Superoxide Generation in Chemically Activated Resistance of Barley in Response to Inoculation with the Powdery Mildew Fungus

In a previous work, a phenotype-specific accumulation of superoxide radical anions (O^??₂) after attack of the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) in near-isogenic barley (Hordeum vulgare L.) lines bearing different Mlx genes for resistance was described (Hückelhoven and Kogel, 1998). We have now a histochemical study of the pathogenesis-related O^??₂ generation in the systemic activated resistance (SAR) response induced in barley cv Pallas by the plant activator 2,6-dichloroisonicotinic acid (DCINA). SAR-specific defence was conducted prevalently characterized by penetration resistance. Fungal arrest was observed before haustorium formation by a highly localized cell wall reinforcement (effective papillae) and, in most cases, by a subsequent hypersensitive cell death (HR). No O^??₂ generation was found in association with these plant defence responses. However, a strong O^??₂ burst in the attacked epidermal cells was detected in the control plants which were not activated by DCINA. This burst coincided with cell wall penetration and subsequent contact of the pathogen with the host plasma membrane. A strong SAR-related O^??₂ burst was induced in the mesophyll tissue beneath the attacked and hypersensitively reacting epidermal cells in plants treated with DCINA. The accumulation of O^??₂ was confined to chloroplasts. The remarkable burst in mesophyll tissue was not followed by mesophyll-HR indicating that chloroplastic O^??₂ generation is not sufficient for the hypersensitive cell death. Since the same pattern of pathogenesis-related O^??₂ accumulation was identified for race-specific response mediated by the Mlg gene for powdery mildew resistance, the present data are consistent with the hypothesis that the SAR phenotype is a phenocopy of the Mlg-type resistance (Kogel et al., 1994).     

Non-host resistance of barley is associated with a hydrogen peroxide burst at sites of attempted penetration by wheat powdery mildew fungus

In barley, non-host resistance against the wheat powdery mildew fungus (Blumeria graminis f.sp. tritici, Bgt) is associated with the formation of cell wall appositions and a hypersensitive reaction in which epidermal cells die rapidly in response to fungal attack. In the interaction of barley with the pathogenic barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh), these defence reactions are also associated with accumulation of H₂O₂. To elucidate the mechanism of non-host resistance, the accumulation of H₂O₂ in response to Bgt was studied in situ by histochemical staining with diaminobenzidine. H₂O₂ accumulation was found in cell wall appositions under appressoria from Bgt and in cells undergoing a hypersensitive reaction. A mutation (mlo5) at the barley Mlo locus, that confers broad spectrum resistance to Bgh, did not influence the barley defence phenotype to Bgt. Significantly, Bgt triggered cell death on mlo5-barley while Bgh did not.     

E3 ubiquitin ligase gene CMPG1–V from Haynaldia villosa L. contributes to powdery mildew resistance in common wheat (Triticum aestivum L.)

Powdery mildew is one of the most devastating wheat fungal diseases. A diploid wheat relative, Haynaldia villosa L., is highly resistant to powdery mildew, and its genetic resource of resistances, such as the Pm21 locus, is now widely used in wheat breeding. Here we report the cloning of a resistance gene from H. villosa, designated CMPG1–V, that encodes a U–box E3 ubiquitin ligase. Expression of the CMPG1–V gene was induced in the leaf and stem of H. villosa upon inoculation with Blumeria graminis f. sp. tritici (Bgt) fungus, and the presence of Pm21 is essential for its rapid induction of expression. CMPG1–V has conserved key residues for E3 ligase, and possesses E3 ligase activity in vitro and in vivo. CMPG1–V is localized in the nucleus, endoplasmic reticulum, plasma membrane and partially in trans‐Golgi network/early endosome vesicles. Transgenic wheat over‐expressing CMPG1–V showed improved broad‐spectrum powdery mildew resistance at seedling and adult stages, associated with an increase in expression of salicylic acid‐responsive genes, H₂O₂ accumulation, and cell‐wall protein cross‐linking at the Bgt infection sites, and the expression of CMPG1–V in H. villosa was increased when treated with salicylic acid, abscisic acid and H₂O₂. These results indicate the involvement of E3 ligase in defense responses to Bgt fungus in wheat, particularly in broad‐spectrum disease resistance, and suggest association of reactive oxidative species and the phytohormone pathway with CMPG1–V‐mediated powdery mildew resistance.     

A high-resolution genetic map and a diagnostic RFLP marker for the Mlg resistance locus to powdery mildew in barley

The semi-dominantly acting Mlg resistance locus in barley confers race-specific resistance to the obligate biotrophic fungus Erysiphe graminis f.sp. hordei. A high-resolution genetic map was constructed at Mlg based on a cross between the near-isogenic barley lines Pallas BC₅ Mlg and Pallas mlg. A total of 2000 F₂ progeny were inspected by cleaved amplified polymorphic sequence (CAPS) analysis, defining a 4.47 cM interval encompassing the resistance locus. Pathogen challenge of the segregants with multiple powdery mildew isolates uncovered a novel resistance specificity in Pallas BC₅ Mlg. Probes from within 4.0 cM of Mlg were mapped in rice, revealing orthologues on five different rice chromosomes and suggesting multiple breaks of chromosomal collinearity in this region between the two grass species. The most tightly Mlg-linked RFLP marker, MWG032, was shown to reliably detect the presence of the resistance allele in a collection of 30 European barley cultivars. Received: 23 March 2000 / Accepted: 20 April 2000     

Differential expression of putative cell death regulator genes in near-isogenic,resistant and susceptible barley lines during interaction with the powdery mildew fungus

We analysed pathogenesis-related expression of genes, that are assumed to be involved in ubiquitous plant defence mechanisms like the oxidative burst, the hypersensitive cell death reaction (HR) and formation of localized cell wall appositions (papillae). We carried out comparative northern blot and RT-PCR studies with near-isogenic barley (Hordeum vulgareL. cv. Pallas) lines (NILs) resistant or susceptible to the powdery mildew fungus race A6 (Blumeria graminis f.sp. hordei, BghA6). The NILs carrying one of the R-genes Mla12, Mlg or the mlo mutant allele mlo5 arrest fungal development by cell wall appositions (mlo5) or a HR (Mla12) or both (Mlg). Expression of an aspartate protease gene, an ascorbate peroxidase gene and a newly identified cysteine protease gene was up-regulated after inoculation with BghA6, whereas the constitutive expression-level of a BAS gene, that encodes an alkyl hydroperoxide reductase, was reduced. Expression of a newly identified barley homologue of a mammalian cell death regulator, Bax inhibitor 1, was enhanced after powdery mildew inoculation. An oxalate oxidase-like protein was stronger expressed in NILS expressing penetration resistance. A so far unknown gene that putatively encodes the large subunit of a superoxide generating NADPH oxidases was constitutively expressed in barley leaves and its expression pattern did not change after inoculation. A newly identified barley Rac1 homologue was expressed constitutively, such as the functionally linked NADPH oxidase gene. Gene expression patterns are discussed with regard to defence mechanisms and signal transduction.     

籽用美洲南瓜叶片对白粉病菌的抗逆生理响应

以籽用美洲南瓜(Cucurbita pepo L.)白粉病抗病品系F2和感病品系M3为试材,在人工气候箱内接种白粉病生理小种2US孢子悬浮液,考察在接种白粉病菌后南瓜幼苗植株与白粉病菌的互作、叶片活性氧代谢及保护酶活性的变化,探讨南瓜抵御白粉病的生理机制。结果表明：(1)与感病品系M3相比,接种白粉病菌后,抗病品系F2叶片上病原菌发育缓慢,较难侵染叶片。(2)抗病品系F2在感病初期叶片H₂O₂、O₂^-·含量迅速升高后逐渐下降,而感病品系在感病初期H₂O₂、O₂^-·含量上升缓慢,在达最大值后始终保持较高水平,且感病品系叶片MDA含量始终高于抗病品系;组织化学染色分析发现,抗病品系叶片着色比感病品系快,之后着色面积有所减少并趋于较低水平。(3)抗病品系F2和感病品系M3叶片抗氧化酶CAT、SOD、POD活性及PAL、PPO活性在接种白粉病菌后均显著增加,但抗病品系的活性及其增幅均高于感病品系。研究发现,籽用美洲南瓜抗病品系叶片上白粉病菌发育缓慢,较难受到侵染,生成菌丝体后叶片上粉状斑点较小;抗病品系在被白粉病菌侵染初期依靠活性氧的增加抵御病原菌的入侵,随着活性氧含量增加抗病品系通过迅速增加自身抗氧化酶活性来防止氧化胁迫;与感病品系相比,抗病品系在受病原菌侵染后能迅速增加PAL、PPO活性以抵御病原菌侵染。     

Specific Targeting of the Arabidopsis Resistance Protein RPW8.2 to the Interfacial Membrane Encasing the Fungal Haustorium Renders Broad-Spectrum Resistance to Powdery Mildew

Powdery mildew fungal pathogens penetrate the plant cell wall and develop a feeding structure called the haustorium to steal photosynthetate from the host cell. Here, we report that the broad-spectrum mildew resistance protein RPW8.2 from Arabidopsis thaliana is induced and specifically targeted to the extrahaustorial membrane (EHM), an enigmatic interfacial membrane believed to be derived from the host cell plasma membrane. There, RPW8.2 activates a salicylic acid (SA) signaling-dependent defense strategy that concomitantly enhances the encasement of the haustorial complex and onsite accumulation of H₂O₂, presumably for constraining the haustorium while reducing oxidative damage to the host cell. Targeting of RPW8.2 to the EHM, however, is SA independent and requires function of the actin cytoskeleton. Natural mutations that impair either defense activation or EHM targeting of RPW8.2 compromise the efficacy of RPW8.2-mediated resistance. Thus, the interception of haustoria is key for RPW8-mediated broad-spectrum mildew resistance.     

Mapping resistance to powdery mildew in barley reveals a large-effect nonhost resistance QTL

Key message

Resistance factors against non-adapted powdery mildews were mapped in barley. Some QTLs seem effective only to non-adapted mildews, while others also play a role in defense against the adapted form.The durability and effectiveness of nonhost resistance suggests promising practical applications for crop breeding, relying upon elucidation of key aspects of this type of resistance. We investigated which genetic factors determine the nonhost status of barley (Hordeum vulgare L.) to powdery mildews (Blumeria graminis). We set out to verify whether genes involved in nonhost resistance have a wide effectiveness spectrum, and whether nonhost resistance genes confer resistance to the barley adapted powdery mildew. Two barley lines, SusBgt_SC and SusBgt_DC, with some susceptibility to the wheat powdery mildew B. graminis f.sp. tritici (Bgt) were crossed with cv Vada to generate two mapping populations. Each population was assessed for level of infection against four B. graminis ff.spp, and QTL mapping analyses were performed. Our results demonstrate polygenic inheritance for nonhost resistance, with some QTLs effective only to non-adapted mildews, while others play a role against adapted and non-adapted forms. Histology analyses of nonhost interaction show that most penetration attempts are stopped in association with papillae, and also suggest independent layers of defence at haustorium establishment and conidiophore formation. Nonhost resistance of barley to powdery mildew relies mostly on non-hypersensitive mechanisms. A large-effect nonhost resistance QTL mapped to a 1.4 cM interval is suitable for map-based cloning.

     

Barley Mla and Rar mutants compromised in the hypersensitive cell death response against Blumeria graminis f.sp. hordei are modified in their ability to accumulate reactive oxygen intermediates at sites of fungal invasion

 The pathogenesis-related accumulation of superoxide radical anions (O^·− ₂) and hydrogen peroxide (H₂O₂) was comparatively analyzed in a barley line (Hordeum vulgare L. cv Sultan-5) carrying the powdery mildew (Blumeria graminis f.sp. hordei, Speer, Bgh) resistance gene Mla12, and in susceptible mutants defective in Mla12 or in genes “required for Mla12-specified disease resistance” (Rar1 and Rar2). In-situ localization of reactive oxygen intermediates was performed both by microscopic detection of azide-insensitive nitroblue tetrazolium (NBT) reduction or diaminobenzidine (DAB) polymerization, and by an NBT-DAB double-staining procedure. The Mla12-mediated hypersensitive cell death occurred either in attacked epidermal cells or adjacent mesophyll cells of wild-type plants. Whole-cell H₂O₂ accumulation was detected in dying cells, while O^·− ₂ emerged in adjacent cells. Importantly, all susceptible mutants lacked these reactions. An oxalate oxidase, which is known to generate H₂O₂ and has been implicated in barley resistance against the powdery mildew fungus, was not differentially expressed between the wild type and all mutants. The results demonstrate that the Rar1 and Rar2 gene products, which are control elements of R-gene-mediated programmed cell death, also control accumulation of reactive oxygen intermediates but not the pathogenesis-related expression of oxalate oxidase. Received: 7 January 2000 / Accepted: 2 June 2000     

ROPGAPs of Arabidopsis limit susceptibility to powdery mildew

The barley ROP GTPase HvRACB is a susceptibility factor of barley to powdery mildew caused by the biotrophic fungus Blumeria graminis f.sp. hordei (Bgh). In a recent publication, we reported about a MICROTUBULE-ASSOCIATED ROP GTPASE-ACTIVATING PROTEIN 1 (HvMAGAP1) of barley. Transient-induced gene silencing or overexpression of HvMAGAP1 resulted in enhanced or reduced susceptibility to Bgh, respectively, indicating a possible HvRACB-antagonistic function of HvMAGAP1 in interaction with Bgh. HvMAGAP1 also influences the polarity of cortical microtubules in interaction with Bgh. In AtROPGAP1 and AtROPGAP4, Arabidopsis homologs of HvMAGAP1, knock-out T-DNA insertions enhanced susceptibility of Arabidopsis to the virulent powdery mildew fungus Erysiphe cruciferarum, indicating functions of ROPGAPs in pathogen interaction of monocots and dicots. Here we discuss the role of AtROPGAP1 and AtROPGAP4 in Arabidopsis pathogenesis of powdery mildew in some more detail.     

A cDNA clone for a pathogenesis-related protein 1 from barley

A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.     

Constitutively activated barley ROPs modulate epidermal cell size, defense reactions and interactions with fungal leaf pathogens

RHO-like monomeric G-proteins of plants (ROPs, also called RACs), are involved in plant development and interaction with the environment. The barley (Hordeum vulgare) ROP protein HvRACB has been shown to be required for entry of the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) into living host cells. To get a deeper insight into evolutionarily conserved functions of ROPs in cell polarity and pathogen responses, we stably expressed constitutively activated (CA) mutant variants of different barley ROPs (HvRACB, HvRAC1, HvRAC3) in barley. CA HvROPs induced epidermal cell expansion and/or abolished polarity in tip growing root hairs. All three CA HvROPs enhanced susceptibility of barley to penetration by Bgh whereas only CA HvRAC1 supported whole cell H₂O₂ production in non-penetrated cells. Despite increasing penetration by Bgh, CA HvRAC1 promoted callose deposition at sites of fungal attack and resistance to penetration by Magnaporthe oryzae. The data show an involvement of ROPs in polar growth processes of the monocot barley and in responses to fungal pathogens with different life style.     

The induction of systemic resistance in barley to powdery mildew infection using salicylates and various phenolic acids

Treatment of the first leaves of barley seedlings with either 5, 10, 15 or 20 mM salicylic acid, sodium salicylate or acetylsalicylic acid resulted in significant reductions in powdery mildew infection on the upper, second leaves. In general, the greatest reduction in mildew infection on the second leaves was obtained by spraying the first leaves with a 15 mM concentration of the compounds. Although the largest reduction in mildew infection of the upper leaves was obtained when the compounds were applied to the first leaves 1–2 days before inoculation, very substantial reductions in infection were still obtained if the first leaves were treated 12 days before inoculation. The three compounds had little direct effect on mildew infection. When ¹⁴C-salicylic acid was fed to first leaves of barley seedlings, uptake was rapid and increased with time. Within 6 h, 0.2% of the salicylic acid appeared in the second leaf and by 24 h after feeding, this had increased to 1.4% (1.1 μmol salicylic acid g^-1 fresh wt). The application of various phenolic acids to first leaves also led to reductions in mildew infection on the second leaves. In particular, treatment of the first leaves with 1 mM vanillic acid, isovanillic acid or syringic acid, reduced mildew infection of the second leaves by 81–87%.     

Influence of Exogenous Salicylic Acid on the Level of Phytohormones in Tissues of Phlox paniculata and Phlox setacea Leaves with Special Reference to Resistance against the Powdery Mildew Causative Agent Erysiphe cichoracearum DC. f. phlogis Jacz

We studied the effects of exogenous salicylic acid on the level of endogenous cytokinins and abscisic and salicylic acids in the tissues of leaves of phloxes contrasting in resistance against the powdery mildew causative agent: susceptible Phlox paniculata L. and resistant Ph. setacea L. Studies were carried out under the conditions of biotic stress. The initial level of salicylic and abscisic acids and cytokinins is the highest in the resistant phlox species. After treatment with salicylic species, the total level of cytokinins and endogenous salicylic acid increased in both species. When the treated phlox species were infected by the powdery mildew causative agent, the level of abscisic and salicylic acids increased in the susceptible Ph. paniculata, while that of cytokinins increased in the resistant Ph. setacea. The role of salicylic acid in the induction of plant defense reactions against phytopathogens is discussed.     

The AtrbohD-mediated oxidative burst elicited by oligogalacturonides in Arabidopsis is dispensable for the activation of defense responses effective against Botrytis cinerea

Oligogalacturonides (OGs) are endogenous elicitors of defense responses released after partial degradation of pectin in the plant cell wall. We have previously shown that, in Arabidopsis (Arabidopsis thaliana), OGs induce the expression of PHYTOALEXIN DEFICIENT3 (PAD3) and increase resistance to the necrotrophic fungal pathogen Botrytis cinerea independently of signaling pathways mediated by jasmonate, salicylic acid, and ethylene. Here, we illustrate that the rapid induction of the expression of a variety of genes by OGs is also independent of salicylic acid, ethylene, and jasmonate. OGs elicit a robust extracellular oxidative burst that is generated by the NADPH oxidase AtrbohD. This burst is not required for the expression of OG-responsive genes or for OG-induced resistance to B. cinerea, whereas callose accumulation requires a functional AtrbohD. OG-induced resistance to B. cinerea is also unaffected in powdery mildew resistant4, despite the fact that callose accumulation was almost abolished in this mutant. These results indicate that the OG-induced oxidative burst is not required for the activation of defense responses effective against B. cinerea, leaving open the question of the role of reactive oxygen species in elicitor-mediated defense.