Assessment and introduction of quantitative resistance to Fusarium head blight in elite spring barley.

Breeding for resistance is a key task to control Fusarium head blight (FHB), a devastating disease of small cereals leading to economic losses and grain contamination with mycotoxins harmful for humans and animals. In the present work, FHB resistance of the six-rowed spring barley 'Chevron' to FHB in Germany was compared with those of adapted German spring barley cultivars. Both under natural infection conditions and after spray inoculation with conidia of Fusarium culmorum, F. sporotrichioides, and F. avenaceum under field conditions, Chevron showed a high level of quantitative resistance to the infection and contamination of grain with diverse mycotoxins. This indicates that Chevron is not only a little susceptible to deoxynivalenol-producing Fusarium spp. but also to Fusarium spp. producing type A trichothecenes and enniatins. Monitoring the initial infection course of F. culmorum on barley lemma tissue by confocal laser-scanning microscopy provided evidence that FHB resistance of Chevron is partially mediated by a preformed penetration resistance, because direct penetration of floral tissue by F. culmorum was observed rarely on Chevron but was common on susceptible genotypes. Alternatively, F. culmorum penetrated Chevron lemma tissue via stomata, which was unusual for susceptible genotypes. We generated double-haploid barley populations segregating for the major FHB resistance quantitative trait loci (QTL) Qrgz-2H-8 of Chevron. Subsequently, we characterized these populations by spray inoculation with conidia of F. culmorum and F. sporotrichioides. This suggested that Qrgz-2H-8 was functional in the genetic background of European elite barley cultivars. However, the degree of achieved resistance was very low when compared with quantitative resistance of the QTL donor Chevron, and the introgression of Qrgz-2H-8 was not sufficient to mediate the cellular resistance phenotype of Chevron in the European backgrounds.

Volatile-mediated signalling in barley induces metabolic reprogramming and resistance against the biotrophic fungus Blumeria hordei.

Plants have evolved diverse secondary metabolites to counteract biotic stress. Volatile organic compounds (VOCs) are released upon herbivore attack or pathogen infection. Recent studies suggest that VOCs can act as signalling molecules in plant defence and induce resistance in distant organs and neighbouring plants. However, knowledge is lacking on the function of VOCs in biotrophic fungal infection on cereal plants. We analysed VOCs emitted by 13 ± 1-day-old barley plants (Hordeum vulgare L.) after mechanical wounding using passive absorbers and TD-GC/MS. We investigated the effect of pure VOC and complex VOC mixtures released from wounded plants on the barley-powdery mildew interaction by pre-exposure in a dynamic headspace connected to a powdery mildew susceptibility assay. Untargeted metabolomics and lipidomics were applied to investigate metabolic changes in sender and receiver barley plants. Green leaf volatiles (GLVs) dominated the volatile profile of wounded barley plants, with (Z)-3-hexenyl acetate (Z3HAC) as the most abundant compound. Barley volatiles emitted after mechanical wounding enhanced resistance in receiver plants towards fungal infection. We found volatile-mediated modifications of the plant-pathogen interaction in a concentration-dependent manner. Pre-exposure with physiologically relevant concentrations of Z3HAC resulted in induced resistance, suggesting that this GLV is a key player in barley anti-pathogen defence. The complex VOC mixture released from wounded barley and Z3HAC induced e.g. accumulation of chlorophyll, linolenic acid and linolenate-conjugated lipids, as well as defence-related secondary metabolites, such as hordatines in receiving plants. Barley VOCs hence induce a complex physiological response and disease resistance in receiver plants.

Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation

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.

A Compromised Mlo Pathway Affects the Response of Barley to the Necrotrophic Fungus Bipolaris sorokiniana (Teleomorph: Cochliobolus sativus) and Its Toxins.

ABSTRACT In search of new durable disease resistance traits in barley to control leaf spot blotch disease caused by the necrotrophic fungus Bipolaris sorokiniana (teleomorph: Cochliobolus sativus), we developed macroscopic and microscopic scales to judge spot blotch disease development on barley. Infection of barley was associated with cell wall penetration and accumulation of hydrogen peroxide. The latter appeared to take place in cell wall swellings under fungal penetration attempts as well as during cell death provoked by the necrotrophic pathogen. Additionally, we tested the influence of a compromised Mlo pathway that confers broad resistance against powdery mildew fungus (Blumeria graminis f. sp. hordei). Powdery mildew-resistant genotypes with mutations at the Mlo locus (mlo genotypes) showed a higher sensitivity to infiltration of toxic culture filtrate of Bipolaris sorokiniana as compared with wild-type barley. Mutants defective in Ror, a gene required for mlo-specified powdery mildew resistance, were also more sensitive to Bipolaris sorokiniana toxins than wild-type barley but showed less symptoms than mlo5 parents. Fungal culture filtrates induced an H2O2 burst in all mutants, whereas wild-type (Mlo) barley was less sensitive. The results support the hypothesis that the barley Mlo gene product functions as a suppresser of cell death. Therefore, a compromised Mlo pathway is effective for control of biotrophic powdery mildew fungus but not for necrotrophic Bipolaris sorokiniana. We discuss the problem of finding resistance traits that are effective against both biotrophic and necrotrophic pathogens with emphasis on the role of the anti-oxidative system of plant cells.

Root-to-shoot signalling: apoplastic alkalinization, a general stress response and defence factor in barley (Hordeum vulgare).

We used a noninvasive microprobe technique to record in substomatal cavities of barley leaves the apoplastic pH response to different stress situations. When K+ (or Na+) activity at the roots of intact plants was increased from 1 to 50 mM, the leaf apoplastic pH increased by 0.4 to 0.6 units within 8 to 12 min when stomata were open, and within 15 to 20 min when stomata were closed. This reaction was accompanied by a correlative increase in K+ activity. Addition of 1 microM abscisic acid caused an apoplastic alkalinization of 0.5 to 0.8 units, and low temperatures (4 degrees C) increased pH by 0.2 to 0.3 units. Addition of 100 mM sorbitol or pH changes in the range 4.0 to 7.9 had no effect, ruling out that osmotic potential and/or pH is the carried signal. On detached leaves, the same treatments yielded qualitatively similar results, suggesting that the xylem is the most likely signal path. Following the attack of powdery mildew, the apoplastic pH of barley leaves substantially increases. We demonstrate that in susceptible barley, pretreatment (soil drench) with the resistance-inducing chemical benzo- (1,2,3)thiadiazole-7-carbothioic acid S-methyl ester markedly enhances this pH response. This is consistent with previous finding that apoplastic alkalinization is related to the degree of resistance towards this fungus.

BAX Inhibitor-1, an ancient cell death suppressor in animals and plants with prokaryotic relatives.

BAX Inhibitor-1 (BI-1) was originally described as testis enhanced gene transcript in mammals. Functional screening in yeast for human proteins that can inhibit the cell death provoking function of BAX, a proapoptotic Bcl-2 family member, led to functional characterisation and renaming of BI-1. The identification of functional homologues of BI-1 in plants and yeast widened the understanding of BI-1 function as an ancient suppressor of programmed cell death. BI-1 is one of the few cell death suppressors conserved in animals and plants. Computer predictions and experimental data together suggest that BI-1 is a membrane spanning protein with 6 to 7 transmembrane domains and a cytoplasmic C-terminus sticking in the endoplasmatic reticulum and nuclear envelope. Proteins similar to BI-1 are present in other eukaryotes, bacteria, and even viruses encode BI-1 like proteins. BI-1 is involved in development, response to biotic and abiotic stress and probably represents an indispensable cell protectant. BI-1 appears to suppress cell death induced by mitochondrial dysfunction, reactive oxygen species or elevated cytosolic Ca(2+) levels. This review focuses on the present understanding about BI-1 and suggests potential directions for further analyses of this increasingly noticed protein.

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 (O2*-) 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 O2*- 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.

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.

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

Summary 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 H(2)O(2). To elucidate the mechanism of non-host resistance, the accumulation of H(2)O(2) in response to Bgt was studied in situ by histochemical staining with diaminobenzidine. H(2)O(2) 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.

Mutations in Ror1 and Ror2 genes cause modification of hydrogen peroxide accumulation in mlo-barley under attack from the powdery mildew fungus.

Abstract Race nonspecific resistance of barley against the barley powdery mildew fungus (Blumeria Graminis f.sp. Hordei, Speer, Bgh) is mediated by recessive mlo alleles and is controlled by at least two additional genes 'required for ml o-specified disease resistance' (Ror1 and Ror2). The pathogenesis-related accumulation of hydrogen peroxide (H(2)O(2)) was comparatively analysed in a susceptible barley line (Hordeum vulgare L. Cv Ingrid, genotype Mlo Ror1, Ror2), a resistant Ingrid backcross line carrying the mutant allele mlo5 (BCIngrid-mlo5, genotype mlo5 Ror1 Ror2), and in the moderately susceptible mutants A44 and A89 (genotypes mlo5 Ror1 ror2 and mlo5 ror1-2 Ror2, respectively). In situ localization of H(2)O(2) was performed by microscopic detection of 3,3-diaminobenzidine (DAB) polymerization. In BCIngrid-mlo5, penetration resistance against Bgh attack was closely correlated to H(2)O(2) accumulation in cytoplasmic aggregates and cell wall appositions beneath the appressorium. In contrast, H(2)O(2) accumulation was almost completely absent in susceptible Ingrid. Lines with mutations in Ror genes showed less H(2)O(2) accumulation beneath appressoria, but more interaction sites with whole cell H(2)O(2) accumulation and hypersensitive cell death response than resistant BCIngrid-mlo5. Thus, mutations in Ror1 or Ror2 genes influence the cellular pattern of H(2)O(2) accumulation in mlo plants attacked by Bgh. The data support the hypothesis that H(2)O(2) accumulation is involved in resistance to fungal penetration.