Bruce Kohorn
Duke University, Biology, Faculty Member
Research Interests: Genetics, Plant Biology, Biology, Plant Cell Walls, Membrane Proteins, and 13 moreCell Biology, Plant Molecular Biology, Extracellular Matrix, Protein Kinases, Arabidopsis, Kinase, Substrate Specificity, Pectins, Cell Wall, Protein Binding, Cytoplasm, Biochemistry and cell biology, and Springer Ebooks
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Research Interests: Biology, Cell Adhesion, Cell Biology, Medicine, Adhesion, and 4 morePlants, Microtubule Dynamics, Pectin, and Cell Wall
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Research Interests: Biology, Cell Biology, Medicine, Cell Division, Signal Transduction, and 13 moreBiological Sciences, Protein-Protein Interaction, Cell Signalling, Signalling, Arabidopsis, Mode of action, Protein Kinase, Cell communication, PLANT PROTEINS, Plasma Membrane, Meristem, Cell Membrane, and Medical and Health Sciences
Plant growth, morphogenesis and development involves cellular adhesion, a process dependent on the composition and structure of the extracellular matrix (ECM) or cell wall. Pectin in the cell wall is thought to play an essential role in... more
Plant growth, morphogenesis and development involves cellular adhesion, a process dependent on the composition and structure of the extracellular matrix (ECM) or cell wall. Pectin in the cell wall is thought to play an essential role in adhesion, and its modification and cleavage are suggested to be highly regulated so as to change adhesive properties. To increase our understanding of plant cell adhesion a population of EMS mutagenized Arabidopsis were screened for hypocotyl adhesion defects using the pectin binding dye Ruthenium Red that penetrates defective but not WT hypocotyl cell walls. Genomic sequencing was used to identify a mutant allele of ELMO1 which encodes a 20 kDa Golgi membrane protein that has no predicted enzymatic domains. ELMO1 colocalizes with several Golgi markers and elmo1−/- plants can be rescued by an ELMO1-GFP fusion. elmo1−/- exhibits reduced mannose content relative to WT but no other cell wall changes and can be rescued to WT phenotype by mutants in ESMER...
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The cellulose- and pectin-rich plant cell wall defines cell structure, mediates defense against pathogens, and facilitates plant cell adhesion. An adhesion mutant screen of Arabidopsis hypocotyls identified a new allele of QUASIMODO2... more
The cellulose- and pectin-rich plant cell wall defines cell structure, mediates defense against pathogens, and facilitates plant cell adhesion. An adhesion mutant screen of Arabidopsis hypocotyls identified a new allele of QUASIMODO2 (QUA2), a gene required for pectin accumulation and whose mutants have reduced pectin content and adhesion defects. A suppressor of qua2 was also isolated and describes a null allele of SABRE (SAB), which encodes a previously described plasma membrane protein required for longitudinal cellular expansion that organizes the tubulin cytoskeleton. sab mutants have increased pectin content, increased levels of expression of pectin methylesterases and extensins, and reduced cell surface area relative to qua2 and Wild Type, contributing to a restoration of cell adhesion.
Research Interests: Biology, Cell Adhesion, Cell Biology, Medicine, Adhesion, and 4 morePlants, Microtubule Dynamics, Pectin, and Cell Wall
Mutations within the signal sequence of cytochrome f (cytf) in Chlamydomonas inhibit thylakoid membrane protein translocation and render cells nonphotosynthetic. Twenty-seven suppressors of the mutant signal sequences were selected for... more
Mutations within the signal sequence of cytochrome f (cytf) in Chlamydomonas inhibit thylakoid membrane protein translocation and render cells nonphotosynthetic. Twenty-seven suppressors of the mutant signal sequences were selected for their ability to restore photoautotrophic growth and these describe six nuclear loci named tip1 through 6 for thylakoid insertion protein. The tip mutations restore the translocation of cytf and are not allele specific, as they suppress a number of different cytf signal sequence mutations. Tip5 and 2 may act early in cytf translocation, while Tip1, 3, 4, and 6 are engaged later. The tip mutations have no phenotype in the absence of a signal sequence mutation and there is genetic interaction between tip4, and tip5 suggesting an interaction of their encoded proteins. As there is overlap in the energetic, biochemical and genetic requirements for the translocation of nuclear and chloroplast-encoded thylakoid proteins, the tip mutations likely identify com...
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Angiosperm cell adhesion is dependent on interactions between pectin polysaccharides which make up a significant portion of the plant cell wall. Cell adhesion in Arabidopsis may also be regulated through a pectin-related signaling cascade... more
Angiosperm cell adhesion is dependent on interactions between pectin polysaccharides which make up a significant portion of the plant cell wall. Cell adhesion in Arabidopsis may also be regulated through a pectin-related signaling cascade mediated by a putative O-fucosyltransferase ESMERALDA1 (ESMD1), and the Epidermal Growth Factor (EGF) domains of the pectin binding Wall associated Kinases (WAKs) are a primary candidate substrate for ESMD1 activity. Genetic interactions between WAKs and ESMD1 were examined using a dominant hyperactive allele of WAK2, WAK2cTAP, and a mutant of the putative O-fucosyltransferase ESMD1. WAK2cTAP expression results in a dwarf phenotype and activation of the stress response and reactive oxygen species (ROS) production, while esmd1 is a suppressor of a pectin deficiency induced loss of adhesion. Here we find that esmd1 suppresses the WAK2cTAP dwarf and stress response phenotype, including ROS accumulation and gene expression. Additional analysis suggests...
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The Wall Associated Kinases (WAKs) bind to both cross-linked polymers of pectin in the plant cell wall, but have a higher affinity for smaller fragmented pectins that are generated upon pathogen attack or wounding. WAKs are required for... more
The Wall Associated Kinases (WAKs) bind to both cross-linked polymers of pectin in the plant cell wall, but have a higher affinity for smaller fragmented pectins that are generated upon pathogen attack or wounding. WAKs are required for cell expansion during normal seedling development and this involves pectin binding and a signal transduction pathway involving MPK3 and invertase induction. Alternatively WAKs bind pathogen generated pectin fragments to activate a distinct MPK6 dependent stress response. Evidence is provided for a model for how newly generated pectin fragments compete for longer pectins to alter the WAK dependent responses.
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Abstract A method for the isolation of cDNAs that encode proteases of known amino acid specificity is presented. The insertion of a cleavable sequence within a functional protein allows the protease to destroy that protein when both are... more
Abstract A method for the isolation of cDNAs that encode proteases of known amino acid specificity is presented. The insertion of a cleavable sequence within a functional protein allows the protease to destroy that protein when both are expressed in Saccharomyces cerevisiae . The yeast transcriptional activator Gal4p, which induces the expression of enzymes required to metabolize galactose, can serve as the cleavable protein since loss of Gal4p activity permits growth of cells on the suicide substrate 2-de-oxygalactose. The insertion of a variety of protease target sites into a region between the transcriptional activation and the DNA-binding domains of Gal4p is permissive for transcriptional activity and, therefore, induction of galactose metabolism. A cDNA library from cells expressing the desired proteolytic activity is introduced into yeast cells expressing the Gal4-target fusion protein. Cells surviving on 2-deoxygalactose should express a cDNA encoding a protease specific to the target. The assay is also useful for the genetic analysis and identification of inhibitors of proteases whose genes have already been cloned.
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ABSTRACT The signal hypothesis, first put forth by G. Blobel and B. Dobberstein over 20 years ago, suggested that all proteins had as part of their primary amino acid sequence an amino-terminal region that defined the destination of... more
ABSTRACT The signal hypothesis, first put forth by G. Blobel and B. Dobberstein over 20 years ago, suggested that all proteins had as part of their primary amino acid sequence an amino-terminal region that defined the destination of proteins carrying that signal. This hypothesis has stood the test of numerous experiments and appears to be pertinent to most cellular compartments and proteins within eukaryotes and prokaryotes, including the chloroplast. Studies with Chlamydomonas reinhardtii, the subject of this chapter, have tested these concepts in live cells and have provided both new findings and the confirmation of in vitro results derived from vascular plants. Proteins synthesized outside the plastid carry chloroplast specific signals, often at their amino terminus. This signal can be removed upon entry of the protein into the chloroplast, but those destined for internal compartments within the plastid also contain additional signals. This sub-organelle targeting information can be removed once the protein is correctly localized. Thus chloroplast sorting is determined by multifunctional signal sequences. Experiments with isolated organelles have defined a number of different energetic parameters that are required for the translocation of proteins with distinct types of signal sequences, and models suggest that multiple mechanisms exist within the chloroplast. C. reinhardtii can be grown either heterotrophically or photoautotrophically thereby allowing for the selection and propagation of mutations that affect the biogenesis of chloroplasts, and thus some that affect protein translocation directly. The genetic analysis of thylakoid protein translocation in C. reinhardtii has revealed at least six loci whose products are involved in the process, and has provided genetic means to dissect those paths in vivo that have been described in isolated organelle studies. While there are likely to be a variety of requirements for the targeting and translocation of proteins to and within the chloroplast, these mechanisms may well share components between apparatuses, and between diverse groups of organisms.
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The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling... more
The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional respo...
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Research Interests: Genetics, Plant Biology, Biology, Plant Cell Walls, Membrane Proteins, and 15 moreCell Biology, Plant Molecular Biology, Extracellular Matrix, Arabidopsis, Kinase, Pectins, Protein Kinase, Plant Cell Wall, Cell Wall, Protein Binding, Cytoplasm, Plasma Membrane, Cell Expansion, Biochemistry and cell biology, and Cell Growth
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Research Interests: Biology, Cell Biology, Medicine, Cell Division, Signal Transduction, and 13 moreBiological Sciences, Protein-Protein Interaction, Cell Signalling, Signalling, Arabidopsis, Mode of action, Protein Kinase, Cell communication, PLANT PROTEINS, Plasma Membrane, Meristem, Cell Membrane, and Medical and Health Sciences
SummaryThe wall‐associated kinases (WAK), a family of five proteins that contain extracellular domains that can be linked to pectin molecules of the cell wall, span the plasma membrane and have a cytoplasmic serine/threonine kinase... more
SummaryThe wall‐associated kinases (WAK), a family of five proteins that contain extracellular domains that can be linked to pectin molecules of the cell wall, span the plasma membrane and have a cytoplasmic serine/threonine kinase domain. Previous work has shown that a reduction in WAK protein levels leads to a loss of cell expansion, indicating that these receptor‐like proteins have a role in cell shape formation. Here it is shown that a single wak2 mutation exhibits a dependence on sugars and salts for seedling growth. This mutation also reduces the expression and activity of vacuolar invertase, often a key factor in turgor and expansion. WAKs may thus provide a molecular mechanism linking cell wall sensing (via pectin attachment) to regulation of solute metabolism, which in turn is known to be involved in turgor maintenance in growing cells.
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SummaryThe angiosperm extracellular matrix, or cell wall, is composed of a complex array of cellulose, hemicelluose, pectins and proteins, the modification and regulated synthesis of which are essential for cell growth and division. The... more
SummaryThe angiosperm extracellular matrix, or cell wall, is composed of a complex array of cellulose, hemicelluose, pectins and proteins, the modification and regulated synthesis of which are essential for cell growth and division. The wall associated kinases (WAKs) are receptor‐like proteins that have an extracellular domain that bind pectins, the more flexible portion of the extracellular matrix, and are required for cell expansion as they have a role in regulating cellular solute concentrations. We show here that both recombinant WAK1 and WAK2 bind pectin in vitro. In protoplasts pectins activate, in a WAK2‐dependent fashion, the transcription of vacuolar invertase, and a wak2 mutant alters the normal pectin regulation of mitogen‐activated protein kinases. Microarray analysis shows that WAK2 is required for the pectin activation of numerous genes in protoplasts, many of which are involved in cell wall biogenesis. Thus, WAK2 plays a major role in signaling a diverse array of cell...
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SummaryPathogen infection of angiosperms must rely on some interaction between the extracellular matrix (ECM) and the invading agent, and may be accompanied by signaling between the ECM and cytoplasm. An Arabidopsis cell wall associated... more
SummaryPathogen infection of angiosperms must rely on some interaction between the extracellular matrix (ECM) and the invading agent, and may be accompanied by signaling between the ECM and cytoplasm. An Arabidopsis cell wall associated receptor kinase (Wak1) has an amino‐terminal domain that is tightly associated with the ECM, spans the plasma membrane and has a cytoplasmic protein kinase domain. Wak1 expression is induced when Arabidopsis plants are infected with pathogen, or when the pathogen response is stimulated either by exogenous salicylate (SA) or its analog 2,2‐dichloroisonicotinic acid (INA). This Wak1 induction requires the positive regulator NPR1/NIM1. Thus Wak1 is a pathogen‐related (PR) protein. Expression of an antisense and a dominant negative allele of Wak1 shows that induced expression of Wak1 is needed for a plant to survive if stimulated by INA. Ectopic expression of the entire Wak1, or the kinase domain alone, can provide resistance to otherwise lethal SA level...
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We used BAL-31 nuclease to delete sequences that surround the transcription initiation site of Drosophila ribosomal DNA. A series of deletions was used as templates for in vitro transcription in a Drosophila cell-free system to identify... more
We used BAL-31 nuclease to delete sequences that surround the transcription initiation site of Drosophila ribosomal DNA. A series of deletions was used as templates for in vitro transcription in a Drosophila cell-free system to identify sequences that influence the activity of RNA polymerase I. Sequences that lie upstream of the site of transcription initiation (nucleotide + 1) affect ribosomal RNA synthesis. We show that the major promoter of polymerase I involves the sequence -43 to -27 and that the region between nucleotides -18 and +20 contains sequences capable of sustaining a low level of accurate transcription.
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Research Interests: Genetics, Plant Biology, Biology, Transmission Electron Microscopy, Plant Molecular Biology, and 15 moreArabidopsis thaliana, Mutation, Chlorophyll, Plants, Arabidopsis, Ultrastructure, Photosystem II, Membrane Protein, Chloroplast, Plant Remains, Light Harvesting, Protein Biosynthesis, Biochemistry and cell biology, Chloroplasts, and photosystem
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The Arabidopsis thaliana wall-associated kinases (WAKs) bind to pectin with an extracellular domain and also contain a cytoplasmic protein kinase domain. WAKs are required for cell elongation and modulate sugar metabolism. This work shows... more
The Arabidopsis thaliana wall-associated kinases (WAKs) bind to pectin with an extracellular domain and also contain a cytoplasmic protein kinase domain. WAKs are required for cell elongation and modulate sugar metabolism. This work shows that in leaf protoplasts a WAK1-GFP fusion protein accumulates in a cytoplasmic compartment that contains pectin. The WAK compartment contains markers for the Golgi, the site of pectin synthesis. The migration of WAK1-GFP to the cell surface is far slower than that of a cell surface receptor not associated with the cell wall, is influenced by the presence of fucose side chains on one or more unidentified molecules that might include pectin, and is dependent upon cellulose synthesis on the plasma membrane. WAK is crosslinked into a detergent-insoluble complex within the cytoplasmic compartment before it appears on the cell surface, and this is independent of fucose modification or cellulose synthesis. Thus, the assembly and crosslinking of WAKs may ...
Research Interests: Biochemistry, Biology, Membrane Proteins, Cell Biology, Medicine, and 15 moreCellulose, Biological Sciences, Protein Kinases, Cell, Arabidopsis, Biological markers, Pectin, Pectins, Cell Wall, Golgi Apparatus, Cytoplasm, Extracellular, Protein Transport, Cell Membrane, and Medical and Health Sciences
Research Interests: Biology, Membrane Proteins, Immunohistochemistry, Cell Biology, Biological Chemistry, and 15 moreMedicine, Biological Sciences, Protein Kinases, Arabidopsis, CHEMICAL SCIENCES, Molecular cloning, Protein Kinase, Species Specificity, Cell Wall, Chlamydomonas Reinhardtii, Cytoplasm, PLANT PROTEINS, Extracellular, Tissue distribution, and Medical and Health Sciences
WAK1 (wall-associated kinase 1) is a cytoplasmic serine/threonine kinase that spans the plasma membrane and extends into the extracellular region to bind tightly to the cell wall. The Wak1 gene was mapped and found to lie in a tight... more
WAK1 (wall-associated kinase 1) is a cytoplasmic serine/threonine kinase that spans the plasma membrane and extends into the extracellular region to bind tightly to the cell wall. The Wak1 gene was mapped and found to lie in a tight cluster of five highly similar genes (Wak1–5) within a 30 kb region. All of the Wak genes encode a cytoplasmic serine/threonine protein
Research Interests: Genetics, Plant Biology, Biology, Membrane Proteins, Cell Biology, and 15 morePlant Molecular Biology, Medicine, Gene expression, Arabidopsis thaliana, Gene Dosage, Arabidopsis, Introns, Isoenzymes, Amino Acid Sequence, Cell Wall, Growth Factor, Chromosomes, Plasma Membrane, Biochemistry and cell biology, and Molecular Sequence Data
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The wall-associated kinases, WAKs, are encoded by five highly similar genes clustered in a 30-kb locus in Arabidopsis. These receptor-like proteins contain a cytoplasmic serine threonine kinase, a transmembrane domain, and a less... more
The wall-associated kinases, WAKs, are encoded by five highly similar genes clustered in a 30-kb locus in Arabidopsis. These receptor-like proteins contain a cytoplasmic serine threonine kinase, a transmembrane domain, and a less conserved region that is bound to the cell wall and contains a series of epidermal growth factor repeats. Evidence is emerging that WAKs serve as pectin receptors, for both short oligogalacturonic acid fragments generated during pathogen exposure or wounding, and for longer pectins resident in native cell walls. This ability to bind and respond to several types of pectins correlates with a demonstrated role for WAKs in both the pathogen response and cell expansion during plant development.