Frank Waller

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.

Systemic Resistance in Arabidopsis Conferred by the Mycorrhizal Fungus Piriformospora indica Requires Jasmonic Acid Signaling and the Cytoplasmic Function of NPR1

We analyzed the requirement of specific defense pathways for powdery mildew (Golovinomyces orontii) resistance induced by the basidiomycete Piriformospora indica in Arabidopsis. Piriformospora indica root colonization reduced G. orontii conidia in wild-type (Col-0), npr1-3 (nonexpressor of PR genes 1-3) and NahG plants, but not in the npr1-1 null mutant. Therefore, cytoplasmic but not nuclear localization of NPR1 is required for P. indica-induced resistance. Two jasmonate signaling mutants were non-responsive to P. indica, and jasmonic acid-responsive vegetative storage protein expression was primed and thus elevated in response to powdery mildew, suggesting that P. indica confers resistance reminiscent of induced systemic resistance (ISR).

Manipulation of plant innate immunity and gibberellin as factor of compatibility in the mutualistic association of barley roots with Piriformospora indica

Fungi of the order Sebacinales (Basidiomycota) are involved in a wide spectrum of mutualistic symbioses with various plants, thereby exhibiting unique potential for biocontrol strategies. Piriformospora indica, a model organism of this fungal order, is able to increase the biomass and grain yield of crop plants, and induces local and systemic resistance to fungal diseases and tolerance to abiotic stress. To elucidate the molecular basis for root colonization, we characterized the interaction of P. indica with barley roots by combining global gene expression profiling, metabolic profiling, and genetic studies. At the metabolic level, we show that fungal colonization reduces the availability of free sugars and amino acids to the root tip. At the transcriptional level, consecutive interaction stages covering pre-penetration-associated events and progressing through to root colonization showed differential regulation of signal perception and transduction components, secondary metabolism, and genes associated with membrane transport. Moreover, we observed stage-specific up-regulation of genes involved in phytohormone metabolism, mainly encompassing gibberellin, auxin and abscisic acid, but salicylic acid-associated gene expression was suppressed. The changes in hormone homoeostasis were accompanied with a general suppression of the plant innate immune system. Further genetic studies showed reduced fungal colonization in mutants that are impaired in gibberellin synthesis as well as perception, and implicate gibberellin as a modulator of the roots basal defence. Our data further reveal the complexity of compatibility mechanisms in host-microbe interactions, and identify gibberellin signaling as potential target for successful fungi.

Lipid Profiling of the Arabidopsis Hypersensitive Response Reveals Specific Lipid Peroxidation and Fragmentation Processes: Biogenesis of Pimelic and Azelaic Acid

Lipid peroxidation (LPO) is induced by a variety of abiotic and biotic stresses. Although LPO is involved in diverse signaling processes, little is known about the oxidation mechanisms and major lipid targets. A systematic lipidomics analysis of LPO in the interaction of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae revealed that LPO is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species and precedes programmed cell death. Singlet oxygen was identified as the major cause of lipid oxidation under basal conditions, while a 13-lipoxygenase (LOX2) and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection. Analysis of lox2 mutants revealed that LOX2 is essential for enzymatic membrane peroxidation but not for the pathogen-induced free jasmonate production. Despite massive oxidative modification of plastid lipids, levels of nonoxidized lipids dramatically increased after infection. Pathogen infection also induced an accumulation of fragmented lipids. Analysis of mutants defective in 9-lipoxygenases and LOX2 showed that galactolipid fragmentation is independent of LOXs. We provide strong in vivo evidence for a free radical-catalyzed galactolipid fragmentation mechanism responsible for the formation of the essential biotin precursor pimelic acid as well as of azelaic acid, which was previously postulated to prime the immune response of Arabidopsis. Our results suggest that azelaic acid is a general marker for LPO rather than a general immune signal. The proposed fragmentation mechanism rationalizes the pathogen-induced radical amplification and formation of electrophile signals such as phytoprostanes, malondialdehyde, and hexenal in plastids.

Root colonization by Piriformospora indica enhances grain yield in barley under diverse nutrient regimes by accelerating plant development

The basidiomycete fungus Piriformospora indica colonizes roots of a broad range of mono- and dicotyledonous plants. It confers enhanced growth, improves resistance against biotic and tolerance to abiotic stress, and enhances grain yield in barley. To analyze mechanisms underlying P. indica-induced improved grain yield in a crop plant, the influence of different soil nutrient levels and enhanced biotic stress were tested under outdoor conditions. Higher grain yield was induced by the fungus independent of different phosphate and nitrogen fertilization levels. In plants challenged with the root rot-causing fungus Fusarium graminearum, P. indica was able to induce a similar magnitude of yield increase as in unchallenged plants. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae, total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. On the other hand, barley plants colonised with the endophyte developed faster, and were characterized by a higher photosynthetic activity at low light intensities. Together with the increased root formation early in development these factors contribute to faster development of ears as well as the production of more tillers per plant. The results indicate that the positive effect of P. indica on grain yield is due to accelerated growth of barley plants early in development, while improved phosphate supply—a central mechanism of host plant fortification by arbuscular mycorrhizal fungi—was not observed in the P. indica-barley symbiosis.

Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap

Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 μl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies.

A role for actin-driven secretion in auxin-induced growth

Summary. In epidermal cells of Zea mays coleoptiles, actin microfilaments are organized in fine strands during cell elongation, but are bundled in response to signals that inhibit growth. This bundling response is accompanied by an increased membrane association of extracted actin. Brefeldin A, an inhibitor of vesicle secretion, increases the membrane association of actin, causes a bundling of cortical actin microfilaments, and reduces the sensitivity of cell elongation to auxin. A model is proposed where auxin controls the dynamics of an actin subpopulation that guides vesicles loaded with components of the auxin-signaling machinery towards the cell poles.

Barley leaf transcriptome and metabolite analysis reveals new aspects of compatibility and Piriformospora indica-mediated systemic induced resistance to powdery mildew.

Colonization of barley roots with the basidiomycete fungus Piriformospora indica (Sebacinales) induces systemic resistance against the biotrophic leaf pathogen Blumeria graminis f. sp. hordei (B. graminis). To identify genes involved in this mycorrhiza-induced systemic resistance, we compared the leaf transcriptome of P. indica-colonized and noncolonized barley plants 12, 24, and 96 h after challenge with a virulent race of B. graminis. The leaf pathogen induced specific gene sets (e.g., LRR receptor kinases and WRKY transcription factors) at 12 h postinoculation (hpi) (prepenetration phase) and vesicle-localized gene products 24 hpi (haustorium establishment). Metabolic analysis revealed a progressing shift of steady state contents of the intermediates glucose-1-phosphate, uridinediphosphate-glucose, and phosphoenolpyruvate 24 and 96 hpi, indicating that B. graminis shifts central carbohydrate metabolism in favor of sucrose biosynthesis. Both B. graminis and P. indica increased glutamine and alanine contents, whereas substrates for starch and nitrogen assimilation (adenosinediphosphate- glucose and oxoglutarate) decreased. In plants that were more B. graminis resistant due to P. indica root colonization, 22 transcripts, including those of pathogenesis-related genes and genes encoding heat-shock proteins, were differentially expressed ?twofold in leaves after B. graminis inoculation compared with non-mycorrhized plants. Detailed expression analysis revealed a faster induction after B. graminis inoculation between 8 and 16 hpi, suggesting that priming of these genes is an important mechanism of P. indica-induced systemic disease resistance.

Activation of a Novel Transcription Factor through Phosphorylation by WIPK, a Wound-Induced Mitogen-Activated Protein Kinase in Tobacco Plants

Wound-induced protein kinase (WIPK) is a tobacco (Nicotiana tabacum) mitogen-activated protein kinase known to play an essential role in defense against wounding and pathogens, although its downstream targets have yet to be clarified. This study identified a gene encoding a protein of 648 amino acids, which directly interacts with WIPK, designated as N. tabacum WIPK-interacting factor (NtWIF). The N-terminal region with approximately 250 amino acids showed a high similarity to the plant-specific DNA binding domain, B3, but no other similarity with known proteins. The C terminus of approximately 200 amino acids appeared to be essential for the interaction with WIPK, and a Luciferase-reporter gene assay using Bright Yellow 2 cells indicated the full-length protein to possess trans-activation activity, located to the middle region of approximately 200 amino acids. In vitro phosphorylation assays indicated that WIPK efficiently phosphorylates the full-length protein and the N terminus but not the C terminus. When full-length NtWIF was coexpressed with WIPK in Bright Yellow 2 cells, the Luciferase transcriptional activity increased up to 5-fold that of NtWIF alone, whereas no effect was observed with a kinase-deficient WIPK mutant. Transcripts of NtWIF began to simultaneously accumulate with those of WIPK 30 min after wounding and 1 h after the onset of hypersensitive response upon tobacco mosaic virus infection. These results suggest that NtWIF is a transcription factor that is directly phosphorylated by WIPK, thereby being activated for transcription of target gene(s) involved in wound and pathogen responses.

Response of actin microfilaments during phytochrome-controlled growth of maize seedlings

SummaryIn seedlings of maize (Zea mays L. cv. Percival), growth is controlled by the plant photoreceptor phytochrome. Whereas coleoptile growth is promoted by continuous far-red light, a dramatic block of mesocotyl elongation is observed. The response of the coleoptile is based entirely upon light-induced stimulation of cell elongation, whereas the response of the mesocotyl involves light-induced inhibition of cell elongation. The light response of actin microfilaments was followed over time in the epidermis by staining with fluorescence-labelled phalloidin. In contrast to the underlying tissue, epidermal cells are characterized by dense longitudinal bundles of microfilaments. These bundles become loosened during phases of rapid elongation (between 2–3 days in irradiated coleoptiles, between 5–6 days in dark-grown coleoptiles). The condensed bundles re-form when growth gradually ceases. The response of actin to light is fast. If etiolated mesocotyls are transferred to far-red light, condensation of microfilaments can be clearly seen 1 h after the onset of stimulation together with an almost complete block of mesocotyl elongation. The observations are discussed in relation to a possible role of actin microfilaments in the signal-dependent control of cell elongation.