Host-related metabolic cues affect colonization strategies of a root endophyte.

The mechanisms underpinning broad compatibility in root symbiosis are largely unexplored. The generalist root endophyte Piriformospora indica establishes long-lasting interactions with morphologically and biochemically different hosts, stimulating their growth, alleviating salt stress, and inducing local and systemic resistance to pathogens. Cytological studies and global investigations of fungal transcriptional responses to colonization of barley and Arabidopsis at different symbiotic stages identified host-dependent colonization strategies and host-specifically induced effector candidates. Here, we show that in Arabidopsis, P. indica establishes and maintains biotrophic nutrition within living epidermal cells, whereas in barley the symbiont undergoes a nutritional switch to saprotrophy that is associated with the production of secondary thinner hyphae in dead cortex cells. Consistent with a diversified trophic behavior and with the occurrence of nitrogen deficiency at the onset of saprotrophy in barley, fungal genes encoding hydrolytic enzymes and nutrient transporters were highly induced in this host but not in Arabidopsis. Silencing of the high-affinity ammonium transporter PiAMT1 gene, whose transcripts are accumulating during nitrogen starvation and in barley, resulted in enhanced colonization of this host, whereas it had no effect on the colonization of Arabidopsis. Increased levels of free amino acids and reduced enzymatic activity for the cell-death marker VPE (vacuolar-processing enzyme) in colonized barley roots coincided with an extended biotrophic lifestyle of P. indica upon silencing of PiAMT1. This suggests that PiAmt1 functions as a nitrogen sensor mediating the signal that triggers the in planta activation of the saprotrophic program. Thus, host-related metabolic cues affect the expression of P. indica's alternative lifestyles.

Evolution of mycoheterotrophy in Polygalaceae: The case of Epirixanthes.

PREMISE OF THE STUDY: The mycoheterotrophic lifestyle has enabled some plant lineages to obtain carbon from their mycorrhizal symbionts. The mycoheterotrophic genus Epirixanthes (Polygalaceae) consists of six species from tropical Asia. Although it is probably closely related to the chlorophyllous genus Salomonia and linked to arbuscular mycorrhizal fungi, lack of DNA sequence data has thus far prevented these hypotheses from being tested. Therefore, the evolutionary history of Epirixanthes remains largely unknown. METHODS: We reconstructed the phylogenetic relationships of Epirixanthes based on nuclear ITS and plastid matK data. Divergence times were inferred using a Bayesian relaxed clock approach, and we phylogenetically analyzed its mycorrhizal symbionts. We furthermore assigned these symbionts to operational taxonomic units, compared them with symbionts of other Polygalaceae, and measured their phylogenetic diversity. KEY RESULTS: We found that Epirixanthes is placed in tribe Polygaleae as sister to Salomonia. Epirixanthes has a Miocene-Oligocene stem age and grows exclusively in symbiosis with fungi of Glomeraceae. Salomonia and some Polygala species are linked to both Glomeraceae and Acaulosporaceae, resulting in higher phylogenetic diversity values. The majority of the symbionts of Epirixanthes are not found in Salomonia or Polygala, although a few shared fungal taxa are found. CONCLUSIONS: Epirixanthes forms a relatively young mycoheterotrophic lineage. The Oligocene-Miocene origin suggests its evolution was influenced by the environmental dynamics in Southeast Asia during this time. Although comparison of fungi from Epirixanthes with those from Salomonia and Polygala suggests some specialization, many other mycoheterotrophic plants are linked to a more narrow set of Glomeraceae.

Combining microtomy and confocal laser scanning microscopy for structural analyses of plant-fungus associations.

The serious problem of extended tissue thickness in the analysis of plant-fungus associations was overcome using a new method that combines physical and optical sectioning of the resin-embedded sample by microtomy and confocal microscopy. Improved tissue infiltration of the fungal-specific, high molecular weight fluorescent probe wheat germ agglutinin conjugated to Alexa Fluor® 633 resulted in high fungus-specific fluorescence even in deeper tissue sections. If autofluorescence was insufficient, additional counterstaining with Calcofluor White M2R or propidium iodide was applied in order to visualise the host plant tissues. Alternatively, the non-specific fluorochrome acid fuchsine was used for rapid staining of both, the plant and the fungal cells. The intricate spatial arrangements of the plant and fungal cells were preserved by immobilization in the hydrophilic resin Unicryl™. Microtomy was used to section the resin-embedded roots or leaves until the desired plane was reached. The data sets generated by confocal laser scanning microscopy of the remaining resin stubs allowed the precise spatial reconstruction of complex structures in the plant-fungus associations of interest. This approach was successfully tested on tissues from ectomycorrhiza (Betula pendula), arbuscular mycorrhiza (Galium aparine; Polygala paniculata, Polygala rupestris), ericoid mycorrhiza (Calluna vulgaris), orchid mycorrhiza (Limodorum abortivum, Serapias parviflora) and on one leaf-fungus association (Zymoseptoria tritici on Triticum aestivum). The method provides an efficient visualisation protocol applicable with a wide range of plant-fungus symbioses.

Hardware Development for Plant Cultivation Allowing In Situ Fluorescence Analysis of Calcium Fluxes in Plant Roots Under Microgravity and Ground-Control Conditions.

Maintaining an optimal leaf and stem orientation to yield a maximum photosynthetic output is accomplished by terrestrial plants using sophisticated mechanisms to balance their orientation relative to the Earth's gravity vector and the direction of sunlight. Knowledge of the signal transduction chains of both gravity and light perception and how they influence each other is essential for understanding plant development on Earth and plant cultivation in space environments. However, in situ analyses of cellular signal transduction processes in weightlessness, such as live cell imaging of signaling molecules using confocal fluorescence microscopy, require an adapted experimental setup that meets the special requirements of a microgravity environment. In addition, investigations under prolonged microgravity conditions require extensive resources, are rarely accessible, and do not allow for immediate sample preparation for the actual microscopic analysis. Therefore, supply concepts are needed that ensure both the viability of the contained plants over a longer period of time and an unhindered microscopic analysis in microgravity. Here, we present a customized supply unit specifically designed to study gravity-induced Ca2+ mobilization in roots of Arabidopsis thaliana. The unit can be employed for ground-based experiments, in parabolic flights, on sounding rockets, and probably also aboard the International Space Station.

The Ustilago maydis forkhead transcription factor Fox1 is involved in the regulation of genes required for the attenuation of plant defenses during pathogenic development.

Ustilago maydis is a plant-pathogenic fungus that establishes a biotrophic relationship with its host plant, Zea mays. The pathogenic stage of U. maydis is initiated by the fusion of two haploid cells, resulting in the formation of a dikaryotic hypha that invades the plant cell. The switch from saprophytic, yeast-like cells to the biotrophic hyphae requires the complex regulation of a multitude of biological processes to constitute the compatible host-fungus interaction. Transcriptional regulators involved in the establishment of the infectious dikaryon and penetration of the host tissue have been identified; however, regulators required during the post-penetration stages remained to be elucidated. In this study, we report the identification of a U. maydis forkhead transcription factor, Fox1, which is exclusively expressed during biotrophic development. Deletion of fox1 results in reduced virulence and impaired tumor development. The Deltafox1 hyphae induce the accumulation of H(2)O(2) in and around infected cells and a maize defense response phenotypically represented by the encasement of proliferating hyphae in a cellulose-containing matrix. The phenotype can be attributed to the fox1-dependent deregulation of several effector genes that are linked to pathogenic development and host defense suppression.

A gravitropic stimulus alters the distribution of EHB1, a negative effector of root gravitropism in Arabidopsis.

In Arabidopsis gravitropism is affected by two antagonistically interacting proteins, AGD12 (ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN) and EHB1 (ENHANCED BENDING 1). While AGD12 enhances gravitropic bending, EHB1 functions as a negative element. To further characterize their cellular function, we analyzed the location of AGD12-GFP and EHB1-GFP fusion proteins in the root apex by confocal laser-scanning microscopy after gravitropic stimulation. For this purpose, a novel method of microscopic visualization was developed with the objective and root axes aligned allowing an improved and comparable discernment of the fluorescence gradient across the columella. In vertical roots, both proteins were localized symmetrically and occurred preferentially in the outer layers of the columella. After reorienting roots horizontally, EHB1-GFP accumulated in the upper cell layers of the columella, that is, opposite to the gravity vector. The gravity-induced EHB1-GFP asymmetry disappeared after reorienting the roots back into the vertical position. No such asymmetry occurred with AGD12-GFP. Our findings reveal that after a gravitropic stimulus the cellular ratio between EHB1 and AGD12 is affected differently in the upper and lower part of the root. Its impact as a significant signaling event that ultimately affects the redirection of the lateral auxin flux toward the lower site of the root is discussed.

EHB1 and AGD12, two calcium-dependent proteins affect gravitropism antagonistically in Arabidopsis thaliana.

The ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN (AGD) 12, a member of the ARF-GAP protein family, affects gravitropism in Arabidopsis thaliana. A loss-of-function mutant lacking AGD12 displayed diminished gravitropism in roots and hypocotyls indicating that both organs are affected by this regulator. AGD12 is structurally related to ENHANCED BENDING (EHB) 1, previously described as a negative effector of gravitropism. In contrast to agd12 mutants, ehb1 loss-of function seedlings displayed enhanced gravitropic bending. While EHB1 and AGD12 both possess a C-terminal C2/CaLB-domain, EHB1 lacks the N-terminal ARF-GAP domain present in AGD12. Subcellular localization analysis using Brefeldin A indicated that both proteins are elements of the trans Golgi network. Physiological analyses provided evidence that gravitropic signaling might operate via an antagonistic interaction of ARF-GAP (AGD12) and EHB1 in their Ca2+-activated states.