Plant Physiol. (1999) 119: 1251-1260

Hypersensitive Cell Death and Papilla Formation in Barley Attacked by the Powdery Mildew Fungus Are Associated with Hydrogen Peroxide but Not with Salicylic Acid Accumulation¹

Ralph Hückelhoven, József Fodor, Christine Preis, and Karl-Heinz Kogel^*

Institute for Phytopathology and Applied Zoology, Ludwigstrasse 23, Justus-Liebig-University Giessen, D-35390 Giessen, Germany (R.H., C.P., K.-H.K.); and Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, H-1525 Budapest, Hungary (J.F.)

	ABSTRACT

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We analyzed the pathogenesis-related generation of H₂O₂ using the microscopic detection of 3,3-diaminobenzidine polymerization in near-isogenic barley (Hordeum vulgare L.) lines carrying different powdery mildew (Blumeria graminis f.sp. hordei) resistance genes, and in a line expressing chemically activated resistance after treatment with 2,6-dichloroisonicotinic acid (DCINA). Hypersensitive cell death in Mla12 and Mlg genotypes or after chemical activation by DCINA was associated with H₂O₂ accumulation throughout attacked cells. Formation of cell wall appositions (papillae) mediated in Mlg and mlo5 genotypes and in DCINA-activated plants was paralleled by H₂O₂ accumulation in effective papillae and in cytosolic vesicles of up to 2 µm in diameter near the papillae. H₂O₂ was not detected in ineffective papillae of cells that had been successfully penetrated by the fungus. These findings support the hypothesis that H₂O₂ may play a substantial role in plant defense against the powdery mildew fungus. We did not detect any accumulation of salicylic acid in primary leaves after inoculation of the different barley genotypes, indicating that these defense responses neither relied on nor provoked salicylic acid accumulation in barley.

	INTRODUCTION

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The role of H₂O₂ and SA in the defense responses of plants against parasites is controversial. Chen et al. (1993) has argued that SA acts via inhibition of a catalase that subsequently results in accumulation of H₂O₂, which may involve cross-linking reactions leading to cell wall toughening (Bradley et al., 1992; Brisson et al., 1994) and/or signaling that results in defense gene activation (Chen et al., 1993; Wu et al., 1997; Chamnongpol et al., 1998). The hypersensitive response, a plant defense reaction that restricts biotrophic and other pathogens (Stakman, 1915), seems to be dependent on the availability of SA (Levine et al., 1994; Shirasu et al., 1997; Tenhaken and Rübel, 1997) and H₂O₂ (Levine et al., 1994; Thordal-Christensen et al., 1997). Bi et al. (1995) and Neuenschwander et al. (1995) have presented data suggesting that systemic acquired resistance does not rely on H₂O₂ accumulation as a downstream event of elevation of SA content. Moreover, van Wees et al. (1997) and Vidal et al. (1998) have provided evidence for SA-independent biological induction of resistance.

We have chosen the interaction of the powdery mildew fungus (Blumeria graminis f.sp. hordei) with barley (Hordeum vulgare L.) to study the role of reactive oxygen species and SA in constitutively expressed and chemically induced resistance. The developmental stages of powdery mildew fungus during its interaction with its host are well defined, and fungal spores show a synchronized growth upon leaf inoculation. After contact of the spore with the cutin layer of a barley leaf, the following fungal structures differentiate within the first 24 hai (Ellingboe, 1972; Aist and Bushnell, 1991): (a) the primary germ tube (1-2 h), (b) the appressorial germ tube with a mature appressorium (10-12 h), and (c) the haustorium initial (16-24 h), which invaginates the epidermal plasma membrane. Formation of aerial mycelium and sporulation are late differentiation events that occur 5 to 7 d post inoculation. The establishment of a mature haustorium represents a key step for successful fungal reproduction, because it is the only organ that serves to feed the pathogen. Quantitative cytological recordings of incompatible interactions have revealed putative host-cell-defense responses conferring arrest of fungal development at distinct stages (Kita et al., 1981; Koga et al., 1988, 1990). Two of these host-cell responses are easily detected: a subcellular, highly localized cell wall reinforcement at sites of attempted penetration (effective papilla) and an active, rapid death of attacked epidermal cells (HR), which can be visualized by whole-cell autofluorescence under UV excitation (Aist and Israel, 1986; Koga et al., 1990; Görg et al., 1993).

In the present study, we used BghA6, which triggers defense responses in NILs bearing the powdery mildew resistance genes mlo5, Mlg, and Mla12 (Hückelhoven and Kogel, 1998). These genes govern fungal arrest at different stages of the interaction: (a) at the penetration stage within papillae, leaving the attacked cell alive (mlo); (b) within papillae of cells that subsequently undergo HR (Mlg); and (c) by HR after penetration of the epidermal host cell (Mla12). A previous publication compared the PR generation of the superoxide anion (O₂^·) in these NILs (Hückelhoven and Kogel, 1998; Kogel and Hückelhoven, 1999). An O₂^· burst was seen at the interaction sites in epidermal cells that had been penetrated by the fungus (compatible interaction and Mla12-mediated resistance), but not at interaction sites showing penetration resistance (Mlg- and mlo5-mediated resistance). These data suggest that the trigger for O₂^· generation in epidermal cells attacked by powdery mildew fungus was the contact of the parasite with the host plasma membrane after host-cell wall penetration, and that O₂^· accumulation was not required or sufficient for HR elicitation.

We show here that H₂O₂, in contrast to O₂^·, is closely associated with HR and the formation of effective papillae, and that these defense responses are not linked to SA accumulation in barley.

	MATERIALS AND METHODS

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Plants, Pathogens, and Inoculation

2

Chemical Induction

1

Histochemical Detection of H₂O₂ at Interaction Sites

1

Extraction of SA and HPLC

1

Residues were resuspended in 400 µL of methanol:water (1:1, v/v), centrifuged for 10 min at 20,000g, and placed in HPLC vials for detection of free SA. For hydrolysis of SA-glycosides the concentrated samples were resuspended in 500 µL of 1 M HCl, incubated at 70°C overnight, concentrated again, and resuspended for HPLC in 500 µL of methanol:water (1:1, v:v). The SA content in the extracts was corrected according to the amount of the internal standard. HPLC was performed with a reverse-phase column (LiChroCart 250-4, LiChroSphere C₁₈, 5 µm, Merck, Darmstadt, Germany) linked to a fluorescence photometer (SFM 25, Kontron, Neufarn, Germany) equipped with a 12-µL flow cell. Excitation and emission wavelengths were 298 and 400 nm, respectively. Separation was at 40°C using a flow rate of 1 mL min¹. Elution was carried out with 0.05 M TCA (pH 2.6):methanol (80:20, v/v) for 1 min, followed by a linear 10-min gradient to TCA:methanol (70:30, v/v), isocratic TCA:methanol (70:30, v/v) for 20 min, 5 min washing with methanol (100%), followed by a return to the first step solvent, and equilibration for 10 min before the next run.

	RESULTS

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Histochemical localization of H₂O₂ using DAB in barley primary leaves attacked by BghA6 showed an accumulation of this active oxygen species in cell wall appositions, as well as in cells undergoing HR, which is in agreement with the results of Thordal-Christensen et al. (1997). To determine the spatial and temporal profile of H₂O₂ generation in these prominent defense reactions, we carried out a comparative kinetic analysis of H₂O₂ formation at early developmental stages of the interaction of BghA6 with NILs bearing the resistance genes Mla12, Mlg, and mlo5. These genes mediate various highly defined defense phenotypes, including the timing and frequency of HR and effective papillae in response to fungal penetration attempts. The quantitatively predominant plant responses governed by these resistance genes were compared with the susceptible phenotype in Figure 1. Figure 1 also displays the defense phenotype mediated by induction of barley with the chemical DCINA. A more detailed quantitative cytological analysis of the interaction phenotypes indicates that the development of BghA6 on the NILs or DCINA-treated cv Pallas was basically the same as had been observed in earlier studies (Table I; see also Hückelhoven and Kogel, 1998; Kogel and Hückelhoven, 1999).

View larger version (21K): [in this window] [in a new window]   Figure 1. Scheme of predominant interaction phenotypes in barley mediated by the powdery mildew resistance genes Mla12, Mlg, and mlo5, or by the resistance-inducing compound DCINA after inoculation with BghA6. Beginning at 16 hai, differences in fungal development are evident on the four NILs. Although the fungus penetrated the epidermal cells of susceptible and Mla12-resistant barley, effective papillae did impede penetration in the Mlg- and the mlo5-mediated response. Mla12 mediated HR in penetrated epidermal cells by 24 to 40 hai. If the fungus was able to establish a differentiated haustorium and branched elongated secondary hyphae, fungal development was arrested by spreading HR of mesophyll cells subjacent to the attacked epidermal cell beginning at 36 hai. The Mlg gene governed effective papilla formation and HR of attacked but noninvaded epidermal cells by 18 to 24 hai. The same phenotype is seen in barley treated with DCINA. The recessive mlo5 gene mediated effective papilla formation, and attacked cells stayed alive. In the compatible interaction, cell wall penetration was followed by formation of a haustorium and elongated secondary hyphae.

H₂O₂ in Compatible Interaction

View larger version (90K): [in this window] [in a new window]   Figure 2. Microscopic, subcellular localization of H2O2 at interaction sites in barley after inoculation with BghA6. Seven-day-old barley primary leaves of NILs were inoculated with 20 conidia mm2 of BghA6. At different time points after inoculation, leaves were cut off, placed in a solution of 1 mg mL1 DAB, and collected for microscopic analysis 8 h later (at the indicated time points). A, Effective papilla (PAPeff) in formation on cv Pallas (22 hai). Papilla and vesicles (V) with DAB polymers are seen beneath the appressorial germ tube (AGT). Scale bar = 8 µm. B, Successful penetration of cv Pallas (24 hai). The fungus had formed a haustorial initial (HI). Whereas the body of the ineffective papilla shows only a faint staining, the anticlinal cell wall close to the penetration site shows typical brownish DAB polymers. Scale bar = 10 µm. C, DAB staining of chloroplasts in healthy mesophyll cells neighboring a transverse vessel in cv Pallas primary leaves. Scale bar = 40 µm. D, BCPmlo5 (30 hai). Autofluorescence under UV-light excitation of effective papilla (PAPeff) halos and vesicles beneath primary and AGTs is indicative of the accumulation of phenolic compounds at a repulsed penetration attempt. Scale bar = 10 µm. E, BCPMla12 (48 hai): mesophyll HR subjacent to a living penetrated epidermal cell (out of focus). Dead cells show DAB staining. Scale bar = 20 µm. F, Epifluorescence microscopy of the interaction site shown in E. The cells in the center have collapsed. Accumulation of polyphenols is indicated by the yellow autofluorescence. Scale bar = 20 µm. G, BCPMla12 (48 hai): epidermal cells surrounding the penetrated, living cell show positive DAB staining, (Legend continues on facing page.)

H₂O₂ in Resistance Mediated by the mlo5 Gene

View larger version (24K): [in this window] [in a new window]   Figure 3. Incidence of interaction sites with H2O2 in papillae of attacked cells of the NILs cv Pallas (Mlo) and BCPmlo5 after inoculation with BghA6. At 14, 22, and 40 hai, leaves were cut off and placed in a solution of 1 mg mL1 DAB. After 8 h (at the indicated time points), leaf segments were analyzed for DAB staining subjacent to appressorial germ tubes (AGT). Each column represents 100 interaction sites per leaf. Three independent experiments gave very similar results.

H₂O₂ in Resistance Mediated by the Mla12 Gene

The frequency of interaction sites with local staining of anticlinal walls of penetrated cells (Fig. 2B) reached a level of 30% by 22 hai, indicating that there was no difference in H₂O₂ formation at this stage of fungal development between compatible and Mla12-incompatible interactions. Later, H₂O₂ accumulated in penetrated cells undergoing HR (Fig. 4; Table I).

View larger version (12K): [in this window] [in a new window]   Figure 4. Incidence of interaction sites with whole-cell DAB staining in NILs bearing the resistance genes Mla12, Mlg, and mlo5, and in cv Pallas expressing chemically induced resistance after inoculation with BghA6. At the indicated time points, leaf segments were microscopically analyzed for positive H2O2 staining. Each column represents 100 interaction sites per leaf. Three independent experiments gave very similar results whereas the attacked cell remained essentially free of dye. ESH, Elongated secondary hyphae; HAU, haustorium. Scale bar = 25 µm. H, BCPMla12 (22 hai): the fungus has penetrated the cell and formed a haustorial initial (HI). A brownish halo (in the focus of the cell wall and plasma membrane) is seen around the penetration site, although the body of the ineffective papilla is only weakly stained. Scale bar = 10 µm. All bright-field microscopic pictures were taken using differential interference contrast.

Mesophyll cell death that was exclusively detected in BCPMla12 with a frequency of 20% of the interaction sites (within 48 hai, see Table I) was closely associated with H₂O₂ accumulation in all of the cells carrying out HR (Fig. 2, E and F). H₂O₂ generation and HR in the mesophyll tissue subjacent to interaction sites was a result of a compatible single-cell interaction in the epidermal layer, leading to successful haustorium formation. By 48 hai, epidermal cells next to cells that had been successfully penetrated by the fungus accumulated H₂O₂ in cell walls, whereas cells containing a haustorium remained essentially free of H₂O₂ (Fig. 2G).

In addition, approximately 10% of all of the interaction sites with successful fungal penetration (cv Pallas and BCPMla12) showed accumulation of H₂O₂ in the cell wall or in the plasma membrane area around the penetration site within 22 hai (seen as a brownish ring in Fig. 2H).

H₂O₂ in Resistance Mediated by the Mlg Gene

The frequency of interaction sites with attacked cells undergoing HR reached a level of 76% by 30 hai (Table I). The number of interaction sites with H₂O₂ accumulation in whole cells was strongly correlated with the number of HRs and, compared with cv Pallas, enhanced in all of the evaluated periods (Fig. 4).

H₂O₂ in Resistance Mediated by the Chemical DCINA

Soil-drench treatment of cv Pallas seedlings with 8 ppm DCINA enhanced the frequency of interaction sites with HR in attacked epidermal cells compared with control plants (Table I). Whole-cell H₂O₂ accumulation was seen in the cells undergoing HR (Fig. 4). The penetration attempt of the fungus was unsuccessful, and effective papillae were associated with a compact brownish DAB staining (not shown). The pattern of H₂O₂ generation was similar to that found in BCPMlg (Fig. 4).

Determination of SA in Compatible and Incompatible Interactions of NILs with BghA6

View larger version (16K): [in this window] [in a new window]   Figure 5. Endogenous levels of total SA in NILs at different time points after inoculation with BghA6. Seven-day-old primary leaves of the barley lines were inoculated with 20 conidia mm2. At the indicated time points, leaves were cut off and levels of total SA were determined by HPLC and fluorescence detection. Each point represents the average of duplicates or triplicates of five leaves. Error bars show the SD of the triplicates. Repetition of the experiments led to similar results. FW, Fresh weight.

View larger version (15K): [in this window] [in a new window]   Figure 6. Exemplary HPLC chromatograms of three analog leaf extracts of BCPmlo5 from independent experiments (different plant batches). Noninoculated samples were taken on the 8th d after seed germination for determination of total SA. For unknown reasons, extracts showed differing contents of SA, although the contents of other compounds did not differ. The middle and the lower chromatogram show low contents of endogenous 2-methoxybenzoic acid (2MBA). The upper line represents an extract containing 2MBA as an internal standard. rel. f., Relative fluorescence.

Basal levels of SA differed significantly between independent experiments (Fig. 6) but not within an experiment (Fig. 5). Figure 6 shows three representative HPLC traces from independent experiments with SA contents amounting to 101, 1122, and 513 ng g¹ fresh weight. The results with the highest contents of SA are shown in Figure 5.

	DISCUSSION

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H₂O₂ Accumulation Is Associated with Barley Defense Phenotypes

A detailed, cell-type-specific investigation revealed a PR H₂O₂ burst in association with the most prominent barley defense responses toward an attacking pathogen, i.e. the formation of effective papillae and HR. Comparative analysis of NILs bearing the resistance genes Mlg, Mla12, and mlo5 demonstrated that these genes affect the frequency, but not the quality, of single-cell defense phenotypes associated with H₂O₂ accumulation. This also applies to the mode of action of the chemical DCINA; i.e. the accumulation pattern of H₂O₂ associated with effective papillae and HR was quantitatively, but not qualitatively, distinguishable from that observed in all interactions of NILs with BghA6, including the susceptible parent cv Pallas.

Aside from its association with HR, H₂O₂ strongly accumulated in the effective papillae that prevented the attacking pathogen from penetration into the epidermal host cells (Figs. 2, A and D, and 3). All of the papillae were encircled by vesicles delivering material for cell wall toughening at the site of fungal attack (McKeen and Rimmer, 1973; Bushnell and Berquist, 1975). In our experiments, these vesicles did not only stain positively for H₂O₂, but also fluoresced under UV excitation, indicating the presence of phenolic material. Because DAB polymerization depends on the presence of H₂O₂ and peroxidase (Thordal-Christensen et al., 1997), our data suggest that the vesicles targeted to effective papillae contained, in addition to H₂O₂, peroxidase, and probably partially polymerized phenolics. Both the papilla-associated H₂O₂ accumulation and the papilla-directed vesicle transport coincided with the arrest of fungal development (Fig. 3; Table I). The absence of H₂O₂ in papillae at early interaction stages correlated with their inefficiency (Figs. 2B and 3; Table I). The unsuccessful penetration attempts by the pathogen may be explained by papilla toughening through cross-linking reactions driven by H₂O₂. Consistently, resistance of papilla-bound phenolics to saponification was found in mlo-resistant barley 2 h earlier than in susceptible plants (von Röpenack et al., 1998). H₂O₂ may also have exerted direct toxic effects on the fungus. Thus, successful penetration may have depended on the ability of the fungus to prevent local H₂O₂ accumulation. Garre et al. (1998) have recently suggested that catalases secreted by Claviceps purpurea during infection of rye may be suppressors of plant defense.

The PR spatial and temporal patterns of H₂O₂ accumulation were slightly different from those of O₂^· in the same NILs (Hückelhoven and Kogel, 1998; Kogel and Hückelhoven, 1999). O₂^· was localized in a subcellular region where host plasma membrane and fungal haustorium came into contact (genotypes cv Pallas and BCPMla12), but not in association with effective papillae (genotypes BCPMlg and BCPmlo5). Thus, O₂^· generation in attacked cells was elicited by the cellular contact of host and pathogen, whereas this was not a prerequisite for H₂O₂ accumulation.

The fact that O₂^· and H₂O₂ accumulation could be spatially distinguished in the same defense phenotype also suggests that H₂O₂ in the cell walls and cytosolic vesicles of attacked epidermal cells did not depend upon preceding O₂^· generation driven by an NADPH oxidase (Mehdy, 1994) but might have been the product of alternative H₂O₂-generating sources. Candidate enzymes for PR H₂O₂ production in the epidermis of barley are peroxidase (Gross et al., 1977; Kerby and Somerville, 1992; Kogel et al., 1994; Scott-Craig et al., 1995), which was inhibited in the O₂^· assay (Hückelhoven and Kogel, 1998), oxalate oxidase (Zhou et al., 1998), and an oxalate-oxidase-like protein. Wei et al. (1998) have recently shown the latter to be involved in papilla resistance to powdery mildew fungus. PR H₂O₂ accumulation in the mesophyll tissue was seen especially in BCPMla12 (Fig. 2E), and it was accompanied by mesophyll cell death occurring exclusively in this line. In contrast, a strong infection-related and partly light-dependent O₂^· burst was found in both BCPMlg and BCPMla12 (Hückelhoven and Kogel, 1998). The O₂^· burst was essentially associated with chloroplasts, whereas H₂O₂ was rarely observed in these organelles. Therefore, PR chloroplastic O₂^· generation did not result in detectable amounts of H₂O₂ at interaction sites. These data indicate that O₂^· and H₂O₂ in the mesophyll tissue were not generated by the same source. We therefore speculate that the H₂O₂ in the mesophyll was generated by an oxalate oxidase recently localized in this tissue of Mla-resistant barley (Zhou et al., 1998).

A transient accumulation of H₂O₂ in cv Pallas and BCPMla12 was also seen at the sites of successful penetration near the plasma membranes around papillae (Fig. 2H). Because O₂^· was detected at the same subcellular region of these NILs (Hückelhoven and Kogel, 1998), H₂O₂ at this site may have stemmed from O₂^· that was generated in response to the cellular contact of host and pathogen. This local H₂O₂ burst was not sufficient to trigger HR. Xu and Heath (1998) recently reported that a Ca²⁺ influx in cowpea cells attacked by the cowpea rust fungus seemed to be elicited by penetration.

Differences in the pattern of H₂O₂ accumulation between BCPMla12 and cv Pallas were detected when epidermal and mesophyll cells of BCPMla12 carried out HR with a higher frequency from 30 and 36 hai onward (Figs. 2E and 4). The mesophyll tissue was not directly attacked by the fungus, implying that a signal spreading from the fungus or the attacked cell was essential for this response.

Because H₂O₂ generation was rarely seen before plant responses became microscopically visible, we suggest that active oxygen species generation in the early stages of incompatible interactions may change the cellular redox state before it becomes detectable by histochemical methods. This interpretation is supported by data from Vanacker et al. (1998), who reported that foliar and apoplastic glutathione contents were increased in resistant barley after inoculation with powdery mildew fungus, although apoplastic glutathione was reduced in susceptible barley. The same study showed that enhanced foliar catalase activity was associated with susceptibility. Our data show that haustorium-invaded cells, even on Mla12-resistant plants (compatible single-cell interaction), were essentially free of H₂O₂, whereas cells encircling the penetrated cell showed an H₂O₂ burst (Fig. 2G). This may indicate that single-cell compatibility was the cause or the consequence of enhanced antioxidative status of the host cell. Because haustorium establishment on the susceptible line cv Pallas was never followed by HR and/or strong H₂O₂ accumulation, we suggest that this was the key event for the achievement of the antioxidative state in the compatible interaction of barley and powdery mildew fungus.

SA Accumulation Is Not Associated with Defense Responses

Basal levels of SA differed significantly between independent experiments, but not within the same kinetic analysis. We avoided analytical mistakes by comparing chromatographs of analog samples, clearly demonstrating that the basal content of SA, but not of the other detected compounds, differed from experiment to experiment (Fig. 6). This may explain the greatly differing data for basal SA levels in barley: Vallélian-Bindschedler et al. (1998) found levels of <150 ng g¹ fresh weight, and Raskin et al. (1990) found levels of 2130 ng g¹ fresh weight.

It is well known that PR gene transcripts accumulate upon inoculation of barley (including the NILs) with powdery mildew fungus (Freialdenhoven et al., 1994; Kogel et al., 1994; Gregersen et al., 1997). Therefore, PR-gene activation is not dependent on elevated SA concentrations regardless of the introgressed resistance genes. This agrees with a study by Vallélian-Bindschedler et al. (1998), who showed that PR gene expression in barley after inoculation with B. graminis f.sp. hordei and B. graminis f.sp. tritici was independent of the accumulation of SA. Furthermore, it was shown that biological induction of resistance in dicotyledonous plants did not necessarily depend on SA accumulation (van Wees et al., 1997; Vidal et al., 1998).

Evidence for an SA-sensitive defense pathway comes from experiments with the synthetic SA derivative 3,5-dichlorosalicylic acid, which induced resistance to powdery mildew fungus in cv Pallas (Kogel et al., 1995); and such evidence also comes from experiments with barley mlo-double mutants (genotype mlo ror1), which are highly sensitive to chemical inducers and show enhanced resistance after treatment with SA (B. Jarosch, P. Schulze-Lefert, and K-H. Kogel, unpublished data). Therefore, as in dicotyledonous plants, there may be SA-dependent as well as SA-independent pathways leading to pathogen resistance in barley.

	FOOTNOTES

   Received October 13, 1998; accepted January 14, 1999.

	ABBREVIATIONS

Abbreviations: DAB, 3,3-diaminobenzidine. DCINA, 2,6-dichloroisonicotinic acid. hai, hour(s) after inoculation. HR, hypersensitive cell death response. NIL, near-isogenic backcross line. PR, pathogenesis-related. SA, salicylic acid.

	ACKNOWLEDGMENTS

The authors are grateful to Drs. Balázs Barna and Ruth Schiffer for fruitful discussions.

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