Symbiotic Proteomics — State of the Art in Plant–Mycorrhizal Fungi Interactions

Mycorrhizae are symbiotic associations between soil fungi belonging to diverse taxa and the roots of about 90% of all terrestrial plant species. The mutualistic nature of these symbioses is based on the nutritional exchanges between the partners. However, the benefits to the plant partner are not limited to an improved mineral nutrition because they also include a general increase in stress tolerance and health. Because of these benefits, mycorrhizae are of great interest in sustainable agriculture and forestry. In the past few years, the development of high-throughput molecular tools, in addition to the advancements in microscopy techniques, has allowed us to gain a deeper insight on the molecular mechanisms underlying the establishment and functioning of these symbioses. In this chapter, we focus on the use of proteomic tools to better understand the molecular bases of cell communication and the regulation of developmental and metabolic pathways in mycorrhizal associations

Ramf: An Open-Source R Package for Statistical Analysis and Display of Quantitative Root Colonization by Arbuscular Mycorrhiza Fungi

Data analysis and graphical representation form an essential part of scientific research dissemination. The life-science community is moving towards a more transparent presentation of single data points or data distributions and away from mean values displayed as bar charts. To facilitate transparent data display to the mycorrhiza community, we present “Ramf” an open-source R package for statistical analysis and preparation of a variety of publication-ready plots, custom-made for analyzing and displaying quantitative root colonization by arbuscular mycorrhiza fungi or any kind of data to be displayed in the same format. Ramf replaces the scripting needed for data analysis and can be readily used by researchers not acquainted with R. In addition, the package is open to improvements by the community. Ramf is available at https://github.com/mchiapello/Ramf

Soil metaproteomics reveals an inter-kingdom stress response to the presence of black truffles

For some truffle species of the Tuber genus, the symbiotic phase is often associated with the presence of an area of scant vegetation, commonly known as the brûlé, around the host tree. Previous metagenomics studies have identified the microorganisms present inside and outside the brûlé of a Tuber melanosporum truffle-ground, but the molecular mechanisms that operate in this ecological niche remain to be clarified. To elucidate the metabolic pathways present in the brûlé, we conducted a metaproteomics analysis on the soil of a characterized truffle-ground and cross-referenced the resulting proteins with a database we constructed, incorporating the metagenomics data for the organisms previously identified in this soil. The soil inside the brûlé contained a larger number of proteins and, surprisingly, more proteins from plants, compared with the soil outside the brûlé. In addition, Fisher’s Exact Tests detected more biological processes inside the brûlé; these processes were related to responses to multiple types of stress. Thus, although the brûlé has a reduced diversity of plant and microbial species, the organisms in the brûlé show strong metabolic activity. Also, the combination of metagenomics and metaproteomics provides a powerful tool to reveal soil functioning

TPLATE recruitment reveals endocytic dynamics at sites of symbiotic interface assembly in arbuscular mycorrhizal interactions

Introduction: Arbuscular mycorrhizal (AM) symbiosis between soil fungi and the majority of plants is based on a mutualistic exchange of organic and inorganic nutrients. This takes place inside root cortical cells that harbor an arbuscule: a highly branched intracellular fungal hypha enveloped by an extension of the host cell membrane—the perifungal membrane—which outlines a specialized symbiotic interface compartment. The perifungal membrane develops around each intracellular hypha as the symbiotic fungus proceeds across the root tissues; its biogenesis is the result of an extensive exocytic process and shows a few similarities with cell plate insertion which occurs at the end of somatic cytokinesis. Materials and Methods: We here analyzed the subcellular localization of a GFP fusion with TPLATE, a subunit of the endocytic TPLATE complex (TPC), a central actor in plant clathrin-mediated endocytosis with a role in cell plate anchoring with the parental plasma membrane. Results: Our observations demonstrate that Daucus carota and Medicago truncatula root organ cultures expressing a 35S::AtTPLATE-GFP construct accumulate strong fluorescent green signal at sites of symbiotic interface construction, along recently formed perifungal membranes and at sites of cell-to-cell hyphal passage between adjacent cortical cells, where the perifungal membrane fuses with the plasmalemma. Discussion: Our results strongly suggest that TPC-mediated endocytic processes are active during perifungal membrane interface biogenesis—alongside exocytic transport. This novel conclusion, which might be correlated to the accumulation of late endosomes in the vicinity of the developing interface, hints at the involvement of TPC-dependent membrane remodeling during the intracellular accommodation of AM fungi

The virome from a collection of endomycorrhizal fungi reveals new viral taxa with unprecedented genome organization

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Soil Fungal Communities Investigated by Metabarcoding Within Simulated Forensic Burial Contexts

Decomposition of animal bodies in the burial environment plays a key role in the biochemistry of the soil, altering the balance of the local microbial populations present before the introduction of the carcass. Despite the growing number of studies on decomposition and soil bacterial populations, less is known on its effects on fungal communities. Shifts in the fungal populations at different post-mortem intervals (PMIs) could provide insights for PMI estimation and clarify the role that specific fungal taxa have at specific decomposition stages. In this study, we buried pig carcasses over a period of 1- to 6-months, and we sampled the soil in contact with each carcass at different PMIs. We performed metabarcoding analysis of the mycobiome targeting both the internal transcribed spacer (ITS) 1 and 2, to elucidate which one was more suitable for this purpose. Our results showed a decrease in the fungal taxonomic richness associated with increasing PMIs, and the alteration of the soil fungal signature even after 6 months post-burial, showing the inability of soil communities to restore their original composition within this timeframe. The results highlighted taxonomic trends associated with specific PMIs, such as the increase of the Mortierellomycota after 4- and 6-months and of Ascomycota particularly after 2 months, and the decrease of Basidiomycota from the first to the last time point. We have found a limited number of taxa specifically associated with the carrion and not present in the control soil, showing that the major contributors to the recorded changes are originated from the soil and were not introduced by the carrion. As this is the first study conducted on burial graves, it sets the baseline for additional studies to investigate the role of fungal communities on prolonged decomposition periods and to identify fungal biomarkers to improve the accuracy of PMI prediction for forensic applications

Powering the ABC multidrug exporter LmrA: How nucleotides embrace the ion-motive force.

LmrA is a bacterial ATP-binding cassette (ABC) multidrug exporter that uses metabolic energy to transport ions, cytotoxic drugs, and lipids. Voltage clamping in a Port-a-Patch was used to monitor electrical currents associated with the transport of monovalent cationic HEPES+ by single-LmrA transporters and ensembles of transporters. In these experiments, one proton and one chloride ion are effluxed together with each HEPES+ ion out of the inner compartment, whereas two sodium ions are transported into this compartment. Consequently, the sodium-motive force (interior negative and low) can drive this electrogenic ion exchange mechanism in cells under physiological conditions. The same mechanism is also relevant for the efflux of monovalent cationic ethidium, a typical multidrug transporter substrate. Studies in the presence of Mg-ATP (adenosine 5'-triphosphate) show that ion-coupled HEPES+ transport is associated with ATP-bound LmrA, whereas ion-coupled ethidium transport requires ATP binding and hydrolysis. HEPES+ is highly soluble in a water-based environment, whereas ethidium has a strong preference for residence in the water-repelling plasma membrane. We conclude that the mechanism of the ABC transporter LmrA is fundamentally related to that of an ion antiporter that uses extra steps (ATP binding and hydrolysis) to retrieve and transport membrane-soluble substrates from the phospholipid bilayer.This research was supported by the Biotechnology and Biological Sciences Research Council grants BB/R00224X/1, BB/I002383/1 and BB/K017713/1, and Medical Research Council grant G0401165 (to H.W.V.V.). We are also grateful for funding by the Human Frontier Science Program (grant RGP0034/2013), Strategic International Cooperative Program (Japan Science and Technology Agency, Japan) and Royal Society (UK) for collaborative research between H.W.V.V. and S.M. C.H.F.L. received a research studentship of Peterhouse, Cambridge. Y.S.K.K. received a Federal Training Award from the Ministry of Health in Malaysia. H.S. and S.R. were supported by the Cambridge Commonwealth, European and International Trust

Efficient 3‐Hydroxybutyrate Production by Quiescent Escherichia coli Microbial Cell Factories is Facilitated by Indole‐Induced Proteomic and Metabolomic Changes

The authors show that quiescent (Q‐Cell) Escherichia coli cultures can maintain metabolic activity in the absence of growth for up to 24 h, leading to four times greater specific productivity of a model metabolite, 3‐hydroxybutyrate (3HB), than a control. Q‐cells can be created by using the proton ionophore indole to halt cell division of an hns mutant strain. This uncouples metabolism from cell growth and allows for more efficient use of carbon feedstocks because less metabolic effort is diverted to surplus biomass production. However, the reason for the increased productivity of cells in the quiescent state was previously unknown. In this study, proteome expression patterns between wild‐type and Q‐cell cultures show that Q‐cells overexpress stress response proteins, which prime them to tolerate the metabolic imbalances incurred through indole addition. Metabolomic data reveal the accumulation of acetyl‐coenzyme A and phosphoenolpyruvate: excellent starting points for high‐value chemical production. We demonstrate the exploitation of these accumulated metabolites by engineering a simple pathway for 3HB production from acetyl‐coenzyme A. Quiescent cultures produced half the cell biomass of control cultures lacking indole, but were still able to produce 39.4 g L−1 of 3HB compared to 18.6 g L−1 in the control. Q‐cells therefore have great potential as a platform technology for the efficient production of a wide range of commodity and high value chemicals