y F I G S . 18±22. Immunogold localization of chitinase in the F. culmorum-infected wheat spikes of the susceptible cv. Agent and the resistant cv. Arina. F I G . 18. Infected lemma from the susceptible cv. Agent, 3 dai. The plant cells were labelled with a low density of gold particles over the cell walls, but not over the cytoplasm and organelles. The hyphal cell wall showed a few gold particles. F I G . 19. Infected ovary from the susceptible cv. Agent, 3 dai. A few gold particles were deposited over the plant cell walls and the hyphal cell wall. F I G . 20. Infected lemma of the resistant cv. Arina, 3 dai. Many gold particles were deposited over the plant cell wall and the wall apposition. The hyphal cell wall in contact with the host cell showed more gold particles (arrowhead). F I G . 21. Infected lemma of the resistant cv. Arina, 3 dai. The electron-dense materials located between the host cell and the hyphal cell showed gold labelling (arrowhead). F I G . 22. Infected ovary of the resistant cv. Arina, 3 dai. Gold particles were observed over the host cell wall, wall appositions (arrowhead) and the hyphal cell walls, whereas the host cytoplasm and the hyphal cytoplasm showed almost no labelling. All bars 0.5 mm. CH: chloroplast; H: hyphal cell; AP: wall apposition.
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tissues (Table 2). Also, wall appositions in the host cells and electron-dense material between the host cell and hyphal cell in the intercellular space of the infected tissues usually showed intense gold labelling (Fig. 21). Five days after inoculation, the labelling patterns and labelling densities for chitinase in the infected lemma and ovary from the resistant cv. Arina and the susceptible cv. Agent did not dier markedly from those of the corresponding infected host tissues three days after inoculation. Gold labelling of the infected rachis tissue was detected over the walls of the cortical cells, xylem vessels and sieve tubes in the vascular bundles, and also over the walls of the hyphal cells. More gold particles accumulated over the cell walls of the infected rachis of the resistant cv. Arina as compared to the susceptible cv. Agent (Figs 23±26, Table 2). Labelling was also often detected over the electron-dense material between host cells and hyphal cells in the intercellular space in the infected rachis of the resistant cv. Arina (Fig. 25). Although the coating material on the secondary thickenings of xylem vessels in the infected rachis of the resistant cv. Arina displayed few gold particles or were free of labelling, the secondary thickenings of xylem vessels in the same tissues were usually labelled with a high density of gold particles (Fig. 26).
Immunocytochemical controls Following incubation of the sections of uninoculated healthy and infected wheat spikes with pre-immune serum and the secondary antibody, or with the secondary antibody alone, no labelling was observed over the host plant tissues and over the hyphal cells (Fig. 27).
DISCUSSION The studies demonstrated the subcellular distribution of two plant hydrolases, b-1,3-glucanase and chitinase in
uninoculated healthy and F. culmorum-infected wheat spikes by means of the immunogold labelling technique. The antibodies used were raised against acidic b-1,3glucanase and acidic chitinase from tobacco. The two hydrolytic enzymes were localized mainly in the cell walls of dierent spike tissues including the lemma, ovary and rachis while the cytoplasm and organelles in these tissues were almost free of labelling. This indicates that the two enzymes accumulate extracellularly in the infected wheat spikes. The described labelling pattern is in accordance with an earlier report that acidic b-1,3-glucanase and acidic chitinase accumulated extracellularly in tomato leaves infected by Cladosporium fulvum, suggesting that they might play a role in the successful defence of the tomato plants [10] against pathogenic fungi. While labelling of the two enzymes in the infected tissues of wheat spikes of the susceptible cv. Agent was only slightly increased compared to the corresponding uninoculated healthy tissues, the number of gold particles in the infected lemma, ovary and rachis of the resistant cv. Arina was signi®cantly higher than in corresponding uninoculated healthy tissues. Thus, the study revealed that the labelling densities for b-1,3-glucanase and chitinase were especially pronounced over the hyphal cell walls where the hyphae were in contact with the host cells. From this it can be concluded that the two hydrolases may diuse from host cells to the hyphal cell surface and may act synergistically in hydrolysing fungal cell wall components and thus interfering with hyphal growth. This situation is similar to ®ndings in tomato and eggplant roots infected by F. oxysporum [3, 4], tomato leaves infected by C. fulvum [10] and wheat leaves infected by Puccinia recondita [9]. Mauch et al. [18] showed that the fungal growth in vitro was inhibited synergistically by combination of plant chitinase and b-1,3-glucanase compared to the single enzyme treatments. Chitin, one of the main components in the fungal walls, is considered to be embedded in a matrix of amorphous material including b-glucans [6], which makes the chitin inaccessible to
Y F I G S . 23±26. Immunogold localization of chitinase in the F. culmorum-infected wheat spikes of the susceptible cv. Agent and resistant cv. Arina. F I G . 23. A cortical cell in the infected rachis from the susceptible cv. Agent, 5 dai. The host cell wall and the hyphal cell wall were labelled with a low density of gold particles. F I G . 24. An infected xylem vessel in the rachis of the susceptible cv. Agent, 5 dai. The secondary thickenings of the vessel showed numerous gold particles. F I G . 25. A cortical cell in the infected rachis from the resistant cv. Arina, 8 dai. Many gold particles were deposited over the plant cell walls and the hyphal wall. The materials located between the plant cell and the hyphal cell were also labelled with gold particles. F I G . 26. An infected xylem vessel in the rachis of the resistant cv. Arina, 8 dai. Numerous gold particles were deposited over the secondary thickenings of the vessel, but not over the electron-dense coating materials on the secondary thickenings. The hyphal cell wall in contact with the host cell showed a dense labelling with gold particles. F I G . 27. Immunocytochemical control test. The infected lemma from the resistant cv. Arina, 3 dai, incubated with pre-immuno antiserum and the secondary antibody. No labelling is found over the plant cell and the hyphal cell. All bars 0.5 mm. H: hyphal cell; ST: secondary thickening; X: xylem vessel.
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chitinase. It has been demonstrated that preliminary treatment with b-1,3-glucanase renders chitin more susceptible to chitinase in Schizophyllum commune [32]. Benhamou et al. [3, 4] also suggested that previous alteration of fungal cell walls by the in¯uence of b-1,3glucanase would make chitin more accessible to plant chitinase in the F. oxysporum-tomato system. To what extent the synthesis of b-1,3-glucanase and chitinase in the infected plant tissues might be aected by the trichothecenes produced and secreted by the pathogens F. culmorum or F. graminearum already at early stages of infection of wheat spikes [11] needs further investigation, since it is known that the trichothecene toxins interfere in protein synthesis of wheat leaves [20]. Besides the accumulation of b-1,3-glucanase and chitinase, further mechanisms might be involved in resistance of wheat spikes to F. culmorum or F. graminearum. Recently, we found that lignin and callose depositions were higher in the F. culmorum-infected wheat spike tissues of the resistant wheat cvs. Frontana and Arina than in the susceptible cv. Agent [14]. Furthermore, Pritsch et al. [22] reported that the defence response genes enconding peroxidase, PR-1, PR-4 and PR-5 (thaumatin-like protein) were also transcribed in F. graminearum-infected wheat spikes at early stages of infection. These ®ndings suggest that b-1,3-glucanase and chitinase as well as the structural cell wall alterations (like depositions of lignin, thick layered appositions and papillae) are implicated in defence reactions and eventually contribute to resistance of wheat spike tissues against F. culmorum. The authors wish to thank Prof. Fritig (C.N.R.S., Institut de Biologie Moleculaire des Plantes, Strasboug, France) for kindly supplying antibodies, and H. Brandl for excellent technical assistance. G. Moll is gratefully acknowledged for preparing photographs. This study has been ®nancially supported by the Deutsche Forschungsgemeinschaft (DFG).
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