A role for the mevalonate pathway in early plant symbiotic signaling

Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca(2+) concentration (Ca(2+) spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca(2+) spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume-rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca(2+) spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca(2+) spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca(2+) spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca(2+) spiking in this heterologous system

Development of a GC/Quadrupole-Orbitrap Mass Spectrometer, Part II: New Approaches for Discovery Metabolomics

Identification of unknown peaks in gas chromatography/mass spectrometry (GC/MS)-based discovery metabolomics is challenging, and remains necessary to permit discovery of novel or unexpected metabolites that may elucidate disease processes and/or further our understanding of how genotypes relate to phenotypes. Here, we introduce two new technologies and an analytical workflow that can facilitate the identification of unknown peaks. First, we report on a GC/Quadrupole-Orbitrap mass spectrometer that provides high mass accuracy, high resolution, and high sensitivity analyte detection. Second, with an “intelligent” data-dependent algorithm, termed molecular-ion directed acquisition (MIDA), we maximize the information content generated from unsupervised tandem MS (MS/MS) and selected ion monitoring (SIM) by directing the MS to target the ions of greatest information content, that is, the most-intact ionic species. We combine these technologies with ¹³C- and ¹⁵N-metabolic labeling, multiple derivatization and ionization types, and heuristic filtering of candidate elemental compositions to achieve (1) MS/MS spectra of nearly all intact ion species for structural elucidation, (2) knowledge of carbon and nitrogen atom content for every ion in MS and MS/MS spectra, (3) relative quantification between alternatively labeled samples, and (4) unambiguous annotation of elemental composition

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant <i>Saccharomyces cerevisiae</i> Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

<div><p>The inability of the yeast <i>Saccharomyces cerevisiae</i> to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of <i>S. cerevisiae</i> to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting <i>S. cerevisiae</i> strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent <i>S. cerevisiae</i> strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in <i>GRE3</i>, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust <i>S. cerevisiae</i> strain with the ability to ferment xylose anaerobically from ACSH.</p></div

Phenotypic screening of wild and domesticated <i>S. cerevisiae</i> strains identifies NRRL YB-210 with tolerance to hydrolysates made from a variety of pretreated lignocellulose.

<p>In (<b>A</b>), 117 <i>S. cerevisiae</i> strains (including some in duplicate) were cultured in 96-well plates and monitored for changes cell density and growth rates calculated as described in Materials and Methods. All strains in each condition were then ranked from 1 (highest growth rate in yellow) to 117 (lowest growth rate, or no growth, in blue) and hierarchically clustered. Arrows indicate clustered rows for BY4741 (green), CEN.PK2 (black) in duplicate microtiter wells, and NRRL YB-210/GLBRCY0 (red). Representative growth data for the YB-210/GLBRCY0 strain in the indicated media from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107499#pone-0107499-g002" target="_blank">Fig. 2A</a> are plotted (<b>B–C</b>). CS, corn stover; SG, switchgrass; YP; Yeast Extract and Peptone supplementation, 6%; 6% glucan loading ACSH, 9%; 9% glucan loading ACSH, Dtx.; Detoxified.</p

Second stage anaerobic adaptation on xylose enabled rapid xylose fermentation by evolved GLBRCY128 isolate.

<p>Average fermentation kinetic profiles of the GLBRCY127 strain cultured in bioreactors containing YPDX media and sparged with nitrogen from biological duplicates are shown (<b>A</b>). Average concentrations with standard deviations of indicated compounds were quantified from media samples at times from initial inoculation. In (<b>B</b>), the percentage of xylose consumed and change in cell density per day is plotted for each transfer during the anaerobic adaptation of Y127 in YP media containing 0.1% glucose and 2% xylose. In the first two transfers (hatched bars), Tween-80 and ergosterol were added to the media. In (<b>C</b>), evolved isolate Y128 was cultured in biological duplicate under the same conditions as in (<b>A</b>), and samples measurements taken in an identical manner.</p

The GLBRCY127 strain developed by directed engineering with xylose isomerase coupled with batch evolution can rapidly consume xylose aerobically.

<p>Average sugar consumption and cell growth of unevolved GLBRCY22-3 strain engineered with <i>ScTAL1</i>, <i>CpxylA</i> and <i>SsXYL3</i> cultured in bioreactors containing YPDX media and sparged with air from biological duplicates is shown (<b>A</b>). Indicated components were quantified from media samples at times from initial inoculation. In (<b>B</b>), the average percentage of xylose consumed and change in cell density per day are plotted for each transfer during the adaption of the Y22-3 strain in YP media containing 0.1% glucose and 2% xylose. The pattern of lower % of xylose consumed and change in cell density per day during every third transfer is due to reaching saturated growth prior to transfer. Average extracellular xylose concentrations and cell density measurements from parental Y22-3 and evolved Y127 strains grown aerobically in culture tubes with YPX media from three independent biological replicates are plotted in (<b>C</b>). In (<b>D</b>), evolved isolate Y127 was cultured in the same conditions as in (<b>A</b>), and samples measurements taken in an identical manner.</p

Fermentation kinetic profiles for engineered and evolved <i>S. cerevisiae</i> strains.

<p>ND, Not Determined for aerobic conditions; ND*, Not Determined – no ethanol produced.</p>1<p>In g xylose consumed/L/h.</p>2<p>In g xylose consumed/g DCW/h.</p>3<p>Calculated from the maximum ethanol concentration produced divided by the consumed xylose concentration at that time.</p>4<p>Calculated from the ethanol concentration produced between two time points after glucose depletion.</p>5<p>Yield of g DCW/g glucose consumed calculated at or near the time of glucose depletion and prior to xylose consumption. No cell growth was observed during xylose consumption.</p>6<p>Yield of g glycerol/g glucose consumed calculated at or near the time of glucose depletion and prior to xylose consumption.</p><p>Fermentation kinetic profiles for engineered and evolved <i>S. cerevisiae</i> strains.</p

GLBRCY128 can anaerobically ferment xylose from ACSH.

<p>A diagram summarizing the engineering and evolution of the YB-210 strain into the evolved Y128 strain is provided in (<b>A</b>). Fermentation kinetic profiles of the Y127 (<b>B</b>) and Y128 (<b>C</b>) strains cultured in bioreactors containing ACSH and sparged with nitrogen from biological duplicates are shown. Average concentrations and standard deviations of indicated components were quantified from media samples at times from initial inoculation. Vertical colored bars indicate time points at which samples were taken for metabolomic analysis described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107499#pone-0107499-g007" target="_blank">Fig. 7A–D</a>.</p