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Publications

Dunker, F.; Trutzenberg, A.; Rothenpieler, J. S.; Kuhn, S.; Pröls, R.; Schreiber, T.; Tissier, A.; Kemen, A.; Kemen, E.; Hückelhoven, R.; Weiberg, A.; Oomycete small RNAs bind to the plant RNA-induced silencing complex for virulence eLife 9, e56096, (2020) DOI: 10.7554/eLife.56096

The exchange of small RNAs (sRNAs) between hosts and pathogens can lead to gene silencing in the recipient organism, a mechanism termed cross-kingdom RNAi (ck-RNAi). While fungal sRNAs promoting virulence are established, the significance of ck-RNAi in distinct plant pathogens is not clear. Here, we describe that sRNAs of the pathogen Hyaloperonospora arabidopsidis, which represents the kingdom of oomycetes and is phylogenetically distant from fungi, employ the host plant's Argonaute (AGO)/RNA-induced silencing complex for virulence. To demonstrate H. arabidopsidis sRNA (HpasRNA) functionality in ck-RNAi, we designed a novel CRISPR endoribonuclease Csy4/GUS reporter that enabled in situ visualization of HpasRNA-induced target suppression in Arabidopsis. The significant role of HpasRNAs together with AtAGO1 in virulence was revealed in plant atago1 mutants and by transgenic Arabidopsis expressing a short-tandem-target-mimic to block HpasRNAs, that both exhibited enhanced resistance. HpasRNA-targeted plant genes contributed to host immunity, as Arabidopsis gene knockout mutants displayed quantitative enhanced susceptibility.
Preprints

Dunker, F.; Trutzenberg, A.; Rothenpieler, J. S.; Kuhn, S.; Pröls, R.; Schreiber, T.; Tissier, A.; Hückelhoven, R.; Weiberg, A.; Oomycete small RNAs invade the plant RNA-induced silencing complex for virulence bioRxiv (2019) DOI: 10.1101/689190

Fungal small RNAs (sRNAs) hijack the plant RNA silencing pathway to manipulate host gene expression, named cross-kingdom RNA interference (ckRNAi). It is currently unknown how conserved and significant ckRNAi is for microbial virulence. Here, we found for the first time that sRNAs of a pathogen representing the oomycete kingdom invade the host plant’s Argonaute (AGO)/RNA-induced silencing complex. To demonstrate the functionality of the plant-invading oomycete Hyaloperonospora arabidopsidis sRNAs (HpasRNAs), we designed a novel CRISPR endoribonuclease Csy4/GUS repressor reporter to visualize in situ pathogen-induced target suppression in Arabidopsis thaliana host plant. By using 5’ RACE-PCR we demonstrated HpasRNAs-directed cleavage of plant mRNAs. The significant role of HpasRNAs together with AtAGO1 in virulence was demonstrated by plant atago1 mutants and by transgenic Arabidopsis expressing a target mimic to block HpasRNAs, that both exhibited enhanced resistance. Individual HpasRNA plant targets contributed to host immunity, as Arabidopsis gene knockout or HpasRNA-resistant gene versions exhibited quantitative enhanced or reduced susceptibility, respectively. Together with previous reports, we found that ckRNAi is conserved among oomycete and fungal pathogens.
Publications

Ried, M. K.; Banhara, A.; Hwu, F.-Y.; Binder, A.; Gust, A. A.; Höfle, C.; Hückelhoven, R.; Nürnberger, T.; Parniske, M.; A set of Arabidopsis genes involved in the accommodation of the downy mildew pathogen Hyaloperonospora arabidopsidis PLOS Pathog. 15, e1007747, (2019) DOI: 10.1371/journal.ppat.1007747

The intracellular accommodation structures formed by plant cells to host arbuscular mycorrhiza fungi and biotrophic hyphal pathogens are cytologically similar. Therefore we investigated whether these interactions build on an overlapping genetic framework. In legumes, the malectin-like domain leucine-rich repeat receptor kinase SYMRK, the cation channel POLLUX and members of the nuclear pore NUP107-160 subcomplex are essential for symbiotic signal transduction and arbuscular mycorrhiza development. We identified members of these three groups in Arabidopsis thaliana and explored their impact on the interaction with the oomycete downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We report that mutations in the corresponding genes reduced the reproductive success of Hpa as determined by sporangiophore and spore counts. We discovered that a developmental transition of haustorial shape occurred significantly earlier and at higher frequency in the mutants. Analysis of the multiplication of extracellular bacterial pathogens, Hpa-induced cell death or callose accumulation, as well as Hpa- or flg22-induced defence marker gene expression, did not reveal any traces of constitutive or exacerbated defence responses. These findings point towards an overlap between the plant genetic toolboxes involved in the interaction with biotrophic intracellular hyphal symbionts and pathogens in terms of the gene families involved.
Publications

Rajaraman, J.; Douchkov, D.; Lück, S.; Hensel, G.; Nowara, D.; Pogoda, M.; Rutten, T.; Meitzel, T.; Brassac, J.; Höfle, C.; Hückelhoven, R.; Klinkenberg, J.; Trujillo, M.; Bauer, E.; Schmutzer, T.; Himmelbach, A.; Mascher, M.; Lazzari, B.; Stein, N.; Kumlehn, J.; Schweizer, P.; Evolutionarily conserved partial gene duplication in the Triticeae tribe of grasses confers pathogen resistance Genome Biol. 19, 116, (2018) DOI: 10.1186/s13059-018-1472-7

BackgroundThe large and highly repetitive genomes of the cultivated species Hordeum vulgare (barley), Triticum aestivum (wheat), and Secale cereale (rye) belonging to the Triticeae tribe of grasses appear to be particularly rich in gene-like sequences including partial duplicates. Most of them have been classified as putative pseudogenes. In this study we employ transient and stable gene silencing- and over-expression systems in barley to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15).ResultsThe partial ARM1 gene is derived from a gene-duplication event in a common ancestor of the Triticeae and contributes to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis. In barley, allelic variants of HvARM1 but not of HvPUB15 are significantly associated with levels of powdery mildew infection. Both HvPUB15 and HvARM1 proteins interact in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. A genome-wide scan for partial gene duplicates reveals further events in barley resulting in stress-regulated, potentially neo-functionalized, genes.ConclusionThe results suggest neo-functionalization of the partial gene copy HvARM1 increases resistance against powdery mildew infection. It further links plastid function with susceptibility to biotrophic pathogen attack. These findings shed new light on a novel mechanism to employ partial duplication of protein-protein interaction domains to facilitate the expansion of immune signaling networks.
Preprints

Rajaraman, J.; Douchkov, D.; Lück, S.; Hensel, G.; Nowara, D.; Pogoda, M.; Rutten, T.; Meitzel, T.; Höfle, C.; Hückelhoven, R.; Klinkenberg, J.; Trujillo, M.; Bauer, E.; Schmutzer, T.; Himmelbach, A.; Mascher, M.; Lazzari, B.; Stein, N.; Kumlehn, J.; Schweizer, P.; The partial duplication of an E3-ligase gene in Triticeae species mediates resistance to powdery mildew fungi bioRxiv (2017) DOI: 10.1101/190728

In plant-pathogen interactions, components of the plant ubiquitination machinery are preferred targets of pathogen-encoded effectors suppressing defense responses or co-opting host cellular functions for accommodation. Here, we employed transient and stable gene silencing-and over-expression systems in Hordeum vulgare (barley) to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15). The partial ARM1 gene was derived from an ancient gene-duplication event in a common ancestor of the Triticeae tribe of grasses comprising the major crop species H. vulgare, Triticum aestivum and Secale cereale. The barley gene HvARM1 contributed to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis, and allelic variants were found to be associated with powdery mildew-disease severity. Both HvPUB15 and HvARM1 proteins interacted in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. The results suggest a neo-functionalization HvARM1 to increase resistance against powdery mildew and provide a link to plastid function in susceptibility to biotrophic pathogen attack.
Publications

Ranf, S.; Gisch, N.; Schäffer, M.; Illig, T.; Westphal, L.; Knirel, Y. A.; Sánchez-Carballo, P. M.; Zähringer, U.; Hückelhoven, R.; Lee, J.; Scheel, D.; A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana Nat. Immunol. 16, 426-433, (2015) DOI: 10.1038/ni.3124

The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor–like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species.
Publications

Dörmann, P.; Kim, H.; Ott, T.; Schulze-Lefert, P.; Trujillo, M.; Wewer, V.; Hückelhoven, R.; Cell-autonomous defense, re-organization and trafficking of membranes in plant-microbe interactions New Phytol. 204, 815-822, (2014) DOI: 10.1111/nph.12978

Plant cells dynamically change their architecture and molecular composition following encounters with beneficial or parasitic microbes, a process referred to as host cell reprogramming. Cell‐autonomous defense reactions are typically polarized to the plant cell periphery underneath microbial contact sites, including de novo cell wall biosynthesis. Alternatively, host cell reprogramming converges in the biogenesis of membrane‐enveloped compartments for accommodation of beneficial bacteria or invasive infection structures of filamentous microbes. Recent advances have revealed that, in response to microbial encounters, plasma membrane symmetry is broken, membrane tethering and SNARE complexes are recruited, lipid composition changes and plasma membrane‐to‐cytoskeleton signaling is activated, either for pre‐invasive defense or for microbial entry. We provide a critical appraisal on recent studies with a focus on how plant cells re‐structure membranes and the associated cytoskeleton in interactions with microbial pathogens, nitrogen‐fixing rhizobia and mycorrhiza fungi.
Publications

Dey, S.; Wenig, M.; Langen, G.; Sharma, S.; Kugler, K. G.; Knappe, C.; Hause, B.; Bichlmeier, M.; Babaeizad, V.; Imani, J.; Janzik, I.; Stempfl, T.; Hückelhoven, R.; Kogel, K.-H.; Mayer, K. F. X.; Vlot, A. C.; Bacteria-Triggered Systemic Immunity in Barley Is Associated with WRKY and ETHYLENE RESPONSIVE FACTORs But Not with Salicylic Acid Plant Physiol. 166, 2133-2151, (2014) DOI: 10.1104/pp.114.249276

Leaf-to-leaf systemic immune signaling known as systemic acquired resistance is poorly understood in monocotyledonous plants. Here, we characterize systemic immunity in barley (Hordeum vulgare) triggered after primary leaf infection with either Pseudomonas syringae pathovar japonica (Psj) or Xanthomonas translucens pathovar cerealis (Xtc). Both pathogens induced resistance in systemic, uninfected leaves against a subsequent challenge infection with Xtc. In contrast to systemic acquired resistance in Arabidopsis (Arabidopsis thaliana), systemic immunity in barley was not associated with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 or the local or systemic accumulation of salicylic acid. Instead, we documented a moderate local but not systemic induction of abscisic acid after infection of leaves with Psj. In contrast to salicylic acid or its functional analog benzothiadiazole, local applications of the jasmonic acid methyl ester or abscisic acid triggered systemic immunity to Xtc. RNA sequencing analysis of local and systemic transcript accumulation revealed unique gene expression changes in response to both Psj and Xtc and a clear separation of local from systemic responses. The systemic response appeared relatively modest, and quantitative reverse transcription-polymerase chain reaction associated systemic immunity with the local and systemic induction of two WRKY and two ETHYLENE RESPONSIVE FACTOR (ERF)-like transcription factors. Systemic immunity against Xtc was further associated with transcriptional changes after a secondary/systemic Xtc challenge infection; these changes were dependent on the primary treatment. Taken together, bacteria-induced systemic immunity in barley may be mediated in part by WRKY and ERF-like transcription factors, possibly facilitating transcriptional reprogramming to potentiate immunity.
Publications

Trujillo, M.; Kogel, K.-H.; Hückelhoven, R.; Superoxide and Hydrogen Peroxide Play Different Roles in the Nonhost Interaction of Barley and Wheat with Inappropriate formae speciales of Blumeria graminis Mol. Plant Microbe Interact. 17, 304-312, (2004) DOI: 10.1094/MPMI.2004.17.3.304

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.
Publications

Trujillo, M.; TROEGER, M.; NIKS, R. E.; Kogel, K.-H.; Hückelhoven, R.; Mechanistic and genetic overlap of barley host and non-host resistance to Blumeria graminis Mol. Plant Pathol. 5, 389-396, (2004) DOI: 10.1111/j.1364-3703.2004.00238.x

Non‐host resistance of barley to Blumeria graminis f.sp. tritici (Bgt ), an inappropriate forma specialis of the grass powdery mildew fungus, is associated with formation of cell wall appositions (papillae) at sites of attempted fungal penetration and a hypersensitive cell death reaction (HR) of single attacked cells. Penetration resistance and HR are also typical features of race‐non‐specific and race‐specific resistance of barley to the appropriate Blumeria graminis f.sp. hordei (Bgh ), raising the question of whether genotypic differences in the cellular response of barley to Bgt are detectable. First, we analysed fungal penetration frequencies and HR in different barley accessions known to show altered non‐host resistance. In genotypes with limited resistance to inappropriate cereal rust fungi, we concomitantly detected low penetration resistance to Bgt and significant differences of HR rates during attack from Bgt . Second, we tested barley mutants known to show altered host responses to Bgh . The rar1‐mutation that suppresses many types of race‐cultivar‐specific resistances did not influence the non‐host response of the Bgt‐isolate used in this study. However, mutants of Ror1 and Ror2 , two genes required for full race non‐specific penetration resistance of mlo‐barley to barley powdery mildew fungus, exhibited altered defence response to Bgt , including higher frequencies of fungal penetration. On these mutants, growth of the inappropriate fungus was arrested subsequent to penetration by HR. Together, the data show that barley defence response to the wheat powdery mildew fungus is determined by similar factors as race‐specific and race‐non‐specific resistance to appropriate Bgh.
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