Biomedical subjects

Ralph Hückelhoven

Publications and source records attributed to Ralph Hückelhoven.

At least 19 recordsLinked to original sources

PubMed2006

The root endophytic fungus Piriformospora indica requires host cell death for proliferation during mutualistic symbiosis with barley.

Fungi of the recently defined order Sebacinales (Basidiomycota) are involved in a wide spectrum of mutualistic symbioses (including mycorrhizae) with various plants, thereby exhibiting a unique potential for biocontrol strategies. The axenically cultivable root endophyte Piriformospora indica is a model organism of this fungal order. It is able to increase biomass and grain yield of crop plants. In barley, the endophyte induces local and systemic resistance to fungal diseases and to abiotic stress. To elucidate the lifestyle of P. indica, we analyzed its symbiotic interaction and endophytic development in barley roots. We found that fungal colonization increases with root tissue maturation. The root tip meristem showed no colonization, and the elongation zone showed mainly intercellular colonization. In contrast, the differentiation zone was heavily infested by inter- and intracellular hyphae and intracellular chlamydospores. The majority of hyphae were present in dead rhizodermal and cortical cells that became completely filled with chlamydospores. In some cases, hyphae penetrated cells and built a meshwork around plasmolyzed protoplasts, suggesting that the fungus either actively kills cells or senses cells undergoing endogenous programmed cell death. Seven days after inoculation, expression of barley BAX inhibitor-1 (HvBI-1), a gene capable of inhibiting plant cell death, was attenuated. Consistently, fungal proliferation was strongly inhibited in transgenic barley overexpressing GFP-tagged HvBI-1, which shows that P. indica requires host cell death for proliferation in differentiated barley roots. We suggest that the endophyte interferes with the host cell death program to form a mutualistic interaction with plants.

Apoptosis↗

PubMed2006

Respiratory burst oxidase homologue A of barley contributes to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei.

Reactive oxygen intermediates (ROI) are closely related to defence reactions of plants against pathogens. A prominent role in the production of ROI has been attributed to the plant respiratory burst oxidase homologues (RBOH) of the human phagocyte GP91(phox). A barley RBOH, which encodes a putative superoxide (O2*-)) producing NADPH oxidase, is described here. Histochemical analysis of the barley-Blumeria graminis f. sp. hordei (Bgh) interaction showed that O(2*-) is produced locally at the site of penetration. In contrast, hydrogen peroxide (H2O2) is produced in non-penetrated cell wall appositions. A barley RBOHA cDNA was isolated and a minor induction of expression of RBOHA was observed during the interactions of barley with Bgh. Transient RNA interference-mediated gene silencing of HvRBOHA during the penetration process of Bgh led to an increase of basal penetration resistance. The results support a potential role of HvRBOHA in cellular accessibility to Blumeria graminis.

Amino Acid Sequence↗

PubMed2006

Endophyte or parasite--what decides?

Symbiosis between a fungus and a plant is a widespread phenomenon in nature. The outcome of such an interaction can vary in a seamless manner from mutualism to parasitism. In most cases, the host plant does not suffer, in fact it often gains an advantage from colonization by a fungus. This benefit is based on a fine-tuned balance between the demands of the invader and the plant response. If the interaction becomes unbalanced, disease symptoms appear or the fungus is excluded by induced host defence reactions. Symbioses of plants with beneficial or neutral endophytes share many common attributes with plant interactions with pathogens. Recent findings emerging from studies of compatible host-fungus interactions have enhanced our understanding of what determines whether the fungus behaves as an endophyte or a parasite and of how plants avoid exploitation by detrimental parasites but benefit from mutualistic endophytes.

Fungi↗

PubMed2006

Metrafenone: studies on the mode of action of a novel cereal powdery mildew fungicide.

Powdery mildew fungi are among the major pathogens causing diseases of cereals in the world. The mode of action of a novel systemic benzophenone fungicide, metrafenone, which is based on a precursor that is discussed in the preceding paper, has been analysed on the powdery mildew fungi of barley (Blumeria graminis Speer f. sp. hordei Marchal) and wheat (Blumeria graminis Speer f. sp. tritici Marchal). Preventive treatments reduced germination and blocked development beyond formation of appressoria, which penetrated less often. Moreover, metrafenone turned out to be an efficient curative fungicide, which rapidly affected fungal survival at low concentrations. The fungicide induced swelling, bursting and collapse of hyphal tips, resulting in the release of globules of cytoplasm. Bifurcation of hyphal tips, secondary appressoria and hyperbranching were also frequently observed. A histochemical analysis showed that metrafenone caused disruption of the apical actin cap and apical vesicle transport as well as weakening of the cell wall at hyphal tips. Finally, metrafenone strongly reduced sporulation. Reduced sporulation was associated with malformation of conidiophores that showed irregular septation, multinucleate cells and delocalisation of actin. Microtubules appeared to be only secondarily affected in metrafenone-treated B. graminis. The results suggest that the mode of action of metrafenone interferes with hyphal morphogenesis, polarised hyphal growth and the establishment and maintenance of cell polarity. Metrafenone likely disturbs a pathway regulating organisation of the actin cytoskeleton.

Ascomycota↗

PubMed2006

Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus.

Localized cell wall modification and accumulation of antimicrobial compounds beneath sites of fungal attack are common mechanisms for plant resistance to fungal penetration. In barley (Hordeum vulgare) leaves, light-microscopically visible vesicle-like bodies (VLBs) containing H(2)O(2) or phenolics frequently accumulate around cell wall appositions (syn. papillae), in which the penetration attempt of the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) is halted. By ultrastructural analyses, we demonstrated that the Bgh-induced VLBs represent different structures. VLBs intensively stained by H(2)O(2)-reactive dyes were actually small papillae instead of cytoplasmic vesicles. Other VLBs were identified as osmiophilic bodies or multivesicular compartments, designated paramural bodies (PMBs) and multivesicular bodies (MVBs). MVBs seemingly followed two distinct pathways: either they were engulfed by the tonoplast for degradation in the vacuole or they fused with the plasma membrane to release their internal vesicles into the paramural space and hence could be the origin of PMBs. MVBs and PMBs appeared to be multicomponent kits possibly containing building blocks to be readily assembled into papilla and antimicrobial compounds to be discharged against fungal penetration. Finally, we propose that released paramural vesicles might be similar to exosomes in animal cells.

Ascomycota↗

PubMed2006

Multivesicular compartments proliferate in susceptible and resistant MLA12-barley leaves in response to infection by the biotrophic powdery mildew fungus.

There is growing evidence that multivesicular bodies and cell wall-associated paramural bodies participate in the enhanced vesicle trafficking induced by pathogen attack. Here, we performed transmission electron microscopy in combination with cytochemical localization of H2O2 to investigate multivesicular compartments during establishment of compatible interaction in susceptible barley (Hordeum vulgare) and during hypersensitive response in resistant MLA12-barley infected by the barley powdery mildew fungus (Blumeria graminis f. sp. hordei). Multivesicular bodies, intravacuolar vesicle aggregates and paramural bodies proliferated in the penetrated epidermal cell during development of the fungal haustorium. These vesicular structures also proliferated at the periphery of intact cells, which were adjacent to the hypersensitive dying cells and deposited cell wall appositions associated with H2O2 accumulation. All plasmodesmata between intact cells and hypersensitive cells were constricted or blocked by cell wall appositions. These results suggest that multivesicular compartments participate in secretion of building blocks for cell wall appositions not only to arrest fungal penetration but also to contain hypersensitive cell death through blocking plasmodesmata. They may also participate in internalization of damaged membranes, deleterious materials, nutrients, elicitors and elicitor receptors.

Ascomycota↗

PubMed2005

Macroarray expression analysis of barley susceptibility and nonhost resistance to Blumeria graminis.

Different formae speciales of the grass powdery mildew fungus Blumeria graminis undergo basic-compatible or basic-incompatible (nonhost) interactions with barley. Background resistance in compatible interactions and nonhost resistance require common genetic and mechanistic elements of plant defense. To build resources for differential screening for genes that potentially distinguish a compatible from an incompatible interaction on the level of differential gene expression of the plant, we constructed eight dedicated cDNA libraries, established 13.000 expressed sequence tag (EST) sequences and designed DNA macroarrays. Using macroarrays based on cDNAs derived from epidermal peels of plants pretreated with the chemical resistance activating compound acibenzolar-S-methyl, we compared the expression of barley gene transcripts in the early host interaction with B. graminis f.sp. hordei or the nonhost pathogen B. graminis f.sp. tritici, respectively. We identified 102 spots corresponding to 94 genes on the macroarray that gave significant B. graminis-responsive signals at 12 and/or 24 h after inoculation. In independent expression analyses, we confirmed the macroarray results for 11 selected genes. Although the majority of genes showed a similar expression profile in compatible versus incompatible interactions, about 30 of the 94 genes were expressed on slightly different levels in compatible versus incompatible interactions.

Ascomycota↗

PubMed2005

The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield.

Disease resistance strategies are powerful approaches to sustainable agriculture because they reduce chemical input into the environment. Recently, Piriformospora indica, a plant-root-colonizing basidiomycete fungus, has been discovered in the Indian Thar desert and was shown to provide strong growth-promoting activity during its symbiosis with a broad spectrum of plants. Here, we report on the potential of P. indica to induce resistance to fungal diseases and tolerance to salt stress in the monocotyledonous plant barley. The beneficial effect on the defense status is detected in distal leaves, demonstrating a systemic induction of resistance by a root-endophytic fungus. The systemically altered "defense readiness" is associated with an elevated antioxidative capacity due to an activation of the glutathione-ascorbate cycle and results in an overall increase in grain yield. Because P. indica can be easily propagated in the absence of a host plant, we conclude that the fungus could be exploited to increase disease resistance and yield in crop plants.

Ascorbic Acid↗

PubMed2005

Ectopic expression of constitutively activated RACB in barley enhances susceptibility to powdery mildew and abiotic stress.

Small RAC/ROP-family G proteins regulate development and stress responses in plants. Transient overexpression and RNA interference experiments suggested that the barley (Hordeum vulgare) RAC/ROP protein RACB is involved in susceptibility to the powdery mildew fungus Blumeria graminis f. sp. hordei. We created transgenic barley plants expressing the constitutively activated RACB mutant racb-G15V under control of the maize (Zea mays) ubiquitin 1 promoter. Individuals of the T1 generation expressing racb-G15V were significantly more susceptible to B. graminis when compared to segregating individuals that did not express racb-G15V. Additionally, racb-G15V-expressing plants showed delayed shoot development from the third leaf stage on, downward rolled leaves, and stunted roots. Expression of racb-G15V decreased photosynthetic CO(2)-assimilation rates and transpiration of nonstressed leaves. In contrast, racb-G15V-expressing barley leaves, when detached from water supply, showed increased water loss and enhanced transpiration. Water loss was associated with reduced responsiveness to abscisic acid in regard to transpiration when compared to segregants not expressing racb-G15V. Hence, RACB might be a common signaling element in response to both biotic and abiotic stress.

Abscisic Acid↗

PubMed2005

Powdery mildew susceptibility and biotrophic infection strategies.

Plants are resistant to most potentially pathogenic microbes. This forces plant pathogens to develop sophisticated strategies to overcome basic plant resistance, either by masking intrusion or by suppression of host defences. This is particularly true for fungal pathogens, which establish long lasting interactions with living host tissue, without causing visible damage to invaded cells. The interactions of cereal crops and Arabidopsis with powdery mildew fungi are model systems for understanding host resistance. Currently, these systems are also promoting the understanding of fungal infection by identifying fungal pathogenicity and virulence factors and host target sites. This minireview focuses on recent findings about host susceptibility and the way powdery mildew fungi might induce it.

Apoptosis↗

PubMed2005

The receptor-like MLO protein and the RAC/ROP family G-protein RACB modulate actin reorganization in barley attacked by the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei.

Cytoskeleton remodelling is a crucial process in determining the polarity of dividing and growing plant cells, as well as during interactions with the environment. Nothing is currently known about the proteins, which regulate actin remodelling during interactions with invading pathogens. The biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) invades susceptible barley (Hordeum vulgare L.) by penetrating epidermal cells, which remain intact during fungal development. In contrast, resistant host plants prevent infection by inhibiting penetration through apoplastic mechanisms, which require focusing defence reactions on the site of attack. We stained actin filaments in a susceptible Mlo-genotype and a near-isogenic race-non-specifically resistant barley mlo5-mutant genotype using fluorescence-labelled phalloidin after chemical fixation. This revealed that the actin cytoskeleton is differentially reorganized in susceptible and resistant hosts challenged by Bgh. Actin filaments were polarized towards the sites of attempted penetration in the resistant host, whereas when susceptible hosts were penetrated, a more subtle reorganization took place around fungal haustoria. Strong actin filament focusing towards sites of fungal attack was closely associated with successful prevention of penetration. Actin focusing was less frequent and seemingly delayed in susceptible wild-type barley expressing the susceptibility factor MLO. Additionally, single cell overexpression of a constitutively activated RAC/ROP G-protein, CA RACB, another potential host susceptibility factor and hypothetical actin cytoskeleton regulator, partly inhibited actin reorganization when under attack from Bgh, whereas knockdown of RACB promoted actin focusing. We conclude that RACB and, potentially, MLO are host proteins involved in the modulation of actin reorganization and cell polarity in the interaction of barley with Bgh.

Actin Cytoskeleton↗

PubMed2004

Apoplastic pH signaling in barley leaves attacked by the powdery mildew fungus Blumeria graminis f. sp. hordei.

To investigate apoplastic responses of barley (Hordeum vulgare L.) to the barley powdery mildew fungus Blumeria graminis f. sp. hordei, noninvasive microprobe techniques were employed. H(+)- and Ca(2+)-selective microprobes were inserted into open stomata of barley leaves inoculated with Blumeria graminis f. sp. hordei race A6 conidia. Resistance gene-mediated responses of barley genotype Ingrid (susceptible parent line) and the near-isogenic resistant Ingrid backcross lines (I-mlo5, I-Mla12, and I-Mlg) were continuously monitored from 20 min to 4 days after inoculation. The main events were categorized as short-term responses around 2 h after inoculation (hai), intermediate responses around 8 and 12 hai, and long-term responses starting between 21 and 24 hai. Short-term responses were rapid transient decreases of apoplastic H(+)- and Ca2+ activities that lasted minutes only. Kinetics were similar for all genotypes tested, and thus, these short-term responses were attributed as nonspecific first encounters of fungal surface material with the host plasma membrane. This is supported by the observation that a microinjected chitin oligomer (GlcNAc)8 yielded similar apoplastic alkalinization. Intermediate responses are trains of H+ (increase) spikes that, being different in susceptible Ingrid and penetration-resistant I-mlo5 (or I-Mlg), were interpreted as accompanying specific events of papillae formation. Long-term events were massive slow and long-lasting alkalinizations up to two pH units above control. Since these latter changes were only observed with near-isogenic hypersensitive reaction (HR)-mounting genotypes I-Mla12 and I-Mlg but not with I-mlo5 or, to a smaller extent, with susceptible Ingrid, both lacking significant rates of HR, they were rated as cell death specific. It is concluded that apoplastic pH changes are important indicators of host-pathogen interactions that correlate with both the different stages of fungal development and the different types of host defense response.

Ascomycota↗

PubMed2004

Superoxide and hydrogen peroxide play different roles in the nonhost interaction of barley and wheat with inappropriate formae speciales of Blumeria graminis.

Nonhost resistance of cereals to inappropriate formae speciales of Blumeria graminis is little understood. However, on the microscopic level, nonhost defense to B. graminis is reminiscent of host defense preventing fungal development by penetration resistance and the hypersensitive cell death response (HR). We analyzed histochemically the accumulation of superoxide anion radicals (O2*-) and hydrogen peroxide (H2O2) at sites of B. graminis attack in nonhost barley and wheat. Superoxide visualized by subcellular reduction of nitroblue tetrazolium accumulated in association with successful fungal penetration in attacked cells and in cells neighboring HR. In contrast, H2O2 accumulated in cell wall appositions beneath fungal penetration attempts or in the entire epidermal cell during HR. The data provide evidence for different roles and sources of superoxide and H2O2 in the nonhost interaction of cereals with inappropriate formae speciales of B. graminis.

Ascomycota↗

PubMed2004

The white barley mutant albostrians shows a supersusceptible but symptomless interaction phenotype with the hemibiotrophic fungus Bipolaris sorokiniana.

Bipolaris sorokiniana (teleomorph: Cochliobolus sativus) is a cereal pathogen of increasing global concern, with most significance in Asiatic cropping systems. In order to gain insight into the mechanism of host resistance, we studied fungal development on the supersusceptible barley mutant albostrians and its parent cv. Haisa. A microscopic dissection of early fungal growth on Haisa and green albostrians leaves revealed a distinct epidermis-localized biotrophic and a mesophyll-based necrotrophic phase. White, green, and striped white-green albostrians leaves showed extreme differences in disease development. When comparing cellular defense responses, we found restriction of fungal spreading after successful infection of host mesophyll tissue to be the most important mechanism limiting outbreak of the disease. Colonization of susceptible green leaves, but not extreme colonization of supersusceptible white albostrians leaves, was associated with macroscopically visible lesion formation and mesophyll accumulation of hydrogen peroxide (H2O2), implying a symptomless growth of the pathogen in supersusceptible host tissue. In contrast, early epidermal papilla-based resistance was closely linked to H2O2 accumulation in all leaf types. In white leaves, ascorbate peroxidase (APX), glutathione-S-transferase (GST), and the cell death regulator Bax-inhibitor-1 (BI-1) showed a stronger constitutive or pathogen responsive activation, whereas glycolate oxidase (GLOX) and catalase (CAT2) expression was stronger in green leaves. We discuss supersusceptibility and symptomless growth on the basis of the histochemical and the gene expression data.

Alcohol Oxidoreductases↗

PubMed2004

The white barley mutant albostrians shows enhanced resistance to the biotroph Blumeria graminis f. sp. hordei.

We performed cytological and molecular analyses of the interaction between the biotrophic barley powdery mildew fungus Blumeria graminis f. sp. hordei and white and green leaves of the barley albostrians mutant. The leaves have the same nuclear genotype but differ from each other in respect to plastid differentiation. White leaves showed enhanced penetration resistance to B. graminis f. sp. hordei, associated with higher epidermal H2O2 accumulation beneath the appressorial germ tubes and protein cross-linking in papillae. Very low basal salicylic acid content was found in white leaves, which further confirmed that H2O2 accumulation and penetration resistance in barley are independent of salicylic acid. Expression analysis of stress and defense-related genes, including such being involved in reactive oxygen species production and cell death regulation, revealed stronger constitutive or pathogen-induced transcript accumulation in white leaves. We discuss the data on the basis of the finding that white albostrians leaves exhibit a supersusceptible interaction phenotype with the hemibiotrophic fungus Bipolaris sorokiniana.

Ascomycota↗

PubMed2004

The barley apoptosis suppressor homologue BAX inhibitor-1 compromises nonhost penetration resistance of barley to the inappropriate pathogen Blumeria graminis f. sp. tritici.

BAX inhibitor-1 (BI-1) proteins have been characterized as suppressors of programmed cell death in mammals and plants. The barley BI-1 is a suppressor of nonspecific background resistance and mlo-mediated penetration resistance to the biotrophic fungal pathogen Blumeria graminis f. sp. hordei when overexpressed in epidermal cells of barley. We report here that BI-1 expression is also slightly up-regulated during interaction with the inappropriate wheat pathogen Blumeria graminis f. sp. tritici. Significantly, overexpression of BI-1 in single epidermal cells of barley by microprojectile-mediated transformation rendered cells susceptible to penetration by inappropriate B. graminis f. sp. tritici. The degree of transgene-induced accessibility to B. graminis f. sp. tritici was thereby similar to the effect achieved by overexpression of the defense suppressor gene Mlo and could not be further enhanced by double expression of both BI-1 and Mlo. Confocal laser scanning microscopy was used to locate a functional green fluorescing GFP:BI-1 fusion protein in endomembranes and the nuclear envelope of barley epidermal cells. Together, enhanced expression of barley BI-1 suppresses penetration resistance to B. graminis f. sp. tritici, linking barley nonhost resistance with cell death regulation.

Apoptosis↗

PubMed2003

SNARE-protein-mediated disease resistance at the plant cell wall.

Failure of pathogenic fungi to breach the plant cell wall constitutes a major component of immunity of non-host plant species--species outside the pathogen host range--and accounts for a proportion of aborted infection attempts on 'susceptible' host plants (basal resistance). Neither form of penetration resistance is understood at the molecular level. We developed a screen for penetration (pen) mutants of Arabidopsis, which are disabled in non-host penetration resistance against barley powdery mildew, Blumeria graminis f. sp. hordei, and we isolated the PEN1 gene. We also isolated barley ROR2 (ref. 2), which is required for basal penetration resistance against B. g. hordei. The genes encode functionally homologous syntaxins, demonstrating a mechanistic link between non-host resistance and basal penetration resistance in monocotyledons and dicotyledons. We show that resistance in barley requires a SNAP-25 (synaptosome-associated protein, molecular mass 25 kDa) homologue capable of forming a binary SNAP receptor (SNARE) complex with ROR2. Genetic control of vesicle behaviour at penetration sites, and plasma membrane location of PEN1/ROR2, is consistent with a proposed involvement of SNARE-complex-mediated exocytosis and/or homotypic vesicle fusion events in resistance. Functions associated with SNARE-dependent penetration resistance are dispensable for immunity mediated by race-specific resistance (R) genes, highlighting fundamental differences between these two resistance forms.

Arabidopsis↗

PubMed2003

Overexpression of barley BAX inhibitor 1 induces breakdown of mlo-mediated penetration resistance to Blumeria graminis.

Cell death regulation is linked to pathogen defense in plants and animals. Execution of apoptosis as one type of programmed cell death in animals is irreversibly triggered by cytochrome c release from mitochondria via pores formed by BAX proteins. This type of programmed cell death can be prevented by expression of BAX inhibitor 1 (BI-1), a membrane protein that protects cells from the effects of BAX by an unknown mechanism. In barley, a homologue of the mammalian BI-1 is expressed in response to inoculation with the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). We found differential expression of BI-1 in response to Bgh in susceptible and resistant plants. Chemical induction of resistance to Bgh by soil drench treatment with 2,6-dichloroisonicotinic acid led to down-regulation of the expression level of BI-1. Importantly, single-cell transient overexpression of BI-1 in epidermal leaf tissue of susceptible barley cultivar Ingrid led to enhanced accessibility, resulting in a higher penetration efficiency of Bgh on BI-1-transformed cells. In Bgh-resistant mlo5 genotypes, which do not express the negative regulator of defense and cell death MLO, overexpression of BI-1 almost completely reconstituted susceptibility to fungal penetration. We suggest that BI-1 is a regulator of cellular defense in barley sufficient to substitute for MLO function in accessibility to fungal parasites.

Amino Acid Sequence↗