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Tissue-Specific Superoxide Generation at Interaction Sites in Resistant and Susceptible Near-Isogenic Barley Lines Attacked by the Powdery Mildew Fungus (Erysiphe graminis f. sp. hordei)

The pathogenesis-related, azide-insensitive generation of superoxide anions (O₂ ^-) was comparatively analyzed in near-isogenic barley (Hordeum vulgare cv. Pallas) lines carrying the powdery mildew (Erysiphe graminis f. sp. hordei) resistance genes Mla12, Mlg, and mlo5, respectively, by the microscopic detection of nitroblue tetra-zolium (NBT) reduction to dark blue formazan dyes. These genes govern fungal arrest at different stages of the interaction: (i) at the penetration stage within cell wall appositions (papillae) leaving the attacked cell alive (mlo); (ii) within papillae of cells that subsequently undergo a hypersensitive cell death (HR) (Mlg); or (iii) after penetration by a subsequent HR (Mla12). The susceptible parent line Pallas showed a transient O₂ ^- generation in penetrated epidermal cells at 18 h after inoculation (hai), whereas epidermal cells of the resistant BCPMla12 produced O₂ ^- over a longer time range (by 18 to 36 hai) preceding cell death. No oxidative burst was detected in association with penetration resistance due to effective papillae (BCPMlg and BCPmlo5) although Mlg specified an HR subsequent to fungal arrest. Hence, O₂ ^- generation in attacked epidermal cells was a result of fungal penetration of the host cell walls and subsequent contact with the host plasma membrane, and not a requirement for HR elicitation. O₂ ^- generation in the mesophyll tissue beneath attacked cells was associated with the response mediated by the genes Mla12 and Mlg. However, only BCPMla12 showed mesophyll cell death. The data indicate that, in barley, O₂ ^- accumulation is not a single key determinant of HR in response to a powdery mildew attack.