Christian Elowsky | University of Nebraska Lincoln (original) (raw)

Papers by Christian Elowsky

The Plant Cell Online, Sep 1, 2011

Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but meas... more Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but measurable demonstration of this intimate interaction is difficult. At the level of cellular metabolism, several biosynthetic pathways involve both mitochondrial-and plastid-localized steps. However, at an environmental response level, it is not clear how the two organelles intersect in programmed cellular responses. Here, we provide evidence, using genetic perturbation of the MutS Homolog1 (MSH1) nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. The mitochondrial form of the protein participates in DNA recombination surveillance, with disruption of the gene resulting in enhanced mitochondrial genome recombination at numerous repeated sequences. The plastid-localized form of the protein interacts with the plastid genome and influences genome stability and plastid development, with its disruption leading to variegation of the plant. These developmental changes include altered patterns of nuclear gene expression. Consistency of plastid and mitochondrial response across both monocot and dicot species indicate that the dual-functioning nature of MSH1 is well conserved. Variegated tissues show changes in redox status together with enhanced plant survival and reproduction under photooxidative light conditions, evidence that the plastid changes triggered in this study comprise an adaptive response to naturally occurring light stress.

Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial... more Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood.

Cell host & microbe, Jan 13, 2016

The bacterial pathogen Pseudomonas syringae depends on effector proteins secreted by its type III... more The bacterial pathogen Pseudomonas syringae depends on effector proteins secreted by its type III secretion system for the pathogenesis of plants. The majority of these effector proteins are known suppressors of immunity, but their plant targets remain elusive. Using Arabidopsis thaliana as a model host, we report that the HopE1 effector uses the host calcium sensor, calmodulin (CaM), as a co-factor to target the microtubule-associated protein 65 (MAP65), an important component of the microtubule network. HopE1 interacted with MAP65 in a CaM-dependent manner, resulting in MAP65-GFP dissociation from microtubules. Transgenic Arabidopsis expressing HopE1 had reduced secretion of the immunity protein PR-1 compared to wild-type plants. Additionally, Arabidopsis map65-1 mutants were immune deficient and were more susceptible to P. syringae. Our results suggest a virulence strategy in which a pathogen effector is activated by host calmodulin to target MAP65 and the microtubule network, th...

We report here the utility of major histocompatibility complex (MHC) class II dextramers for in s... more We report here the utility of major histocompatibility complex (MHC) class II dextramers for in situ detection of self-reactive CD4 T cells in two target organs, the brain and heart. We optimized the conditions for in situ detection of antigen-specific CD4 T cells using brain sections obtained from SJL mice immunized with myelin proteolipid protein (PLP) 139-151; the sections were costained with IA s /PLP 139-151 (specific) or Theiler's murine encephalomyelitis virus (TMEV) 70-86 (control) dextramers and anti-CD4. Analysis of sections by laser scanning confocal microscope revealed detection of cells positive for PLP 139-151 but not for TMEV 70-86 dextramers to be colocalized with CD4-expressing T cells, indicating that the staining was specific to PLP 139-151 dextramers. Further, we devised a method to reliably enumerate the frequencies of antigenspecific T cells by counting the number of dextramer + CD4 + T cells in the 'Z' serial images acquired sequentially. We next extended these observations to detect cardiac myosin-specific T cells in autoimmune myocarditis induced in A/J mice by immunizing with cardiac myosin heavy chain-a (Myhc) 334-352. Heart sections prepared from immunized mice were costained with Myhc 334-352 (specific) or bovine ribonuclease 43-56 (control) dextramers together with anti-CD4; the sections showed the infiltrations of Myhc-specific CD4 T cells. The data suggest that MHC class II dextramers are useful tools for enumerating the frequencies of antigen-specific CD4 T cells in situ by direct staining without having to amplify the fluorescent signals, an approach commonly employed with conventional MHC tetramers.

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 i... more As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein–protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochon-dria results in 7%–10% of plants altered in leaf morphology, heat tolerance, and mitochondrial genome stability. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH1. MSH1 depletion alters non-photoactive redox behavior in plastids and a subset of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.

The parental species and hybrid swarm of native Ulmus rubra Muhl. and the introduced, naturalized... more The parental species and hybrid swarm of native Ulmus rubra Muhl. and the introduced, naturalized and weedy U. pumila L. were investigated in a 65-km transect in Lancaster, Saunders, and Butler counties in Nebraska. Thirty-two trees of U. rubra, 32 of U. pumila, and 50 of the hybrid swarm were sampled for leaves and buds and subsampled for flowers and fruits. Leaves were measured for petiole length, blade length, width, primary and secondary teeth per cm, number of secondary teeth per primary tooth, and texture. Buds were scored for color and distribution of trichomes. Flowers were sampled for stamen counts and pollen size. Fruits were measured for length, width, and color and distribution of trichomes. Statistically significant differences (P<.05) were detected between the two parental species in all characters except fruit lengths. The hybrid swarm was also significantly different from both parental species, except in such characters as leaf length-to-width ratios, numbers of teeth, petiole length, and pollen size. A formal description of the hybrid swarm is given, and its name is proposed as Ulmus × intermedia Elowsky.

The Journal of biological chemistry, Jan 8, 2015

Hyaluronan turnover accelerates metastatic progression of prostate cancer partly by increasing ra... more Hyaluronan turnover accelerates metastatic progression of prostate cancer partly by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed Hyal1 as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of hyaluronan and the endocytic recycling marker transferrin. Live imaging of Hyal1, sucrose gradient centrifugation, and specific co-localization of Rab GTPases defined the subcellular distribution of Hyal1 as early and late endosomes, lysosomes, and recycling vesicles. Manipulation of vesicular trafficking by chemical inhibitors or with constitutively active and dominant negative Rab expression constructs, caused atypical localization of Hyal1. Using the catalytically inactive point mutant Hyal1-E131Q, we found enzymatic activity of Hyal1 was necessary for normal localization within the cell, as Hyal1-E131Q was mainly found withi...

The Plant journal : for cell and molecular biology, 2014

The pathogen Pseudomonas syringae requires a type-III protein secretion system and the effector p... more The pathogen Pseudomonas syringae requires a type-III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type-III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type-III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector-triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis, and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP-triggered immune responses compared with wild-type plants. An N-terminal region of HopK1 shares similarity with the corresponding region in the well-studied type-III effector AvrRps4; however, their C-terminal regions are dissimilar, indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The ...

Trinexapac-ethyl and mefluidide are synthetic plant growth regulators (PGRs) which inhibit gibber... more Trinexapac-ethyl and mefluidide are synthetic plant growth regulators (PGRs) which inhibit gibberellic acid (GA) biosynthesis. Two greenhouse experiments using tall fescue cv. Mustang (Festuca arundinacea Schreb.) and St. Augustinegrass cv. Raleigh [Stenotaphrum secundatum S. (Walt.) Kuntze] were conducted with a randomized complete block experimental design with 4 replications. Chemical applications were trinexapac-ethyl at 0.286 kg ha -1 for tall fescue and 0.145 kg ha -1 for St. Augustinegrass and mefluidide at 0.137 kg ha -1 for both species. Leaf cell density and arrangement were sampled 4, 8, 16, 32, and 48 days after treatment (DAT) using a confocal microscope. Leaf elongation rates were measured three times per week for seven weeks after chemical application. Trinexapac-ethyl and mefluidide decreased leaf elongation rate of both species by 30%. Tall fescue and St. Augustinegrass cell density increased 15 and 20%, respectively, from both PGR treatments relative to the control. These results indicate that these PGRs inhibit cell elongation. Increased cell density resulted in greater cell wall content. This has been correlated with increased wear tolerance in turfgrasses, however, PGR treated turf also has a longer recover period than nontreated turf. This may be utilized under certain cases were increased wear tolerance is more important than recovery.

Journal of Visualized Experiments, 2014

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers fo... more This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-α (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers + cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IA s /PLP 139-151 dextramers (specific)/anti-CD4 and IA s /Theiler's murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IA k /Myhc 334-352 dextramers/

Toxins, 2010

Shiga toxin (Stx) produced by the invasive Shigella dysenteriae serotype 1 (S. dysenteriae1) caus... more Shiga toxin (Stx) produced by the invasive Shigella dysenteriae serotype 1 (S. dysenteriae1) causes gastrointestinal and kidney complications. It has been assumed that Stx is released intracellularly after enterocyte invasion by S. dysenteriae1. However, there is little information about Stx distribution inside S. dysenteriae1-infected enterocytes. Here, we use intestinal epithelial T84 cells to characterize the trafficking of Stx delivered into the cytosol, in ways that mimic aspects of S. dysenteriae1 infection. We find that cytoplasmic Stx is transported into nucleoli. Stx nucleolar movement is carrier-and energy-dependent. Stx binding to the nucleoli of normal human enterocytes in vitro supports possible roles for nucleolar trafficking in toxin-induced intestinal pathology.

The Plant Journal, 2012

Ubiquinone (coenzyme Q) is the generic name of a class of lipid-soluble electron carriers formed ... more Ubiquinone (coenzyme Q) is the generic name of a class of lipid-soluble electron carriers formed of a redox active benzoquinone ring attached to a prenyl side chain. The length of the latter varies among species, and depends upon the product specificity of a trans-long-chain prenyl diphosphate synthase that elongates an allylic diphosphate precursor. In Arabidopsis, this enzyme is assumed to correspond to an endoplasmic reticulum-located solanesyl diphosphate synthase, although direct genetic evidence was lacking. In this study, the reconstruction of the functional network of Arabidopsis genes linked to ubiquinone biosynthesis singled out an unsuspected solanesyl diphosphate synthase candidate -product of gene At2g34630 -that, extraordinarily, had been shown previously to be targeted to plastids and to contribute to the biosynthesis of gibberellins. Green fluorescent protein (GFP) fusion experiments in tobacco and Arabidopsis, and complementation of a yeast coq1 knockout lacking mitochondrial hexaprenyl diphosphate synthase demonstrated that At2g34630 is also targeted to mitochondria. At2g34630 is the main -if not solecontributor to solanesyl diphosphate synthase activity required for the biosynthesis of ubiquinone, as demonstrated by the dramatic (75-80%) reduction of the ubiquinone pool size in corresponding RNAi lines. Overexpression of At2g34630 gave up to a 40% increase in ubiquinone content compared to wild-type plants. None of the silenced or overexpressing lines, in contrast, displayed altered levels of plastoquinone. Phylogenetic analyses revealed that At2g34630 is the only Arabidopsis trans-long-chain prenyl diphosphate synthase that clusters with the Coq1 orthologs involved in the biosynthesis of ubiquinone in other eukaryotes.

The Plant Journal, 2012

It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA),... more It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA), a necessary step to form the naphthoquinone ring of phylloquinone (vitamin K 1 ). In fact, only recently has the hydrolysis of DHNA-CoA been demonstrated to be enzyme driven in vivo, and the cognate thioesterase characterized in the cyanobacterium Synechocystis. With a few exceptions in certain prokaryotic (Sorangium and Opitutus) and eukaryotic (Cyanidium, Cyanidioschyzon and Paulinella) organisms, orthologs of DHNA-CoA thioesterase are missing outside of the cyanobacterial lineage. In this study, genomic approaches and functional complementation experiments identified two Arabidopsis genes encoding functional DHNA-CoA thioesterases. The deduced plant proteins display low percentages of identity with cyanobacterial DHNA-CoA thioesterases, and do not even share the same catalytic motif. GFP-fusion experiments demonstrated that the Arabidopsis proteins are targeted to peroxisomes, and subcellular fractionations of Arabidopsis leaves confirmed that DHNA-CoA thioesterase activity occurs in this organelle. In vitro assays with various aromatic and aliphatic acyl-CoA thioester substrates showed that the recombinant Arabidopsis enzymes preferentially hydrolyze DHNA-CoA. Cognate T-DNA knock-down lines display reduced DHNA-CoA thioesterase activity and phylloquinone content, establishing in vivo evidence that the Arabidopsis enzymes are involved in phylloquinone biosynthesis. Extraordinarily, structure-based phylogenies coupled to comparative genomics demonstrate that plant DHNA-CoA thioesterases originate from a horizontal gene transfer with a bacterial species of the Lactobacillales order.

The Plant Cell, 2014

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. H... more It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent b-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch.

THE PLANT CELL ONLINE, 2009

Postendosymbiotic evolution has given rise to proteins that are multiply targeted within the cell... more Postendosymbiotic evolution has given rise to proteins that are multiply targeted within the cell. Various mechanisms have been identified to permit the expression of proteins encoding distinct N termini from a single gene. One mechanism involves alternative translation initiation (aTI). We previously showed evidence of aTI activity within the Arabidopsis thaliana organellar DNA polymerase gene POLg2. Translation initiates at four distinct sites within this gene, two non-AUG, to produce distinct plastid and mitochondrially targeted forms of the protein. To understand the regulation of aTI in higher plants, we used Polg2 as a model to investigate both cis-and trans-acting features of the process. Here, we show that aTI in Polg2 and other plant genes involves ribosome scanning dependent on sequence context at the multiple initiation sites to condition specific binding of at least one trans-acting factor essential for site recognition. Multiple active translation initiation sites appear to operate in several plant genes, often to expand protein targeting. In plants, where the mitochondrion and plastid must share a considerable portion of their proteomes and coordinate their functions, leaky ribosome scanning behavior provides adaptive advantage in the evolution of protein dual targeting and translational regulation.

THE PLANT CELL ONLINE, 2011

Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but meas... more Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but measurable demonstration of this intimate interaction is difficult. At the level of cellular metabolism, several biosynthetic pathways involve both mitochondrial-and plastid-localized steps. However, at an environmental response level, it is not clear how the two organelles intersect in programmed cellular responses. Here, we provide evidence, using genetic perturbation of the MutS Homolog1 (MSH1) nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. The mitochondrial form of the protein participates in DNA recombination surveillance, with disruption of the gene resulting in enhanced mitochondrial genome recombination at numerous repeated sequences. The plastid-localized form of the protein interacts with the plastid genome and influences genome stability and plastid development, with its disruption leading to variegation of the plant. These developmental changes include altered patterns of nuclear gene expression. Consistency of plastid and mitochondrial response across both monocot and dicot species indicate that the dual-functioning nature of MSH1 is well conserved. Variegated tissues show changes in redox status together with enhanced plant survival and reproduction under photooxidative light conditions, evidence that the plastid changes triggered in this study comprise an adaptive response to naturally occurring light stress.

The Plant Cell Online, Sep 1, 2011

Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but meas... more Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but measurable demonstration of this intimate interaction is difficult. At the level of cellular metabolism, several biosynthetic pathways involve both mitochondrial-and plastid-localized steps. However, at an environmental response level, it is not clear how the two organelles intersect in programmed cellular responses. Here, we provide evidence, using genetic perturbation of the MutS Homolog1 (MSH1) nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. The mitochondrial form of the protein participates in DNA recombination surveillance, with disruption of the gene resulting in enhanced mitochondrial genome recombination at numerous repeated sequences. The plastid-localized form of the protein interacts with the plastid genome and influences genome stability and plastid development, with its disruption leading to variegation of the plant. These developmental changes include altered patterns of nuclear gene expression. Consistency of plastid and mitochondrial response across both monocot and dicot species indicate that the dual-functioning nature of MSH1 is well conserved. Variegated tissues show changes in redox status together with enhanced plant survival and reproduction under photooxidative light conditions, evidence that the plastid changes triggered in this study comprise an adaptive response to naturally occurring light stress.

Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial... more Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood.

Cell host & microbe, Jan 13, 2016

The bacterial pathogen Pseudomonas syringae depends on effector proteins secreted by its type III... more The bacterial pathogen Pseudomonas syringae depends on effector proteins secreted by its type III secretion system for the pathogenesis of plants. The majority of these effector proteins are known suppressors of immunity, but their plant targets remain elusive. Using Arabidopsis thaliana as a model host, we report that the HopE1 effector uses the host calcium sensor, calmodulin (CaM), as a co-factor to target the microtubule-associated protein 65 (MAP65), an important component of the microtubule network. HopE1 interacted with MAP65 in a CaM-dependent manner, resulting in MAP65-GFP dissociation from microtubules. Transgenic Arabidopsis expressing HopE1 had reduced secretion of the immunity protein PR-1 compared to wild-type plants. Additionally, Arabidopsis map65-1 mutants were immune deficient and were more susceptible to P. syringae. Our results suggest a virulence strategy in which a pathogen effector is activated by host calmodulin to target MAP65 and the microtubule network, th...

We report here the utility of major histocompatibility complex (MHC) class II dextramers for in s... more We report here the utility of major histocompatibility complex (MHC) class II dextramers for in situ detection of self-reactive CD4 T cells in two target organs, the brain and heart. We optimized the conditions for in situ detection of antigen-specific CD4 T cells using brain sections obtained from SJL mice immunized with myelin proteolipid protein (PLP) 139-151; the sections were costained with IA s /PLP 139-151 (specific) or Theiler's murine encephalomyelitis virus (TMEV) 70-86 (control) dextramers and anti-CD4. Analysis of sections by laser scanning confocal microscope revealed detection of cells positive for PLP 139-151 but not for TMEV 70-86 dextramers to be colocalized with CD4-expressing T cells, indicating that the staining was specific to PLP 139-151 dextramers. Further, we devised a method to reliably enumerate the frequencies of antigenspecific T cells by counting the number of dextramer + CD4 + T cells in the 'Z' serial images acquired sequentially. We next extended these observations to detect cardiac myosin-specific T cells in autoimmune myocarditis induced in A/J mice by immunizing with cardiac myosin heavy chain-a (Myhc) 334-352. Heart sections prepared from immunized mice were costained with Myhc 334-352 (specific) or bovine ribonuclease 43-56 (control) dextramers together with anti-CD4; the sections showed the infiltrations of Myhc-specific CD4 T cells. The data suggest that MHC class II dextramers are useful tools for enumerating the frequencies of antigen-specific CD4 T cells in situ by direct staining without having to amplify the fluorescent signals, an approach commonly employed with conventional MHC tetramers.

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 i... more As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein–protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochon-dria results in 7%–10% of plants altered in leaf morphology, heat tolerance, and mitochondrial genome stability. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH1. MSH1 depletion alters non-photoactive redox behavior in plastids and a subset of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.

The parental species and hybrid swarm of native Ulmus rubra Muhl. and the introduced, naturalized... more The parental species and hybrid swarm of native Ulmus rubra Muhl. and the introduced, naturalized and weedy U. pumila L. were investigated in a 65-km transect in Lancaster, Saunders, and Butler counties in Nebraska. Thirty-two trees of U. rubra, 32 of U. pumila, and 50 of the hybrid swarm were sampled for leaves and buds and subsampled for flowers and fruits. Leaves were measured for petiole length, blade length, width, primary and secondary teeth per cm, number of secondary teeth per primary tooth, and texture. Buds were scored for color and distribution of trichomes. Flowers were sampled for stamen counts and pollen size. Fruits were measured for length, width, and color and distribution of trichomes. Statistically significant differences (P<.05) were detected between the two parental species in all characters except fruit lengths. The hybrid swarm was also significantly different from both parental species, except in such characters as leaf length-to-width ratios, numbers of teeth, petiole length, and pollen size. A formal description of the hybrid swarm is given, and its name is proposed as Ulmus × intermedia Elowsky.

The Journal of biological chemistry, Jan 8, 2015

Hyaluronan turnover accelerates metastatic progression of prostate cancer partly by increasing ra... more Hyaluronan turnover accelerates metastatic progression of prostate cancer partly by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed Hyal1 as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of hyaluronan and the endocytic recycling marker transferrin. Live imaging of Hyal1, sucrose gradient centrifugation, and specific co-localization of Rab GTPases defined the subcellular distribution of Hyal1 as early and late endosomes, lysosomes, and recycling vesicles. Manipulation of vesicular trafficking by chemical inhibitors or with constitutively active and dominant negative Rab expression constructs, caused atypical localization of Hyal1. Using the catalytically inactive point mutant Hyal1-E131Q, we found enzymatic activity of Hyal1 was necessary for normal localization within the cell, as Hyal1-E131Q was mainly found withi...

The Plant journal : for cell and molecular biology, 2014

The pathogen Pseudomonas syringae requires a type-III protein secretion system and the effector p... more The pathogen Pseudomonas syringae requires a type-III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type-III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type-III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector-triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis, and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP-triggered immune responses compared with wild-type plants. An N-terminal region of HopK1 shares similarity with the corresponding region in the well-studied type-III effector AvrRps4; however, their C-terminal regions are dissimilar, indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The ...

Trinexapac-ethyl and mefluidide are synthetic plant growth regulators (PGRs) which inhibit gibber... more Trinexapac-ethyl and mefluidide are synthetic plant growth regulators (PGRs) which inhibit gibberellic acid (GA) biosynthesis. Two greenhouse experiments using tall fescue cv. Mustang (Festuca arundinacea Schreb.) and St. Augustinegrass cv. Raleigh [Stenotaphrum secundatum S. (Walt.) Kuntze] were conducted with a randomized complete block experimental design with 4 replications. Chemical applications were trinexapac-ethyl at 0.286 kg ha -1 for tall fescue and 0.145 kg ha -1 for St. Augustinegrass and mefluidide at 0.137 kg ha -1 for both species. Leaf cell density and arrangement were sampled 4, 8, 16, 32, and 48 days after treatment (DAT) using a confocal microscope. Leaf elongation rates were measured three times per week for seven weeks after chemical application. Trinexapac-ethyl and mefluidide decreased leaf elongation rate of both species by 30%. Tall fescue and St. Augustinegrass cell density increased 15 and 20%, respectively, from both PGR treatments relative to the control. These results indicate that these PGRs inhibit cell elongation. Increased cell density resulted in greater cell wall content. This has been correlated with increased wear tolerance in turfgrasses, however, PGR treated turf also has a longer recover period than nontreated turf. This may be utilized under certain cases were increased wear tolerance is more important than recovery.

Journal of Visualized Experiments, 2014

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers fo... more This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-α (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers + cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IA s /PLP 139-151 dextramers (specific)/anti-CD4 and IA s /Theiler's murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IA k /Myhc 334-352 dextramers/

Toxins, 2010

Shiga toxin (Stx) produced by the invasive Shigella dysenteriae serotype 1 (S. dysenteriae1) caus... more Shiga toxin (Stx) produced by the invasive Shigella dysenteriae serotype 1 (S. dysenteriae1) causes gastrointestinal and kidney complications. It has been assumed that Stx is released intracellularly after enterocyte invasion by S. dysenteriae1. However, there is little information about Stx distribution inside S. dysenteriae1-infected enterocytes. Here, we use intestinal epithelial T84 cells to characterize the trafficking of Stx delivered into the cytosol, in ways that mimic aspects of S. dysenteriae1 infection. We find that cytoplasmic Stx is transported into nucleoli. Stx nucleolar movement is carrier-and energy-dependent. Stx binding to the nucleoli of normal human enterocytes in vitro supports possible roles for nucleolar trafficking in toxin-induced intestinal pathology.

The Plant Journal, 2012

Ubiquinone (coenzyme Q) is the generic name of a class of lipid-soluble electron carriers formed ... more Ubiquinone (coenzyme Q) is the generic name of a class of lipid-soluble electron carriers formed of a redox active benzoquinone ring attached to a prenyl side chain. The length of the latter varies among species, and depends upon the product specificity of a trans-long-chain prenyl diphosphate synthase that elongates an allylic diphosphate precursor. In Arabidopsis, this enzyme is assumed to correspond to an endoplasmic reticulum-located solanesyl diphosphate synthase, although direct genetic evidence was lacking. In this study, the reconstruction of the functional network of Arabidopsis genes linked to ubiquinone biosynthesis singled out an unsuspected solanesyl diphosphate synthase candidate -product of gene At2g34630 -that, extraordinarily, had been shown previously to be targeted to plastids and to contribute to the biosynthesis of gibberellins. Green fluorescent protein (GFP) fusion experiments in tobacco and Arabidopsis, and complementation of a yeast coq1 knockout lacking mitochondrial hexaprenyl diphosphate synthase demonstrated that At2g34630 is also targeted to mitochondria. At2g34630 is the main -if not solecontributor to solanesyl diphosphate synthase activity required for the biosynthesis of ubiquinone, as demonstrated by the dramatic (75-80%) reduction of the ubiquinone pool size in corresponding RNAi lines. Overexpression of At2g34630 gave up to a 40% increase in ubiquinone content compared to wild-type plants. None of the silenced or overexpressing lines, in contrast, displayed altered levels of plastoquinone. Phylogenetic analyses revealed that At2g34630 is the only Arabidopsis trans-long-chain prenyl diphosphate synthase that clusters with the Coq1 orthologs involved in the biosynthesis of ubiquinone in other eukaryotes.

The Plant Journal, 2012

It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA),... more It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA), a necessary step to form the naphthoquinone ring of phylloquinone (vitamin K 1 ). In fact, only recently has the hydrolysis of DHNA-CoA been demonstrated to be enzyme driven in vivo, and the cognate thioesterase characterized in the cyanobacterium Synechocystis. With a few exceptions in certain prokaryotic (Sorangium and Opitutus) and eukaryotic (Cyanidium, Cyanidioschyzon and Paulinella) organisms, orthologs of DHNA-CoA thioesterase are missing outside of the cyanobacterial lineage. In this study, genomic approaches and functional complementation experiments identified two Arabidopsis genes encoding functional DHNA-CoA thioesterases. The deduced plant proteins display low percentages of identity with cyanobacterial DHNA-CoA thioesterases, and do not even share the same catalytic motif. GFP-fusion experiments demonstrated that the Arabidopsis proteins are targeted to peroxisomes, and subcellular fractionations of Arabidopsis leaves confirmed that DHNA-CoA thioesterase activity occurs in this organelle. In vitro assays with various aromatic and aliphatic acyl-CoA thioester substrates showed that the recombinant Arabidopsis enzymes preferentially hydrolyze DHNA-CoA. Cognate T-DNA knock-down lines display reduced DHNA-CoA thioesterase activity and phylloquinone content, establishing in vivo evidence that the Arabidopsis enzymes are involved in phylloquinone biosynthesis. Extraordinarily, structure-based phylogenies coupled to comparative genomics demonstrate that plant DHNA-CoA thioesterases originate from a horizontal gene transfer with a bacterial species of the Lactobacillales order.

The Plant Cell, 2014

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. H... more It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent b-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch.

THE PLANT CELL ONLINE, 2009

Postendosymbiotic evolution has given rise to proteins that are multiply targeted within the cell... more Postendosymbiotic evolution has given rise to proteins that are multiply targeted within the cell. Various mechanisms have been identified to permit the expression of proteins encoding distinct N termini from a single gene. One mechanism involves alternative translation initiation (aTI). We previously showed evidence of aTI activity within the Arabidopsis thaliana organellar DNA polymerase gene POLg2. Translation initiates at four distinct sites within this gene, two non-AUG, to produce distinct plastid and mitochondrially targeted forms of the protein. To understand the regulation of aTI in higher plants, we used Polg2 as a model to investigate both cis-and trans-acting features of the process. Here, we show that aTI in Polg2 and other plant genes involves ribosome scanning dependent on sequence context at the multiple initiation sites to condition specific binding of at least one trans-acting factor essential for site recognition. Multiple active translation initiation sites appear to operate in several plant genes, often to expand protein targeting. In plants, where the mitochondrion and plastid must share a considerable portion of their proteomes and coordinate their functions, leaky ribosome scanning behavior provides adaptive advantage in the evolution of protein dual targeting and translational regulation.

THE PLANT CELL ONLINE, 2011

Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but meas... more Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but measurable demonstration of this intimate interaction is difficult. At the level of cellular metabolism, several biosynthetic pathways involve both mitochondrial-and plastid-localized steps. However, at an environmental response level, it is not clear how the two organelles intersect in programmed cellular responses. Here, we provide evidence, using genetic perturbation of the MutS Homolog1 (MSH1) nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. The mitochondrial form of the protein participates in DNA recombination surveillance, with disruption of the gene resulting in enhanced mitochondrial genome recombination at numerous repeated sequences. The plastid-localized form of the protein interacts with the plastid genome and influences genome stability and plastid development, with its disruption leading to variegation of the plant. These developmental changes include altered patterns of nuclear gene expression. Consistency of plastid and mitochondrial response across both monocot and dicot species indicate that the dual-functioning nature of MSH1 is well conserved. Variegated tissues show changes in redox status together with enhanced plant survival and reproduction under photooxidative light conditions, evidence that the plastid changes triggered in this study comprise an adaptive response to naturally occurring light stress.