The endoplasmic reticulum in plant immunity and cell death

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane, or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programed cell death (PCD) in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signaling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants

LIFEGUARD proteins support plant colonization by biotrophic powdery mildew fungi

Pathogenic microbes manipulate eukaryotic cells during invasion and target plant proteins to achieve host susceptibility. BAX INHIBITOR-1 (BI-1) is an endoplasmic reticulum-resident cell death suppressor in plants and animals and is required for full susceptibility of barley to the barley powdery mildew fungus Blumeria graminis f.sp. hordei. LIFEGUARD (LFG) proteins resemble BI-1 proteins in terms of predicted membrane topology and cell-death-inhibiting function in metazoans, but display clear sequence-specific distinctions. This work shows that barley (Hordeum vulgare L.) and Arabidopsis thaliana genomes harbour five LFG genes, HvLFGa–HvLFGe and AtLFG1–AtLFG5, whose functions are largely uncharacterized. As observed for HvBI-1, single-cell overexpression of HvLFGa supports penetration success of B. graminis f.sp. hordei into barley epidermal cells, while transient-induced gene silencing restricts it. In penetrated barley epidermal cells, a green fluorescent protein-tagged HvLFGa protein accumulates at the site of fungal entry, around fungal haustoria and in endosomal or vacuolar membranes. The data further suggest a role of LFG proteins in plant–powdery mildew interactions in both monocot and dicot plants, because stable overexpression or knockdown of AtLFG1 or AtLFG2 also support or delay development of the powdery mildew fungus Erysiphe cruciferarum on the respective Arabidopsis mutants. Together, this work has identified new modulators of plant–powdery mildew interactions, and the data further support functional similarities between BI-1 and LFG proteins beyond cell death regulation

Growth versus immunity : a redirection of the cell cycle?

Diseases caused by plant pathogens significantly reduce growth and yield in agricultural crop production. Raising immunity in crops is therefore a major aim in breeding programs. However, efforts to enhance immunity are challenged by the occurrence of growth inhibition triggered by immunity that can be as detrimental as diseases. In this review, we will propose molecular models to explain the inhibitory growth-immunity crosstalk. We will briefly discuss why the resource reallocation model might not represent the driving force for the observed growth-immunity trade-offs. We suggest a model in which immunity redirects and initiates hormone signalling activities that can impair plant growth by antagonising cell cycle regulation and meristem activities

Impact of spatial organization on a novel auxotrophic interaction among soil microbes

A key prerequisite to achieve a deeper understanding of microbial communities and to engineer synthetic ones is to identify the individual metabolic interactions among key species and how these interactions are affected by different environmental factors. Deciphering the physiological basis of species–species and species–environment interactions in spatially organized environments requires reductionist approaches using ecologically and functionally relevant species. To this end, we focus here on a defined system to study the metabolic interactions in a spatial context among the plant-beneficial endophytic fungus Serendipita indica, and the soil-dwelling model bacterium Bacillus subtilis. Focusing on the growth dynamics of S. indica under defined conditions, we identified an auxotrophy in this organism for thiamine, which is a key co-factor for essential reactions in the central carbon metabolism. We found that S. indica growth is restored in thiamine-free media, when co-cultured with B. subtilis. The success of this auxotrophic interaction, however, was dependent on the spatial and temporal organization of the system; the beneficial impact of B. subtilis was only visible when its inoculation was separated from that of S. indica either in time or space. These findings describe a key auxotrophic interaction in the soil among organisms that are shown to be important for plant ecosystem functioning, and point to the potential importance of spatial and temporal organization for the success of auxotrophic interactions. These points can be particularly important for engineering of minimal functional synthetic communities as plant seed treatments and for vertical farming under defined conditions

Polyclonal Aptamer Libraries from a FluRoot-SELEX for the Specific Labeling of the Apical and Elongation/Differentiation Zones of Arabidopsis thaliana Roots

In more than 30 years of aptamer research, it has become widely accepted that aptamers are fascinating binding molecules for a vast variety of applications. However, the majority of targets have been proteins, although special variants of the so-called SELEX process for the molecular evolution of specific aptamers have also been developed, allowing for the targeting of small molecules as well as larger structures such as cells and even cellular networks of human (tumor) tissues. Although the provocative thesis is widely accepted in the field, that is, in principle, any level of complexity for SELEX targets is possible, the number of studies on whole organs or at least parts of them is limited. To pioneer this thesis, and based on our FluCell-SELEX process, here, we have developed polyclonal aptamer libraries against apices and the elongation/differentiation zones of plant roots as examples of organs. We show that dedicated libraries can specifically label the respective parts of the root, allowing us to distinguish them in fluorescence microscopy. We consider this achievement to be an initial but important evidence for the robustness of this SELEX variant. These libraries may be valuable tools for plant research and a promising starting point for the isolation of more specific individual aptamers directed against root-specific epitopes

The formation of a camalexin-biosynthetic metabolon

Arabidopsis thaliana efficiently synthesizes the antifungal phytoalexin camalexin without apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting channeling of the biosynthetic pathway by formation of an enzyme complex. To identify such protein interactions, two independent untargeted co49 immunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B1 and CYP71A13 as baits were performed and the camalexin biosynthetic P450 enzymes were shown to co-purify. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, interaction of CYP71A13 and Arabidopsis P450 Reductase 1 (ATR1) was observed. An increased substrate affinity of CYP79B2 in presence of CYP71A13 was shown, indicating allosteric interaction. Camalexin biosynthesis involves glutathionylation of an intermediary indole-3-cyanohydrin, synthesized by CYP71A12 and especially CYP71A13. It was demonstrated by FRET-FLIM and co-IP, that the glutathione transferase GSTU4, which is co-expressed with tryptophan- and camalexin-specific enzymes, was physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knock-out and reduced in GSTU4 overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather has a role in a competing mechanism

Symbiont-host interactome mapping reveals effector-targeted modulation of hormone networks and activation of growth promotion

Plants have benefited from interactions with symbionts for coping with challenging environments since the colonisation of land. The mechanisms of symbiont-mediated beneficial effects and similarities and differences to pathogen strategies are mostly unknown. Here, we use 106 (effector-) proteins, secreted by the symbiont Serendipita indica (Si) to modulate host physiology, to map interactions with Arabidopsis thaliana host proteins. Using integrative network analysis, we show significant convergence on target-proteins shared with pathogens and exclusive targeting of Arabidopsis proteins in the phytohormone signalling network. Functional in planta screening and phenotyping of Si effectors and interacting proteins reveals previously unknown hormone functions of Arabidopsis proteins and direct beneficial activities mediated by effectors in Arabidopsis. Thus, symbionts and pathogens target a shared molecular microbe-host interface. At the same time Si effectors specifically target the plant hormone network and constitute a powerful resource for elucidating the signalling network function and boosting plant productivity

Linkage of murine (T,G)-A- -L-specific idiotypic determinants to the heavy chain constant region allotypic markers

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46735/1/251_2005_Article_BF01567784.pd

Molekulare Untersuchungen zum Mechanismus der Nichtwirtresistenz von Gerste (Hordeum vulgare L.) gegen den Weizenmehltaupilz (Blumeria graminis f.sp. tritici)

Every plant pathogen has only a limited range of host species on which it can cause disease. The remaining plants are nonhost plants to this pathogen and can resist the attacker due to a multitude of different mechanisms that collectively contribute to nonhost resistance. Due to its durable effectiveness, nonhost resistance has moved into the focus of scientific interest, since it promises to be of use for the generation of resistant crop plants. Considerable effort has been made to elucidate signal transduction processes of plant defense. In spite of a number of investigations, which examined the transcriptome of plants during the interaction with pathogens, particularly powdery mildew fungi, we are still far from understanding the nature of nonhost resistance especially in terms of its constancy. Hence, this study aimed at the advanced understanding of the mechanisms that underlie nonhost resistance and its counterpart, basic compatibility. For this purpose, the model system of barley (Hordeum vulare L.) interacting with appropriate or inappropriate formae speciales of the biotrophic fungal pathogen Blumeria graminis was used. Since colonization-success of the powdery mildew fungus depends upon the living host cell, it was also part of this work to investigate the role of a putative cell death inhibitor in cell death and defense regulation in barley. A macroarray based approach was followed to comparatively analyze the expression of 1,536 barley gene transcripts in the early host interaction with Blumeria graminis f.sp. hordei (Bgh) and the nonhost pathogen Blumeria graminis f.sp. tritici (Bgt), respectively. The cDNA fragments on the macroarray mainly derived from epidermal peels of plants pre-treated with the chemical resistance activating compound acibenzolar-S-methyl, and were therefore expected to be enriched with defense-related transcripts. 102 spots corresponding to 94 genes repeatedly gave B. graminis-responsive signals on the macroarray at 12 and/or 24 hours after inoculation. In independent expression analyses, the differential expression of 18 arbitrarily selected genes could be confirmed. The temporal expression profile of the majority of the genes was similar in the compatible and the incompatible interaction. The data support the view that common genetic and mechanistic elements of plant defense underlie background resistance in compatible interactions and nonhost resistance. BAX INHIBITOR-1 (BI-1) proteins are negative regulators of programmed cell death in mammals and plants. When overexpressed in epidermal cells of barley, BI-1 suppressed non-specific background resistance and mlo-mediated penetration resistance to the biotrophic fungal pathogen Bgh. It could be demonstrated that overexpression of BI-1 partially protected barley cells from cell death and breaks nonhost resistance of barley epidermal cells to the nonhost pathogen Bgt. The degree of transgene-induced accessibility was thereby similar to the effect achieved by overexpression of the defense suppressor gene MLO and could not be further enhanced by simultaneous expression of both BI-1 and MLO. Furthermore, results indicate that during defense suppression, BI-1 modulates defense-associated hydrogen peroxide accumulation underneath the site of attempted fungal penetration. In barley epidermal cells, a functional green fluorescing GFP-BI-1 fusion protein accumulated in endomembranes and the nuclear envelope and was found in the vicinity of the site of fungal attack and/or around intracellular fungal structures. Together, enhanced expression of barley BI-1 suppresses nonhost resistance to Bgt, linking barley nonhost penetration resistance with cell death regulation.Jedes Pflanzenpathogen hat nur ein limitiertes Spektrum an Wirtspflanzen, auf dem es eine Krankheit verursachen kann. Alle anderen Pflanzen sind so genannte Nichtwirtpflanzen für dieses Pathogen, d.h. sie können einer Attacke mit Hilfe unterschiedlicher Abwehrmechanismen widerstehen, die gemeinsam zur Nichtwirtresistenz beitragen. Aufgrund ihrer dauerhaften Wirksamkeit ist die Nichtwirtresistenz in den Fokus wissenschaftlicher Untersuchungen gerückt, weil die Ausnutzung der zugrunde liegenden Mechanismen möglicherweise für die Erzeugung resistenter Kulturpflanzen von Nutzen sein könnte. Die Aufklärung von Signaltransduktionsprozessen der pflanzlichen Abwehr ist seit langem Gegenstand intensiver Forschung. Trotz einer Vielzahl von Untersuchungen, die sich mit dem Transkriptom von Pflanzen während der Interaktion mit Phytopathogenen, insbesondere Mehltaupilzen, befasst haben, ist ein vollständiges Verständnis der Natur der Nichtwirtresistenz vor allem mit Blick auf ihre Konstanz noch in weiter Ferne. Die vorliegende Arbeit sollte zur Aufklärung der zellulären Prozesse und Mechanismen beitragen, die die Nichtwirtresistenz bestimmen. Für diesen Zweck wurde die Interaktion von Gerste (Hordeum vulgare L.) mit passenden und unpassenden formae speciales des Getreidemehltaupilzes (Blumeria graminis) untersucht. Das Pathosystem gilt als Modellsystem für die Interaktion einer Pflanze mit einem biotrophen Pathogen. Da der Erfolg des Mehltaupilzes an die lebende Wirtszelle gebunden ist, wurde außerdem die Rolle eines potenziellen Zelltodinhibitors in Zelltod- und Abwehrregulation in der Gerste überprüft. Für eine vergleichende Expressionsanalyse von 1.536 Genen in der frühen Wirtinteraktion der Gerste mit dem Echten Gerstenmehltaupilz (Blumeria graminis f.sp. hordei, Bgh) und der Nichtwirtinteraktion mit dem Echten Weizenmehltaupilz (Blumeria graminis f.sp. tritici, Bgt) wurde ein auf der Makroarraytechnik basierender Versuchsansatz verfolgt. Die auf die Arraymembranen aufgebrachten cDNA Fragmente stammten hauptsächlich aus Epidermisgewebe von Pflanzen, die mit dem Resistenzinduktor Acibenzolar-S-methyl vorbehandelt worden waren und daher mit abwehrrelevanten Gentranskripten angereichert sein sollten. 102 cDNA Fragmente von 94 Genen zeigten zu den Zeitpunkten 12 und/oder 24 Stunden nach Inokulation Responsivität auf Infektion mit B. graminis. In unhabhängigen Expressionsstudien konnte die differentielle Expression von 18 zufällig ausgewählten Genen bestätigt werden. Die Mehrheit der Gene zeigte sowohl in der kompatiblen als auch in der inkompatiblen Interaktion ein ähnliches Expressionsmuster. Die Ergebnisse unterstützen die Annahme, dass sowohl die Hintergrundresistenz in der kompatiblen Interaktion als auch die Nichtwirtresistenz von ähnlichen genetischen Elementen bestimmt werden. BAX INHIBITOR-1 (BI-1) Proteine sind als negative Regulatoren des Programmierten Zelltods in Tieren und Pflanzen bekannt. Die Überexpression des BI-1 Gens in Gerste beeinträchtigte sowohl die unspezifische Hintergrundresistenz als auch die mlo-vermittelte Penetrationsresistenz gegenüber Bgh. Neben der zellschützenden Wirkung von Gersten BI-1 konnte in dieser Arbeit gezeigt werden, dass durch die Überexpression des BI-1 Gens sogar die Nichtwirtresistenz von Gerstenepidermiszellen gegenüber Bgt gebrochen werden kann. Durch Einschleusen des Transgens wurde dabei ein ähnliches Suszeptibilitätsniveau erreicht, wie durch die Überexpression des Abwehrsuppressorgens MLO, wobei die gleichzeitige Überexpression der beiden Gene keinen weiteren Resistenz-supprimierenden Einfluss hatte. Weitere Ergebnisse zeigten, dass BI-1 im Zuge der Abwehrsuppression die lokale Akkumulation von Wasserstoffperoxid am Ort der pilzlichen Attacke zu beeinflussen scheint. In Epidermiszellen von Gerste akkumulierte ein funktionelles grün fluoreszierendes GFP-BI-1 Fusionsprotein in Endomembranen und in der Hülle des Zellkerns. Es war ebenfalls am Ort der pilzlichen Attacke und/oder an intrazellulären pilzlichen Strukturen zu finden. Die erhöhte Expression des Zelltodsuppressorgens BI-1 beeinträchtigte somit auch die Nichtwirtresistenz von Gerste gegenüber Bgt und verknüpft damit die Regulation von Nichtwirtinteraktion und Programmiertem Zelltod