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Browsing by Author "Pilot, Guillaume"

Now showing 1 - 20 of 23
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    Amino Acids Are an Ineffective Fertilizer for Dunaliella spp. Growth
    Murphree, Colin A.; Dums, Jacob T.; Jain, Siddharth K.; Zhao, Chengsong; Young, Danielle Y.; Khoshnoodi, Nicole; Tikunov, Andrey; Macdonald, Jeffrey; Pilot, Guillaume; Sederoff, Heike (Frontiers, 2017-05-26)
    Autotrophic microalgae are a promising bioproducts platform. However, the fundamental requirements these organisms have for nitrogen fertilizer severely limit the impact and scale of their cultivation. As an alternative to inorganic fertilizers, we investigated the possibility of using amino acids from deconstructed biomass as a nitrogen source in the genus Dunaliella. We found that only four amino acids (glutamine, histidine, cysteine, and tryptophan) rescue Dunaliella spp. growth in nitrogen depleted media, and that supplementation of these amino acids altered the metabolic profile of Dunaliella cells. Our investigations revealed that histidine is transported across the cell membrane, and that glutamine and cysteine are not transported. Rather, glutamine, cysteine, and tryptophan are degraded in solution by a set of oxidative chemical reactions, releasing ammonium that in turn supports growth. Utilization of biomass-derived amino acids is therefore not a suitable option unless additional amino acid nitrogen uptake is enabled through genetic modifications of these algae.
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    Arabidopsis UMAMIT24 and 25 are amino acid exporters involved in seed loading
    Besnard, Julien; Zhao, Chengsong; Avice, Jean-Christophe; Vitha, Stanislav; Hyodo, Ayumi; Pilot, Guillaume; Okumoto, Sakiko (Oxford University Press, 2018-10-12)
    Phloem-derived amino acids are the major source of nitrogen supplied to developing seeds. Amino acid transfer from the maternal to the filial tissue requires at least one cellular export step from the maternal tissue prior to the import into the symplasmically isolated embryo. Some members of UMAMIT (usually multiple acids move in an out transporter) family (UMAMIT11, 14, 18, 28, and 29) have previously been implicated in this process. Here we show that additional members of the UMAMIT family, UMAMIT24 and UMAMIT25, also function in amino acid transfer in developing seeds. Using a recently published yeast-based assay allowing detection of amino acid secretion, we showed that UMAMIT24 and UMAMIT25 promote export of a broad range of amino acids in yeast. In plants, UMAMIT24 and UMAMIT25 are expressed in distinct tissues within developing seeds; UMAMIT24 is mainly expressed in the chalazal seed coat and localized on the tonoplast, whereas the plasma membrane-localized UMAMIT25 is expressed in endosperm cells. Seed amino acid contents of umamit24 and umamit25 knockout lines were both decreased during embryogenesis compared with the wild type, but recovered in the mature seeds without any deleterious effect on yield. The results suggest that UMAMIT24 and 25 play different roles in amino acid translocation from the maternal to filial tissue; UMAMIT24 could have a role in temporary storage of amino acids in the chalaza, while UMAMIT25 would mediate amino acid export from the endosperm, the last step before amino acids are taken up by the developing embryo.
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    Characterization of signaling pathways underlying key growth and development processes in Populus trichocarpa
    Rigoulot, Stephen Bradley (Virginia Tech, 2018-09-05)
    The project goals for this dissertation were to manipulate Populus trichocarpa source-sink relationships to optimize this woody crop species for specific agricultural traits such as increased growth rate, stress tolerance and/or improvements in overall biomass accumulation. We targeted specific tissues such as xylem, where alterations in the relationship of source and sink tissues can lead to the control of xylem cell deposition or of various wood properties. This led to the characterization of 165 protein-protein interactions and 20 protein-DNA interaction which constitute numerous woody tissue related subnetworks. One such network, centered on the DIVARACATA and RADIALIS INTERACTING FACTOR (PtrDRIF), identified PtrWOX13c as an interacting protein. Characterization of PtrWOX13c shows that it displays the ability to control promoters related to lignin biosynthesis genes and overexpression phenotypes show alterations in axillary branch activity. Genes which control the differentiation and specialization of cells such as members of the WOX family are also highly responsive to abiotic stress which can force major changes in plant metabolism and nutrient mobilization. ABA, a prominent plant phytohormone with known roles in the adaptation to stress has shown novel connections in the regulation of growth promoting complexes such as TOR through antagonistic regulatory actions of the SnRK2 protein kinase in Arabidopsis. Characterization of the core ABA signaling in P. trichocarpa has identified a regulatory clade A protein phosphatase which interacts with numerous PtrSnRK2 proteins and when overexpressed in hybrid poplar results in increased height and node production potentially by indirect control of growth promoting complexes like TOR through SnRK2 inhibition. This work has also demonstrated that in addition to the involvement of phytohormones in the regulation of plant development, sugar phosphates such as T6P can exert significant control of plant architecture. Together, these studies comprise the discovery and subsequent characterization of novel wood associated networks, hormone pathways and sugar signaling in the manipulation of P. trichocarpa source-sink relationships for the promotion of biomass accumulation.
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    Characterization of the amino acid transporter AAP1 in Arabidopsis thaliana
    Boyd, Shelton Roosevelt (Virginia Tech, 2018-01-22)
    Amino acids are essential molecules in plant metabolism. Amino acids carry reduced nitrogen while serving as precursors for protein synthesis and secondary metabolites. Translocation of amino acids in the cell is mediated by amino acid transporters. While about 100 transporters have been identified, only a dozen have been fully characterized. The regulation of amino acid transporters is not fully understood and stands as the basis of this study. Previous toxicity-based screenings of Arabidopsis thaliana mutants led to the isolation of a loss-of-function line and the phenylalanine insensitive growth (pig1) mutant capable of growth on toxic concentrations of phenylalanine (1). The pig1-1 mutants also displayed a deregulated metabolism (1). We followed this work with a similar forward genetic screening of Arabidopsis thaliana that led to the identification of 18 mutants capable of growth in the presence of amino acids at toxic concentrations. From this screen, seven mutations were confirmed to affect the amino acid transporter AAP1. Here I demonstrate that, when expressed in yeast deficient for endogenous amino acid transporters, three variant aap1 proteins restored growth similar to yeast complemented by wild type AAP1. Transport of radiolabeled Pro was abolished by variant aap1 proteins while deletion of an intracellular loop spanning the 8th and 9th transmembrane domains reduced Pro transport in yeast. Site directed mutagenesis of this loop conferred a variant aap1 protein which augmented Pro transport in yeast. Amino acid transport in loss-of-function aap1 plants display decreased uptake and increased efflux. In addition, aap1 mutant plants accumulated between 2 and 8 times more free amino acids in the leaves than the wild type. These observations are not fully compatible with the accepted role of AAP1 in transport by the root. The present work describes how the amino acid transporter AAP1 could play a role in regulating amino acid metabolism. We hypothesize that the amino acid transporter AAP1 functions as a senor that is involved in amino acid homeostasis in addition to its established role as a transporter. Is true, this would make AAP1 the first identified amino acid sensor in plants. Knowledge of the mechanism of amino acid sensing would enable us to engineer crops for improved nutrition in a more efficient way than affecting metabolic enzymes.
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    Characterization of the Arabidopsis glutamine dumper1 mutant reveals connections between amino acid homeostasis and plant stress responses
    Yu, Shi (Virginia Tech, 2015-04-15)
    Amino acids constitute the major organic form of transported nitrogen in plants, elements for protein synthesis, and precursors of many plant secondary metabolites, such as lignin, hormones, and flavonoids. Furthermore, amino acid metabolism lies at the crossroad of carbon and nitrogen metabolism. The Arabidopsis glutamine dumper1 (gdu1) mutant secretes glutamine from hydathodes, a phenotype caused by the overexpression of Glutamine Dumper1 (GDU1). GDU1 is a small transmembrane protein presents only in higher plants. The gdu1-1D mutant shows a pleiotropic phenotype: perturbed amino acid metabolism, tolerance to exogenous toxic concentrations of amino acids, elevated amino acid export, and activated stress/defense responses, lesions, and smaller rosettes. The biochemical function of GDU1 remains elusive. To better elucidate the biological processes leading to the complex Gdu1D phenotype, two approaches were conducted: (1) An ethyl methanesulfonate suppressor screening of the Gdu1D phenotype, which led to the isolation of intragenic mutations in GDU1 and mutations in the ubiquitin ligase LOG2 (Loss Of Gdu1D 2). Study of the intragenic mutations in GDU1 helped to characterize its structure-function relationships. Characterization of LOG2 showed that LOG2 interacts with GDU1 and is necessary for the Gdu1D phenotype. (2) The responses of the plant to the dexamethasone-induced expression of GDU1 were studied over time. This experiment identified major signaling pathways contributing to different components of the Gdu1D phenotype and the early events triggered by the perturbation of amino acid homeostasis. Our results showed that GDU1 overexpression first increases amino acid export, which is followed by amino acid imbalance and stress responses. This study sheds light on how amino acid imbalance interacts with various plant signaling pathways and stress responses, and suggests that LOG2 is involved in this process.
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    Characterizing the role in amino acid sensing and signaling of Amino Acid Permease 1 in Arabidopsis
    Shelley, Brett A. (Virginia Tech, 2021-07-28)
    Amino acids are necessary for protein synthesis and specialized metabolism in plants. Yet very little is known about how plants sense and regulate when and where to allocate amino acids to meet the demand for nitrogen in growing tissues. In particular, while characterized in yeast and mammals, no amino acid sensor has been identified in plants. Amino Acid Permease 1 (AAP1) has been previously characterized and was shown to mediate amino acid uptake from the soil. aap1 knockout plants and several EMS mutants affected in AAP1 sequence display enhanced tolerance to toxic concentrations of amino acids. Yet, two of the corresponding variant proteins appear to be functional transporters, effectively dissociating amino acid transport and phenotype. To understand this apparent discrepancy, I precisely studied AAP1 localization of expression at the plant and cellular level, and in specific tissue types of the root where AAP1 function is required for the tolerance phenotype and the amino acid uptake activity. I showed that AAP1 protein is present in the endoplasmic reticulum of the cortex in wild type plants Yet, its ectopic expression in root tip and phloem increased amino acid uptake, while expression in cortex could not. This and other of my results do not support the current model of AAP1 functioning in amino acid uptake by the root. I propose that the main effect of mutations in AAP1 is a disturbance in amino acid metabolism, possibly triggered by altered amino acid sensing. In this new model, AAP1 would be necessary for sensing amino acid status of cortex cells, possibly in the endoplasmic reticulum, and adjust amino acid metabolic activity and uptake to current availability. In effect, disruption of the sensing function, either by complete loss of AAP1 function (knockout) or by uncoupling the transport and sensing function (EMS mutants), would lead to the various characteristics of the phenotype of the aap1 mutants I observed. My main hypothesis is that AAP1 is a transporter endowed with sensing function, i.e., an amino acid transceptor.
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    Comparative Functional Genomics Characterization of Low Phytic Acid Soybeans and Virus Resistant Soybeans
    DeMers, Lindsay Carlisle (Virginia Tech, 2020-06-02)
    The field of functional genomics aims to understand the complex relationship between genotype and phenotype by integrating genome-wide approaches, such as transcriptomics, proteomics, and metabolomics. Large-scale "-omics" research has been made widely possible by the advent of high-throughput techniques, such as next-generation sequencing and mass-spectrometry. The vast data generated from such studies provide a wealth of information on the biological dynamics underlying phenotypes. Though functional genomics approaches are used extensively in human disease research, their use also spans organisms as miniscule as mycoplasmas to as great as sperm whales. In particular, functional genomics is instrumental in agricultural advancements for the improvement of productivity and sustainability in crop and livestock production. Improvement in soybean production is especially imperative, as soybeans are a primary source of oil and protein for human and livestock consumption, respectively. The research presented here employs functional genomics approaches – transcriptomics and metabolomics – to discern the transcriptional regulation and metabolic events underlying two economically important agronomic traits in soybean: seed phytic acid content and Soybean mosaic virus resistance. At normal levels, seed phytic acid content inhibits mineral absorption in humans and livestock, acting as an antinutrient and contributing to phosphorus pollution; however, the development of low phytic acid soybeans has helped mitigate these issues, as their seeds increase nutrient bioavailability and reduce environmental impact. Despite these desirable qualities, low phytic acid soybeans exhibit poor seed performance, which negatively affects germination rates and yield and has prevented their large-scale commercial production. Thus, part of the focus of this research was investigating the effects of mutations conferring the low phytic acid phenotype on seed germination. Comparative studies between low and normal phytic acid soybean seeds were carried out and revealed distinct differences in metabolite profiles and in the transcriptional regulation of biological pathways that may be vital for successful seed germination. The final part of this research concerns Rsv3-mediated extreme resistance, a unique mode of resistance that is effective against the most virulent strains of Soybean mosaic virus. The molecular mechanisms governing this type of resistance are poorly characterized. Therefore, the research presented here attempts to elucidate the regulatory elements responsible for the induction of the Rsv3-mediated extreme resistance response. Utilizing a comparative transcriptomic time series approach on Soybean mosaic virus-inoculated Rsv3 (resistant) and rsv3 (susceptible) soybean lines, this final study provides gene candidates putatively functioning in the regulation of biological pathways demonstrated to be crucial for Rsv3-mediated resistance.
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    Degenerate oligonucleotide primed amplification of genomic DNA for combinatorial screening libraries and strain enrichment
    Freedman, Benjamin Gordon (Virginia Tech, 2014-12-22)
    Combinatorial approaches in metabolic engineering can make use of randomized mutations and/or overexpression of randomized DNA fragments. When DNA fragments are obtained from a common genome or metagenome and packaged into the same expression vector, this is referred to as a DNA library. Generating quality DNA libraries that incorporate broad genetic diversity is challenging, despite the availability of published protocols. In response, a novel, efficient, and reproducible technique for creating DNA libraries was created in this research based on whole genome amplification using degenerate oligonucleotide primed PCR (DOP-PCR). The approach can produce DNA libraries from nanograms of a template genome or the metagenome of multiple microbial populations. The DOP-PCR primers contain random bases, and thermodynamics of hairpin formation was used to design primers capable of binding randomly to template DNA for amplification with minimal bias. Next-generation high-throughput sequencing was used to determine the design is capable of amplifying up to 98% of template genomic DNA and consistently out-performed other DOP-PCR primers. Application of these new DOP-PCR amplified DNA libraries was demonstrated in multiple strain enrichments to isolate genetic library fragments capable of (i) increasing tolerance of E. coli ER2256 to toxic levels of 1-butanol by doubling the growth rate of the culture, (ii) redirecting metabolism to ethanol and pyruvate production (over 250% increase in yield) in Clostridium cellulolyticum when consuming cellobiose, and (iii) enhancing L-arginine production when used in conjunction with a new synthetic gene circuit.
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    Detailed characterization of the UMAMIT proteins provides insight into their evolution, amino acid transport properties, and role in the plant
    Zhao, Chengsong; Pratelli, Rejane; Yu, Shi; Shelley, Brett; Collakova, Eva; Pilot, Guillaume (Oxford University Press, 2021-09-30)
    Amino acid transporters play a critical role in distributing amino acids within the cell compartments and between plant organs. Despite this importance, relatively few amino acid transporter genes have been characterized and their role elucidated with certainty. Two main families of proteins encode amino acid transporters in plants: the amino acid-polyamine-organocation superfamily, containing mostly importers, and the UMAMIT (usually multiple acids move in and out transporter) family, apparently encoding exporters, totaling 63 and 44 genes in Arabidopsis, respectively. Knowledge of UMAMITs is scarce, based on six Arabidopsis genes and a handful of genes from other species. To gain insight into the role of the members of this family and provide data to be used for future characterization, we studied the evolution of the UMAMITs in plants, and determined the functional properties, the structure, and localization of the 47 Arabidopsis UMAMITs. Our analysis showed that the AtUMAMITs are essentially localized at the tonoplast or the plasma membrane, and that most of them are able to export amino acids from the cytosol, confirming a role in intra- and intercellular amino acid transport. As an example, this set of data was used to hypothesize the role of a few AtUMAMITs in the plant and the cell.
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    Development of novel approaches to study Cuscuta campestris biology
    Bernal Galeano, Vivian Angelica (Virginia Tech, 2021-09-16)
    Cuscuta campestris is an obligate parasitic plant that lacks expanded leaves and roots and requires a host to complete its lifecycle. Parasite-host connections occur via an haustorium, a unique organ that acts as a bridge for the exchange of water, nutrients, macromolecules like mRNA, microRNA, and proteins, and microorganisms. Studies of Cuscuta spp. are challenging due to its dependence on the host and other host influences on the parasite. Recent research has shown intriguing aspects of Cuscuta biology like exchange genetic material with its hosts and loss of genes involved in processes such as high photosynthetic rates and defense. We developed new tools and methodologies that allow us to explore C. campestris biology in an unprecedent way. Foremost of these is an axenic method to grow C. campestris on an Artificial Host System (AHS). The AHS allows C. campestris to display its entire life cycle in vitro, including seed production. Using the AHS, we studied haustorial function, determining the role of nutrients and phytohormones on parasite haustorium development and growth, and found genes involved in haustorial function. The AHS allowed us to demonstrate the positive effect of light on C. campestris growth in the absence of a photosynthetic host and to investigate carotenoid- and ABA- related processes in the haustorial regions. We also wanted to understand how C. campestris defenses work independently from a plant host, so we studied the parasite responses to the bacterial epitope flg22 and the bacteria Peudomonas syringe. Our findings indicate that C. campestris is able to sense flg22, but its response differs from those observed in other non-parasite plants. Transcriptomic analysis revealed up-regulation of genes related to biotic and abiotic stresses, and downregulation of genes related to cuticle development. Our study contributes to understanding the C. campestris immune response in the absence of a host plant. Taken together, this research contributes novel methodologies that enable insights into C. campestris biology without the interference of a plant host on the parasite.
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    Functional Analysis of Plant Glutamate Receptors
    Price, Michelle B. (Virginia Tech, 2013-10-02)
    The plant glutamate receptors (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) and are hypothesized to be potential amino acid sensors in plants. Since their first discovery in 1998, the members of plant GLRs have been implicated in diverse processes such as C/N ratio sensing, root formation, pollen germination and plant-pathogen interaction. However, the exact properties of these channels, such as the spectrum of ligands, ion specificities, and subunit compositions are still not well understood. It is well established that animal iGluRs form homo- or hetero-tetramers in order to form ligand-gated cation channels. The first aspect of this research was to determine if plant GLRs likewise require different subunits to form functional channels. A modified yeast-2-hybrid system approach was initially taken and applied to 14 of the 20 AtGLRs to identify a number of candidate interactors in yeast. Forster resonance energy transfer (FRET), which measures the transfer of energy between interacting molecules, was performed in mammalian cells to confirm interaction between a few of those candidates. Interestingly, despite an abundance of overlapping co-localization between heteromeric combinations, only homomeric interactions were identified between GLRs 1.1 and 3.4 in HEK293 cells. Further, amino acids have been implicated in signaling between plants and microbes, but the mechanisms for amino acid perception in defense responses are far from being understood. Recently it was demonstrated that calcium responses initiated by bacterial and fungal microbe-associated molecular patterns (MAMPs) were diminished in seedlings treated with known agonists and antagonists of mammalian iGluRs, suggesting potential roles of GLRs in pathogen responses. Analysis of publicly available microarray data shows altered gene expression of a sub-fraction of GLRs in response to pathogen infection and bacterial elicitors. Thus, the second goal of my PhD research was aimed at determining whether GLRs are involved in the interaction between plants and pathogens. Gene expression changes of a number of candidate GLRs as well as pathogen growth was examined in response to the plant pathogen Pseudomonas syringae pv. tomato DC3000. Interestingly, single gene and multi-gene deficient plants responded differently with regards to pathogen susceptibility, likely as a result of functional compensation between GLRs.
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    Functional analysis of Poplar genes regulating flowering and vegetative growth
    Mahendra, Rienzy Ayeshan Rangajeewa (Virginia Tech, 2019-06-24)
    Poplar (Populus spp. and hybrids) are used for pulp, paper and solid wood products. Furthermore, poplar is being developed as a dedicated biomass crop for biofuels and biomaterials. Thus, methods to accelerate genetic improvement to improve woody biomass yield, quality and optimal growth on marginal lands are of considerable interest. One approach is to identify genes that could be manipulated through breeding or biotechnology to achieve these goals. I studied two sets of candidate genes for improving biomass, growth and manipulating flowering time. First, I studied the functions of PopNAC154 and PopNAC156, co-orthologs of Arabidopsis SECONDARY CELL WALL NAC DOMAIN2 (SND2), which are putative regulators of wood cell wall synthesis, the source of lignocellulosic biomass. Second, I studied PopCEN1, PopCEN2, and PopBFT, members of the TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS (CEN) gene family that act as flowering repressors in Arabidopsis and many plants. I studied INRA 717-1B (P. tremula x P. alba) transgenics with an artificial microRNA (AmiRNA) downregulating PopNAC154 and PopNAC156 (AmiSND2 trees). In a field trial, AmiSND2 trees showed higher mean height and diameter than wild-type (WT). We also observed that AmiSND2 transgenics showed delayed leaf senescence and leaf drop. After conducting controlled environment studies with AmiSND2 trees, I was able to confirm that downregulation of PopNAC154 and PopNAC156 genes does not alter the short daylength-induced bud set and growth cessation but it delays the low temperature induced leaf senescence and leaf drop. Further I was able to show that down regulation of the PopNAC154 and PopNAC156 genes resulted in significantly higher mean plant heights and delayed bud set compared to the WT plants under low soil nutrient conditions. Wood chemistry data analysis of field grown AmiSND2 trees showed that they have a significantly higher cellulose content a lower lignin content compared to that of the WT. Thus, these results show that downregulating the PopNAC154 and PopNAC156 genes has the potential to increase biomass yield and quality. In a previous study, simultaneous downregulation of PopCEN1 and PopCEN2 genes using RNA interference (RNAi) method caused poplar trees to flower only after two years of growth in the field. I used CRISPR/CAS9 method to knock-out each paralog individually as well as the related gene, PopBFT. The popcen1 mutant trees developed flowers even under in vitro conditions, but popcen2 mutants did not show an obvious phenotype. popbft mutant trees also did not show an obvious phenotype under standard growing conditions. However, when soil nutrient availability was allowed to deplete, the popbft mutants showed lower mean plant height compared to the WT and also showed lower root length and root volume under low Nitrogen conditions in an in vitro assay compared to the WT. These results prove that PopCEN1 gene is directly involved in repressing flowering in poplar and allele-specific mutation should be tested as an approach to accelerate breeding. PopCEN2 and PopBFT might not have a role in regulation of flowering time, and though additional studies are needed, PopBFT appears to have a role in regulating growth in response to nutrient availability.
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    The Impact of Iron Deficiency on Plant-Oomycete Interactions
    Herlihy, John H. (Virginia Tech, 2020-04-08)
    Plants are sessile organisms adapted to cope with dynamic changes in their environment. Abiotic stresses, such as heat, drought, or nutrient deficiency must be overcome simultaneously with biotic threats such as pathogens and herbivores. Oomycete pathogens represent a significant threat to global food production and natural ecosystems. Novel modes of oomycete disease control could increase crop yield and reduce pesticide application. Overlaps between the plant response to iron deficiency and pathogens have been documented, but the impact of simultaneous imposition of both stresses on the plant have not been studied. Additionally, nothing is known about the impact of iron deficiency on oomycete infection, or mechanisms of oomycete iron uptake. We adapted a hydroponic system to simultaneously impose iron deficiency and monitor pathogen infection. The oomycete pathogens Hyaloperonospora arabidopsidis, and Phytophthora capsici grew less well on iron-deficient Arabidopsis thaliana, at least in part because of observed activation of immunity due to iron stress. We screened A. thaliana T-DNA insertion mutants defective in iron metabolism and transport and identified potential mechanisms of H. arabidopsidis iron acquisition. We conducted RNA sequencing to understand how A. thaliana responds to iron deficiency and root infection of P. capsici. 323 genes were differentially upregulated in iron-starved plants over three days, irrespective of pathogen infection, representing a core iron deficiency response. This group of core genes included the primary A. thaliana iron uptake pathway and genes for coumarin biosynthesis. Salicylic acid responsive genes were observed in both treatments consistent with this defense hormone's previously identified role in iron deficiency. Genes related to glucosinolate production – shown to be important in defense against P. capsici – were down regulated during infection, potentially due to the activity of virulence effectors. Our work demonstrates crosstalk between the iron deficiency response and plant immunity, and that iron acquisition remains important to the plant even after pathogen invasion. These new insights provide a first step in developing novel resistance strategies to control oomycetes in agronomically important crops.
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    Increased Expression of UMAMIT Amino Acid Transporters Results in Activation of Salicylic Acid Dependent Stress Response
    Besnard, Julien; Sonawala, Unnati; Maharjan, Bal; Collakova, Eva; Finlayson, Scott A.; Pilot, Guillaume; McDowell, John M.; Okumoto, Sakiko (2021-01-26)
    In addition to their role in the biosynthesis of important molecules such as proteins and specialized metabolites, amino acids are known to function as signaling molecules through various pathways to report nitrogen status and trigger appropriate metabolic and cellular responses. Moreover, changes in amino acid levels through altered amino acid transporter activities trigger plant immune responses. Specifically, loss of function of major amino acid transporter, over-expression of cationic amino acid transporter, or over-expression of the positive regulators of membrane amino acid export all lead to dwarfed phenotypes and upregulated salicylic acid (SA)-induced stress marker genes. However, whether increasing amino acid exporter protein levels lead to similar stress phenotypes has not been investigated so far. Recently, a family of transporters, namely USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTERS (UMAMITs), were identified as amino acid exporters. The goal of this study was to investigate the effects of increased amino acid export on plant development, growth, and reproduction to further examine the link between amino acid transport and stress responses. The results presented here show strong evidence that an increased expression of UMAMIT transporters induces stress phenotypes and pathogen resistance, likely due to the establishment of a constitutive stress response via a SA-dependent pathway.
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    Inference of Transcription Regulatory Network in Low Phytic Acid Soybean Seeds
    Redekar, Neelam R.; Pilot, Guillaume; Raboy, Victor; Li, S.; Saghai-Maroof, Mohammad A. (Frontiers, 2017-11-30)
    A dominant loss of function mutation in myo-inositol phosphate synthase (MIPS) gene and recessive loss of function mutations in two multidrug resistant protein type-ABC transporter genes not only reduce the seed phytic acid levels in soybean, but also affect the pathways associated with seed development, ultimately resulting in low emergence. To understand the regulatory mechanisms and identify key genes that intervene in the seed development process in low phytic acid crops, we performed computational inference of gene regulatory networks in low and normal phytic acid soybeans using a time course transcriptomic data and multiple network inference algorithms. We identified a set of putative candidate transcription factors and their regulatory interactions with genes that have functions in myo-inositol biosynthesis, auxin-ABA signaling, and seed dormancy. We evaluated the performance of our unsupervised network inference method by comparing the predicted regulatory network with published regulatory interactions in Arabidopsis. Some contrasting regulatory interactions were observed in low phytic acid mutants compared to non-mutant lines. These findings provide important hypotheses on expression regulation of myo-inositol metabolism and phytohormone signaling in developing low phytic acid soybeans. The computational pipeline used for unsupervised network learning in this study is provided as open source software and is freely available at https://lilabatvt.github.io/LPANetwork/.
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    New Tools to Understand Mechanisms of Nutrient Transfer from Plants to Biotrophic Pathogens
    Dinkeloo, Kasia (Virginia Tech, 2018-10-12)
    The interaction between Arabidopsis and its natural downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa), provides a model for understanding how oomycetes colonize plants. Hpa is a model organism for many highly destructive oomycete pathogens and transcriptomics of this interaction have been well-documented. However, the material in these studies has been derived from infected leaves that contain a mix of pathogen-proximal and pathogen-distal plant cells. The most direct interactions between Arabidopsis and Hyaloperonospora arabidopsidis occur in haustoriated cells- where the pathogen can secrete effectors and acquire nutrients needed for successful colonization and reproduction. These cells are difficult to isolate due to their limited number and ephemeral nature. I have developed a method to isolate the translatome (i.e., mRNAs associated with ribosomes) of pathogen-proximal cells. This method utilizes translating ribosome immuno-purification technology (TRAP), regulated by both pathogen-responsive and tissue-specific promoters, to isolate mRNAs that are being translated in pathogen-proximal cells. Compared to "bulk" transcriptomics of material isolated from homogenized leaves, this method will enrich for transcripts that are differentially expressed, and translated, in pathogen-proximal cells. From this method, RNA was isolated in amount and quality sufficient for sequencing. This sequencing data will enable the discovery of plant genes that may be manipulated by the pathogen to suppress defense responses and extract nutrients.
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    Plasticity of Primary Metabolism in Parasitic Orobanchaceae
    Clermont, Kristen Renee (Virginia Tech, 2018-11-20)
    Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. Species in this family span the spectrum of host nutrient dependency, allowing comparisons that provide insight into parasite adaptation. A key aspect of this is the relationship between parasite metabolism and the metabolite profile of its host. To what extent does the metabolite profile of the parasite depend on that of the host? Do parasites that differ in host-dependency also differ in their metabolism or do they use common metabolic strategies? These questions were addressed using comparative profiling of primary metabolites to gain insight into carbon and nitrogen assimilation by the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor. First, metabolite profiles of these parasites and their hosts were compared during the key life stages before and after haustorial attachment. Second, the impact of specific variations in host metabolism was analyzed for P. aegyptiaca growing on Arabidopsis thaliana hosts that had mutations in amino acid metabolism but otherwise identical genetic backgrounds. Comparison of P. aegyptiaca and T. versicolor metabolite profiles identified substantial differences in the stages spanning the transition from pre-haustorial development through post-haustorial feeding. Each parasite species is distinct from the other and from their hosts. For parasites growing on host lines that differ in amino acid content, the size of P. aegyptiaca tubercles decreased when grown on the aap6 mutant line, which has decreased levels of asparagine in the phloem sap compared to the wild type. However, altered amino acid levels in other lines did not impact P. aegyptiaca growth, indicating that this parasite has ability to compensate for variation in host metabolic composition. This research highlights the importance of aspartate and asparagine to early post-attachment metabolism in both P. aegyptiaca and T. versicolor and through host deficiencies possibly associated with decreased growth in P. aegyptiaca. Overall, this work provides insights both into the metabolism of parasitic plants and lays the foundation for the development of new metabolism-based control strategies.
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    A split green fluorescent protein system to enhance spatial and temporal sensitivity of translating ribosome affinity purification
    Dinkeloo, Kasia; Pelly, Zoe; McDowell, John M.; Pilot, Guillaume (Wiley, 2022-04-18)
    Translating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity co-purification of ribosomes and the mRNAs that they are translating. This population of actively translated mRNAs (translatome) can be interrogated by quantitative PCR or RNA sequencing. Condition- or cell-specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell-specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: One is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split green fluorescent protein (GFP); the second is controlled by a stimulus-specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi-molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre-existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant-pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies.
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    Suppressor mutations in the Glutamine Dumper1 protein dissociate disturbance in amino acid transport from other characteristics of the Gdu1D phenotype
    Yu, Shi; Pratelli, Rejane; Denbow, Cynthia J.; Pilot, Guillaume (Frontiers, 2015-08-04)
    Intracellular amino acid transport across plant membranes is critical for metabolic pathways which are often split between different organelles. In addition, transport of amino acids across the plasma membrane enables the distribution of organic nitrogen through the saps between leaves and developing organs. Amino acid importers have been studied for more than two decades, and their role in this process is well-documented. While equally important, amino acid exporters are not well-characterized. The over-expression of GDU1, encoding a small membrane protein with one transmembrane domain, leads to enhancement of amino acid export by Arabidopsis cells, glutamine secretion at the leaf margin, early senescence and size reduction of the plant, possibly caused by the stimulation of amino acid exporter(s). Previous work reported the identification of suppressor mutations of the GDU1 over-expression phenotype, which affected the GDU1 and LOG2 genes, the latter encoding a membrane-bound ubiquitin ligase interacting with GDU1. The present study focuses on the characterization of three additional suppressor mutations affecting GDU1. Size, phenotype, glutamine transport and amino acid tolerance were recorded for recapitulation plants and over-expressors of mutagenized GDU1 proteins. Unexpectedly, the over-expression of most mutated GDU1 led to plants with enhanced amino acid export, but failing to display secretion of glutamine and size reduction. The results show that the various effects triggered by GDU1 over-expression can be dissociated from one another by mutagenizing specific residues. The fact that these residues are not necessarily conserved suggests that the diverse biochemical properties of the GDU1 protein are not only born by the characterized transmembrane and VIMAG domains. These data provide a better understanding of the structure/function relationships of GDU1 and may enable modifying amino acid export in plants without detrimental effects on plant fitness.
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    Testing the efficiency of plant artificial microRNAs by transient expression in Nicotiana benthamiana reveals additional action at the translational level
    Yu, Shi; Pilot, Guillaume (Frontiers, 2014-11-19)
    Artificial microRNAs (amiRNAs) have become an important tool to assess gene functions due to their high efficiency and specificity to decrease target gene expression. Based on the observed degree of complementarity between microRNAs (miRNAs) and their targets, it was widely accepted that plant miRNAs act at the mRNA stability level, while the animal miRNAs act at the translational level. Contrary to these canonical dogmas, recent evidence suggests that both plant and animal miRNAs act at both levels. Nevertheless, it is still impossible to predict the effect of an artificial miRNA on the stability or translation of the target mRNA in plants. Consequently, identifying and discarding inefficient amiRNAs prior to stable plant transformation would help getting suppressed mutants faster and at reduced cost. We designed and tested a method using transient expression of amiRNAs and the corresponding target genes in Nicotiana benthamiana leaves to test the efficacy of amiRNAs for suppression of the target protein accumulation. The ability of the amiRNAs to suppress the target gene expression in N. benthamiana was then compared to that in stably transformed Arabidopsis. It was found that the efficacy of 16 amiRNAs, targeting a total of four genes, varied greatly. The effects of amiRNAs on target mRNA accumulation did not always correlate with target protein accumulation or the corresponding phenotypes, while a similar trend of the silencing efficacy of amiRNAs could be observed between N. benthamiana and stably transformed Arabidopsis. Our results showed that, similar to endogenous plant miRNAs, plant amiRNAs could act at the translational level, a property needed to be taken into account when testing the efficacy of individual amiRNAs. Preliminary tests in N. benthamiana can help determine which amiRNA would be the most likely to suppress target gene expression in stably transformed plants.