assaying-proteasomal-degradation-in-a-cell-free-system-in-plants

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Arabidopsis yellow variegated1 (VAR1) and VAR2 are separate loci that encode similar chloroplast FtsH proteases. To date, FtsH is the best-characterized protease in thylakoid membranes involved in the turnover of photosynthetic protein complexes. It comprises a protein family that is encoded by 12 different nuclear genes in Arabidopsis. We show here that nine FtsH proteins are located in the chloroplasts. Mutations in either VAR1 or VAR2 cause typical leaf variegation and sensitivity to photoinhibition. By contrast, none of these phenotypes was observed in T-DNA insertion mutants in other ftsH genes (ftsh1, ftsh6, and ftsh8) closely related to VAR1 and VAR2. This finding suggests that VAR1 and VAR2 play a predominant role in the photosystem II repair cycle in thylakoid membranes. By generating VAR1- and VAR2-specific antibodies, we found that loss of either VAR1 or VAR2 results in the decreased accumulation of the other. Thus, the genetic nonredundancy between VAR1 and VAR2 could be attributed to their coordinated regulation at the protein level. These observations led us to examine whether VAR1 and VAR2 form a complex. Sucrose density gradient and gel filtration analyses revealed a complex of approximately 400 to 450 kD, probably representing a hexamer. Furthermore, VAR1 and VAR2 were shown to coprecipitate by immunoprecipitation using VAR1- and VAR2-specific antibodies. The majority of VAR1 appears to exist as heterocomplexes with VAR2, whereas VAR2 may be present as homocomplexes as well. Based on these results, we conclude that VAR1 and VAR2 are the major components of an FtsH complex involved in the repair of photodamaged proteins in thylakoid membranes.

Coordinated regulation and complex formation of yellow variegated1 and yellow variegated2, chloroplastic FtsH metalloproteases involved in the repair cycle of photosystem II in Arabidopsis thylakoid membranes.

The proteolytic machinery of chloroplasts and mitochondria in Arabidopsis consists primarily of three families of ATP-dependent proteases, Clp, Lon, and FtsH, and one family of ATP-independent proteases, DegP. However, the functional significance of the multiplicity of their genes is not clear. To test whether expression of specific isomers could be differently affected by growth conditions, we analyzed transcript abundance following short-term exposure to different environmental stimuli, using 70-mer oligonucleotide arrays. This analysis revealed variability in the response to high light and different temperatures within members of each family. Thirty out of the 41 tested genes were up-regulated in response to high light, including both chloroplast and mitochondrial isozymes, whereas only six and five genes responded to either high or low temperature, respectively. The extent of response was variable, ranging from 2- to 20-fold increase in the steady-state levels. Absolute transcript levels of the tested genes, compiled from one-channel arrays, were also variable. In general, transcripts encoding mitochondrial isozymes were accumulated to a lower level than chloroplastic ones. Within the FtsH family, transcript abundance of most genes correlated with the severity of mutant phenotypes in the relevant genes. This correlation was also evident at the protein level. Analysis of FtsH isozymes revealed that FtsH2 was the most abundant species, followed by FtsH5 and 8, with FtsH1 being accumulated to only 10% of FtsH2 level. These results suggest that, unlike previous expectations, the relative importance of different chloroplast protease isozymes, evidenced by mutant phenotypes at least in the FtsH family, is determined by their abundance, and not necessarily by different specific functions or specialized expression under certain conditions.

Expression in multigene families. Analysis of chloroplast and mitochondrial proteases.

The chloroplast ATP-dependent metalloprotease FtsH is involved in the degradation of unassembled proteins, the repair of photosystem II (PSII) from photoinhibition, and, apparently, the formation of thylakoids. In Arabidopsis, it is encoded by a family of 12 genes. However, the products of only four of them, FtsH1, 2, 5 and 8, have been found in chloroplasts to date. Mutations in two of these, FtsH2 and 5, demonstrate a visible phenotype of variegated leaves, with the phenotype of the FtsH2 mutant being more pronounced. Moreover, the degree of variegation appears to be dependent on developmental stage and environmental factors, suggesting an intricate relationship between the different gene products. To explore this, developmental and light effects on the accumulation of FtsH protease were studied in wild-type (WT) and FtsH2-mutant plants. Whereas cotyledons of the mutant were indistinguishable from those of the WT, the first true leaves were almost completely white. Subsequent leaves contained increasing proportions of green sectors. Analysis of the mRNA of the four FtsH genes, in cotyledons, first and second leaves of WT and mutant plants, revealed that: (i) transcript level increases during development, and (ii) transcript level in the mutant is higher than in the WT. FtsH protein level in the mutant was ca. 50% of that found in the WT, whereas the levels of other thylakoid proteins were the same. In individual leaves, the level of FtsH protein increased during development as well. Exposure of seedlings to different light intensities did not affect the degree of variegation, suggesting that it is due to a defect in chloroplast development rather than photobleaching. Examination of FtsH protein during exposure to high light revealed a decrease in its level, concomitant with a decrease in PSII potential, suggesting that the kinetics of photoinhibition reflects not only photodamage to PSII and induction of protective mechanisms, but also a decrease in repair capacity due to a reduction in the level of FtsH protease.

Developmental and light effects on the accumulation of FtsH protease in Arabidopsis chloroplasts--implications for thylakoid formation and photosystem II maintenance.

FtsH protease is important in chloroplast biogenesis and thylakoid maintenance. Although bacteria contain only one essential FTSH gene, multiple genes exist in cyanobacteria and higher plants. However, the functional significance of FTSH multiplication in plants is unclear. We hypothesized that some FTSH genes may be redundant. To test this hypothesis, we generated double mutant combinations among the different FTSH genes in Arabidopsis thaliana. A double mutant of ftsh1 and ftsh8 showed no obvious phenotypic alterations, and disruption of either FTSH1 or FTSH5 enhanced the phenotype of the ftsh2 mutant. Unexpectedly, new phenotypes were recovered from crosses between ftsh2 and ftsh8 and between ftsh5 and ftsh1, including albinism, heterotrophy, disruption of flowering, and severely reduced male fertility. These results suggest that the duplicated genes, FTSH1 and FTSH5 (subunit type A) and FTSH2 and FTSH8 (subunit type B), are redundant. Furthermore, they reveal that the presence of two types of subunits is essential for complex formation, photosystem II repair, and chloroplast biogenesis.

Two types of FtsH protease subunits are required for chloroplast biogenesis and Photosystem II repair in Arabidopsis.

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.

Pollen-specific pectin methylesterase involved in pollen tube growth.

Pollen Rohr Dehnung in der Stempel ist ein entscheidender Schritt in der geschlechtlichen Fortpflanzung der Pflanzen. Da die Wand der Pollen Rohr Spitze aus einer einzigen Schicht von Pektin besteht und, im Gegensatz zu den meisten anderen pflanzlichen Zellw&auml;nde, keine Zellulose oder Callose enth&auml;lt, Rolle Pektin Methylesterases (PMEs) wahrscheinlich eine zentrale im Pollen Rohr Wachstum und der Bestimmung der Pollen Rohr Morphologie. Funktionellen Untersuchungen der Bl&uuml;tenstaub-spezifische PMEs, die noch in den Kinderschuhen, sind daher wichtig f&uuml;r das Verst&auml;ndnis der Bl&uuml;tenstaub-Entwicklung. Wir bestimmt ein neues Arabidopsis Bl&uuml;tenstaub-spezifische PME, AtPPME1, charakterisiert seiner native Ausdrucksmuster und Reverser verwendet, um seine Beteiligung an der Festlegung der Form des Rohres Pollen und die Rate der seine Dehnung zu demonstrieren.

Bl&uuml;tenstaub-spezifische Pektin Methylesterase Pollen Rohr Wachstum beteiligt.

Sexual reproduction of flowering plants depends on delivery of the sperm to the egg, which occurs through a long, polarized projection of a pollen cell, called the pollen tube. The pollen tube grows exclusively at its tip, and this growth is distinguished by very fast rates and reaches extended lengths. Thus, one of the most fascinating aspects of pollen biology is the question of how enough cell wall material is produced to accommodate such rapid extension of pollen tube, and how the cell wall deposition and structure are regulated to allow for rapid changes in the direction of growth. This review discusses recent advances in our understanding of the mechanism of pollen tube growth, focusing on such basic cellular processes as control of cell shape and growth by a network of cell wall-modifying enzymes, molecular motor-mediated vesicular transport, and intracellular signaling by localized gradients of second messengers.

How pollen tubes grow.

Geschlechtliche Fortpflanzung der Bl&uuml;tenpflanzen richtet sich nach der Lieferung der Spermien in das Ei, das durch eine lange und polarisierenden Projektion einer Pollen-Zelle, die Pollen-R&ouml;hre genannt auftritt. Das Pollen-Rohr w&auml;chst ausschlie&szlig;lich an der Spitze, und dieses Wachstum zeichnet sich durch sehr schnelle Preise und erweiterte L&auml;ngen erreicht. Einer der faszinierendsten Aspekte der Bl&uuml;tenstaub Biologie deshalb die Frage, wie gen&uuml;gend Zellwand-Material produziert wird, um solche rasche Ausweitung der Pollen Rohr aufnehmen zu k&ouml;nnen, und wie die Zellwand Ablagerung und Struktur reguliert werden, um schnelle Ver&auml;nderungen in Richtung Wachstum zu erm&ouml;glichen. Diesen Bericht erl&auml;utert die j&uuml;ngsten Fortschritte im Verst&auml;ndnis des Mechanismus der Pollen Rohr Wachstum, Konzentration auf grundlegende zellul&auml;re Prozesse wie Zelle Form und Wachstum durch ein Netz von Zellwand-&auml;ndern Enzyme, molekulare Motor-vermittelte vesikul&auml;ren Transport und des intrazellul&auml;ren Signalisierens von lokalisierten Farbverl&auml;ufe der zweiten boten.

Wie Pollen Rohre wachsen.

Genetic transformation of plants by Agrobacterium, which in nature causes neoplastic growths, represents the only known case of trans-kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant and diverse species? The answer to this question lies in the ability of Agrobacterium to hijack fundamental cellular processes which are shared by most eukaryotic organisms. Our knowledge of these host cellular functions is critical for understanding the molecular mechanisms that underlie genetic transformation of eukaryotic cells. This review outlines the bacterial virulence machinery and provides a detailed discussion of seven major biological systems of the host cell-cell surface receptor arrays, cellular motors, nuclear import, chromatin targeting, targeted proteolysis, DNA repair, and plant immunity--thought to participate in the Agrobacterium-mediated genetic transformation.

Biological systems of the host cell involved in Agrobacterium infection.

Genetische Transformation von Pflanzen durch Agrobacterium, wodurch in der Natur neoplastische Wucherungen, stellt den einzigen bekannten Fall des Trans-K&ouml;nigreich DNA &Uuml;bertragung. Dar&uuml;ber hinaus kann unter Laborbedingungen Agrobacterium auch diverse andere eukaryontischer Arten, aus Pilzen, Seeigel auf menschliche Zellen umwandeln. Wie kann die Agrobacterium Virulenz Maschinerie in solch einer Vielzahl von evolution&auml;r weit entfernten und vielf&auml;ltige Arten funktionieren? Die Antwort auf diese Frage liegt in der F&auml;higkeit von Agrobacterium grundlegende zellul&auml;re Prozesse zu entf&uuml;hren, die von den meisten eukaryotischen Organismen geteilt werden. Unser Wissen &uuml;ber diese Host-Zellfunktionen ist entscheidend f&uuml;r das Verst&auml;ndnis der molekularen Mechanismen, die genetische Transformation der eukaryotischen Zellen zugrunde liegen. Diesen Bericht beschreibt die bakterielle Virulenz-Maschinerie und stellt eine ausf&uuml;hrliche Diskussion der sieben wichtigsten biologischen Systemen die Host-Zell-Zell-Oberfl&auml;che-Rezeptor-Arrays, zellul&auml;re Motoren, nukleare Import Zielen Chromatin, gezielten Proteolyse, DNA-Reparatur und Pflanze Immunit&auml;t--dachte zur Teilnahme an der Agrobakterium-vermittelte genetischen Transformations.

Biologische Systeme der Wirtszelle Agrobacterium Infektion beteiligt.

Nuclear proteins are involved in many critical biological processes within plant cells and, therefore, are in the focus of studies that usually begin with demonstrating that the protein of interest indeed exhibits nuclear localization. Thus, studies of plant nuclear proteins would be facilitated by a convenient experimental system for identification of proteins that are actively imported into the cell nucleus and visualization of their nuclear accumulation in vivo. To this end, we developed a system of vectors that allows screening for cDNAs coding for nuclear proteins in a simple genetic assay in yeast cells, and verification of nuclear accumulation in planta following one-step transfer and autofluorescent tagging of the identified clones into a multiple cloning site-compatible and reading frame-compatible plant expression vector. In a recommended third experimental step, the plant expression cassette containing the identified clone can be transferred, also by a one-step cloning, into a binary multigene expression vector for transient or stable coexpression with any other proteins.

Yeast-plant coupled vector system for identification of nuclear proteins.

Nukleare Proteine sind viele wichtige biologische Prozesse innerhalb Pflanzenzellen beteiligt und daher stehen im Mittelpunkt der Studien, die in der Regel beginnen mit Nachweis, dass das Protein des Interesses in der Tat nukleare Lokalisierung aufweist. Somit w&uuml;rden Studien &uuml;ber nukleare Pflanzeneiwei&szlig; durch eine komfortable experimentelle System zur Identifizierung von Proteinen, die aktiv in den Zellkern importiert werden und Visualisierung zu nuklearen Anh&auml;ufung in-vivo erleichtert werden. Zu diesem Zweck entwickelten wir ein System von Vektoren, Screening f&uuml;r cDNA Kodierung f&uuml;r nukleare Proteine in einer einfachen genetischen Probe in Hefezellen und Verifizierung von nuklearen Anreicherung im Planta nach einstufigen Transfer und Eigenfluoreszenz tagging der identifizierten Klone in einen mehrere Klonen Website-kompatible und lesen-Frame-kompatible Werk Ausdruck Vektor erm&ouml;glicht. In einem empfohlenen dritten experimentelle Schritt kann die Pflanze Ausdruck Kassette mit den identifizierten Klon, auch durch ein einstufiges, Klonen, in einen bin&auml;ren multigene Ausdruck Vektor f&uuml;r transiente oder stabile Coexpression mit jeder anderen Proteinen &uuml;bertragen werden.

Hefe-Anlage gekoppelt Vektor-System f&uuml;r die Identifizierung des nuklearen Proteine.

Transcriptional repression by histone modification represents a universal mechanism that underlies critical biological processes, such as neurogenesis and hematopoietic differentiation, in animals. In plants, however, the extent to which these regulatory pathways are involved in development and morphogenesis is not well defined. SWP1/LDL1 is a component of a plant corepressor complex that is involved in regulation of flower timing. Here, we report that SWP1 also plays a role in the regulation of root elongation by repressing a root-specific gene lateral root primordium 1 (LRP1) via histone deacetylation. A null mutation in SWP1 results in hyperacetylation of histones H3 and H4 in LRP1 chromatin, elevation of LRP1 expression, and increased root elongation. This effect of SWP1 knockout on the root phenotype is mimicked by transgenic expression of LRP1, which bypasses the SWP1-mediated suppression of the native gene. Thus, SWP1 likely functions as a regulator of developmental events both in the shoot and in the root meristem.

Regulation of root elongation by histone acetylation in Arabidopsis.

Transcriptional Repression von Histon-&Auml;nderung stellt einen universellen Mechanismus, der wichtige biologische Prozesse, wie Neurogenese und h&auml;matopoetischen Differenzierung, bei Tieren zugrunde liegt. In Pflanzen jedoch ist das Ausma&szlig;, zu dem diese Regulationsmechanismen in Entwicklung und Morphogenese eingebunden sind, nicht klar definiert. SWP1/LDL1 ist eine Komponente von einer Pflanze Corepressor komplexe, die in der Verordnung von Blume Timing beteiligt ist. Hier berichten wir, dass SWP1 auch eine Rolle bei der Regulierung der Wurzel Dehnung spielt durch ein Wurzel-spezifische gen seitliche Wurzel Urzeit 1 (LRP1) &uuml;ber Histon-Deacetylierung verdr&auml;ngen. Eine null-Mutation in SWP1 f&uuml;hrt Hyperacetylation der Histone H3 und H4 in LRP1 Chromatin, Erh&ouml;hung von LRP1 Ausdruck und erh&ouml;hte Stamm Dehnung. Dieser Effekt der SWP1 k.o. auf der Stamm-Ph&auml;notyp ist durch transgene Ausdruck des LRP1, nachgeahmt, die die SWP1-vermittelte Unterdr&uuml;ckung des native-Gens umgeht. So funktioniert SWP1 wahrscheinlich als ein Regulator der entwicklungspolitischen Veranstaltungen sowohl in die Dreharbeiten der Wurzel Meristem.

Verordnung der Wurzel Dehnung von Histon-Acetylierung in Arabidopsis.

The ubiquitin/26S proteasome pathway is a basic biological mechanism involved in the regulation of a multitude of cellular processes. Increasing evidence indicates that plants utilize the ubiquitin/26S proteasome pathway in their immune response to pathogen invasion, emphasizing the role of this pathway during plant-pathogen interactions. The specific functions of proteasomal degradation in plant-pathogen interactions are diverse, and do not always benefit the host plant. Although in some cases, proteasomal degradation serves as an effective barrier to help plants ward off pathogens, in others, it is used by the pathogen to enhance the infection process. This review discusses the different roles of the ubiquitin/26S proteasome pathway during interactions of plants with pathogenic viruses, bacteria, and fungi.

Proteasomal degradation in plant-pathogen interactions.

Die Ubiquitin/26S Proteasom-Pathway ist eine grundlegende biologische Mechanismus der Regulierung einer Vielzahl zellul&auml;rer Prozesse beteiligt. Zunehmende Beweise gibt, dass Pflanzen die Ubiquitin/26S Proteasom-Pathway in ihre Immunantwort auf Krankheitserreger Invasion, betonen die Rolle von diesem Weg w&auml;hrend Pflanze-Pathogen Interaktionen nutzen. Die spezifischen Funktionen des Proteasomal Abbaus in Pflanze-Pathogen Interaktionen sind vielf&auml;ltig und nicht immer die Gastgeber-Pflanze profitieren. Obwohl in einigen F&auml;llen Proteasomal Abbau als effektiver Schutz dient, Pflanzen Ward aus Krankheitserreger, in anderen zu helfen dient sie zur Verbesserung des Infektion-Prozess von der Erreger. Diesen Bericht erl&auml;utert die verschiedenen Rollen des Ubiquitin/26S Proteasom Signalweges w&auml;hrend Interaktionen von Pflanzen mit pathogene Viren, Bakterien und Pilze.

Proteasomal-Abbau in Pflanze-Pathogen Interaktionen.

Agrobacterium exports DNA into plant cells, eliciting neoplastic growths on many plant species. During this process, a Skp1-Cdc53-cullin-F-box (SCF) complex that contains the bacterial virulence F-box protein VirF facilitates genetic transformation by targeting for proteolysis proteins, the Agrobacterium protein VirE2 and the host protein VIP1, that coat the transferred DNA. However, some plant species do not require VirF for transformation. Here, we show that Agrobacterium induces expression of a plant F-box protein, which we designated VBF for VIP1-binding F-box protein, that can functionally replace VirF, regulating levels of the VirE2 and VIP1 proteins via a VBF-containing SCF complex. When expressed in Agrobacterium and exported into the plant cell, VBF functionally complements tumor formation by a strain lacking VirF. VBF expression is known to be induced by diverse pathogens, suggesting that Agrobacterium has co-opted a plant defense response and that bacterial VirF and plant VBF both contribute to targeted proteolysis that promotes plant genetic transformation.

Agrobacterium induces expression of a host F-box protein required for tumorigenicity.

Agrobacterium exportiert DNA in Pflanzenzellen, technologieorientierter neoplastische Wucherungen auf viele Pflanzenarten. W&auml;hrend dieses Prozesses, ein Skp1-Cdc53-Michel cullin-F-Box (SCF)-Komplex, der die bakterielle Virulenz F-Box Protein VirF genetische Transformation erleichtert enth&auml;lt durch targeting f&uuml;r Proteolyse Proteine, das Agrobakterium Protein VirE2 und das Host-Protein VIP1, Mantel, die die &uuml;bertragene DNA. Jedoch erfordern einige Pflanzenarten keine VirF f&uuml;r Transformation. Hier zeigen wir, dass Agrobacterium Ausdruck einer Pflanze F-Box-Protein induziert, die wir VBF f&uuml;r VIP1-Bindung F-Box Protein, benannt, die funktionell VirF, Regulierung der Ebenen der Proteine VirE2 und VIP1 &uuml;ber eine komplexe VBF-haltigen SCF ersetzen k&ouml;nnen. Wenn in Agrobacterium ausgedr&uuml;ckt und in die Pflanzenzelle exportiert, erg&auml;nzt VBF funktional Tumorbildung durch einen Stamm fehlt VirF. VBF Ausdruck bekannterma&szlig;en werden durch unterschiedliche Krankheitserreger, darauf hindeutet, dass Agrobacterium eine Pflanze-Abwehr-Reaktion und die bakterielle VirF kooptiert hat induziert und Anlagenbau VBF sowohl zur gezielten Proteolyse beitragen, die genetische Transformation der Pflanze f&ouml;rdert.

Agrobacterium induziert Ausdruck eines Hosts F-Box Protein f&uuml;r Tumorigenicity erforderlich.

Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this review we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications, ranging from generation of commercial crops with valuable new phenotypes to 'bioreactor' plants for large-scale production of recombinant proteins to research model plants expressing various reporter proteins.

Nuclear and plastid genetic engineering of plants: comparison of opportunities and challenges.

The soil phytopathogen Agrobacterium has the unique ability to introduce single-stranded transferred DNA (T-DNA) from its tumor-inducing (Ti) plasmid into the host cell in a process known as horizontal gene transfer. Following its entry into the host cell cytoplasm, the T-DNA associates with the bacterial virulence (Vir) E2 protein, also exported from Agrobacterium, creating the T-DNA nucleoprotein complex (T-complex), which is then translocated into the nucleus where the DNA is integrated into the host chromatin. VirE2 protects the T-DNA from the host DNase activities, packages it into a helical filament, and interacts with the host proteins, one of which, VIP1, facilitates nuclear import of the T-complex and its subsequent targeting to the host chromatin. Although the VirE2 and VIP1 protein components of the T-complex are vital for its intracellular transport, they must be removed to expose the T-DNA for integration. Our recent work demonstrated that this task is aided by an host defense-related F-box protein VBF that is induced by Agrobacterium infection and that recognizes and binds VIP1. VBF destabilizes VirE2 and VIP1 in yeast and plant cells, presumably via SCF-mediated proteasomal degradation. VBF expression in and export from the Agrobacterium cell lead to increased tumorigenesis. Here, we discuss these findings in the context of the "arms race" between Agrobacterium infectivity and plant defense.

Plant defense pathways subverted by Agrobacterium for genetic transformation.

Die Boden-Phytopathogen Agrobacterium hat die einzigartige F&auml;higkeit, einzelstr&auml;ngige &uuml;bertragenen DNA (T-DNA) von seiner Tumor-induzierende (Ti)-Plasmid in der Wirtszelle in einem Proze&szlig; bekannt als Horizontaler Gentransfer einzuf&uuml;hren. Nach seinem Inkrafttreten Host Zelle Zytoplasma ordnet die T-DNA der bakteriellen Virulenz (Vir) E2 Protein, ebenfalls aus Agrobacterium, erstellen die T-DNA-Nukleoprotein komplexe exportiert (T-Komplex), das dann in den Zellkern transloziert ist wo die DNA in die Host-Chromatin integriert ist. VirE2 sch&uuml;tzt die T-DNA aus dem Host DNase, packt es in eine spiralf&ouml;rmige Filament und interagiert mit der Host-Proteine, eine der erleichtert, VIP1, die nukleare Import von der T-komplex und seine anschlie&szlig;ende targeting an das Host-Chromatin. Obwohl die VirE2 und VIP1-Protein-Komponenten der T-Komplex f&uuml;r den intrazellul&auml;ren Transport von entscheidender Bedeutung sind, m&uuml;ssen sie entfernt werden, um die T-DNA f&uuml;r die Integration verf&uuml;gbar zu machen. Unsere j&uuml;ngsten Arbeiten gezeigt, dass diese Aufgabe durch einen Host im Zusammenhang mit der Verteidigung F-Box Protein VBF unterst&uuml;tzt wird, wird durch Agrobacterium Infektion induziert und das erkennt und bindet VIP1. VBF destabilisiert VirE2 und VIP1 in Hefe und pflanzlichen Zellen, vermutlich &uuml;ber Verschlechterung der SCF-vermittelte Proteasomal. VBF Ausdruck im und Export aus der Agrobakterium-Zelle f&uuml;hren zu erh&ouml;hten Tumorentstehung. Hier diskutieren wir diese Erkenntnisse im Zusammenhang mit dem &quot;Wettr&uuml;sten&quot; zwischen Agrobacterium Infektiosit&auml;t und Verteidigung der Pflanze.

Pflanzen Sie Verteidigung Wege Massenverhaftung von Agrobacterium f&uuml;r genetische Transformation.

Covalent modifications of histones, such as acetylation, methylation and ubiquitination, are central for regulation of gene expression. Heterochromatic gene silencing, for example, is associated with hypoacetylation, methylation and demethylation, and deubiquitination of specific amino acid residues in histone molecules. Many of these changes can be effected by histone-modifying repressor complexes that include histone lysine demethylases, such as KDM1 in animals and KDM1C in plants. However, whereas KDM1-containing repressor complexes have been implicated in histone demethylation, methylation and deacetylation, whether or not they can also mediate histone deubiquitination remains unknown. We identify an Arabidopsis otubain-like deubiquitinase OTLD1 which directly interacts with the Arabidopsis KDM1C in planta, and use one target gene to exemplify that both OTLD1 and KDM1C are involved in transcriptional gene repression via histone deubiquitination and demethylation. We also show that OTLD1 binds plant chromatin and has enzymatic histone deubiquitinase activity, specific for the H2B histone. Thus, we suggest that, during gene repression, lysine demethylases can directly interact and function in a protein complex with histone deubiquitinases.

Involvement of KDM1C histone demethylase-OTLD1 otubain-like histone deubiquitinase complexes in plant gene repression.

Kovalente Modifikationen der Histone, wie Acetylierung, Methylierung und Ubiquitination, sind von zentraler Bedeutung f&uuml;r die Regulierung der Genexpression. Heterochromatic gen-silencing, z. B. ist Hypoacetylation, Methylierung und Demethylierung und Deubiquitination bestimmten Aminos&auml;ure R&uuml;ckst&auml;nde in Histon-Molek&uuml;le zugeordnet. Viele dieser &Auml;nderungen k&ouml;nnen erfolgen durch &Auml;ndern von Histon-Verdr&auml;nger-komplexe, die Histon-Lysin-Demethylasen, wie KDM1 bei Tieren und KDM1C in Pflanzen enthalten. Doch w&auml;hrend KDM1-haltigen Verdr&auml;nger-komplexe in Histon-Demethylierung, Methylierung und Deacetylierung verwickelt haben, k&ouml;nnen ob sie auch Histon Deubiquitination bleibt unbekannt schl&uuml;pfen. Wir identifizieren ein Arabidopsis Otubain-wie Deubiquitinase OTLD1 die direkt interagiert mit der Arabidopsis KDM1C in Planta, und verwenden Sie ein Ziel-gen um zu veranschaulichen, die sowohl OTLD1 als auch KDM1C transkriptionelle gen Unterdr&uuml;ckung &uuml;ber Histon Deubiquitination und Demethylierung beteiligt sind. Wir zeigen auch, dass OTLD1 Pflanze Chromatin bindet und enzymatische Histon Deubiquitinase Aktivit&auml;t, hat speziell f&uuml;r die H2B Histon. Daher empfehlen wir, w&auml;hrend gen Unterdr&uuml;ckung, Lysin Demethylasen direkt interagieren und Funktion in einem Protein Komplex mit Histon-Deubiquitinases.

Beteiligung der KDM1C Histon Demethylase-OTLD1 Otubain-artigen Histon Deubiquitinase komplexe Anlage gen Unterdr&uuml;ckung.

Plant genetic engineering emerged as a methodology to introduce only few transgenes into the plant genome. Following fast-paced developments of the past few decades, engineering of much larger numbers of transgenes became a reality, allowing to introduce full metabolic pathways from other organisms into plants and generate transgenics with startling new traits. From the advent of the classical plant genetic engineering, the transgenes were introduced into the nuclear genome of the plant cell, and this strategy still is quite successful when applied to few transgenes. However, for introducing large number of transgenes, we advocate that the chloroplast genome is a superior choice, especially for engineering of new complete metabolic pathways into plants. The ability to genetically engineer plants with complex and fully functional metabolic pathways from other organisms bears a substantial promise in generation of pharmaceuticals, i.e., biopharming, and new agricultural crops with that traits never existed before, leading to enhancement in quality of human life.

Expression of complete metabolic pathways in transgenic plants.

One the most intriguing, yet least studied, aspects of the bacterium-host plant interaction is the role of the host ubiquitin/proteasome system (UPS) in the infection process. Increasing evidence indicates that pathogenic bacteria subvert the host UPS to facilitate infection. Although both mammalian and plant bacterial pathogens are known to use the host UPS, the first prokaryotic F-box protein, an essential component of UPS, was identified in Agrobacterium. During its infection, which culminates in genetic modification of the host cell, Agrobacterium transfers its T-DNA--as a complex (T-complex) with the bacterial VirE2 and host VIP1 proteins--into the host cell nucleus. There the T-DNA is uncoated from its protein components before undergoing integration into the host genome. It has been suggested that the host UPS mediates this uncoating process, but there is no evidence indicating that this activity can unmask the T-DNA molecule. Here we provide support for the idea that the plant UPS uncoats synthetic T-complexes via the Skp1/Cullin/F-box protein VBF pathway and exposes the T-DNA molecule to external enzymatic activity.

Article	Year
Coordinated regulation and complex formation of yellow variegated1 and yellow variegated2, chloroplastic FtsH metalloproteases involved in the repair cycle of photosystem II in Arabidopsis t... The Plant cell\| PubMed ID: 14630971	2003
Expression in multigene families. Analysis of chloroplast and mitochondrial proteases. Plant physiology\| PubMed ID: 15266057	2004
Developmental and light effects on the accumulation of FtsH protease in Arabidopsis chloroplasts--implications for thylakoid formation and photosystem II maintenance. The Plant journal : for cell and molecular biology\| PubMed ID: 15918877	2005
Two types of FtsH protease subunits are required for chloroplast biogenesis and Photosystem II repair in Arabidopsis. The Plant cell\| PubMed ID: 16126834	2005
Pollen-specific pectin methylesterase involved in pollen tube growth. Developmental biology\| PubMed ID: 16564517	2006
How pollen tubes grow. Developmental biology\| PubMed ID: 17214979	2007
Biological systems of the host cell involved in Agrobacterium infection. Cellular microbiology\| PubMed ID: 17222189	2007
Yeast-plant coupled vector system for identification of nuclear proteins. Plant physiology\| PubMed ID: 17704231	2007
Regulation of root elongation by histone acetylation in Arabidopsis. Journal of molecular biology\| PubMed ID: 18835563	2009
Proteasomal degradation in plant-pathogen interactions. Seminars in cell & developmental biology\| PubMed ID: 19505586	2009
Agrobacterium induces expression of a host F-box protein required for tumorigenicity. Cell host & microbe\| PubMed ID: 20227663	2010
Nuclear and plastid genetic engineering of plants: comparison of opportunities and challenges. Biotechnology advances\| PubMed ID: 20685387	2010
Plant defense pathways subverted by Agrobacterium for genetic transformation. Plant signaling & behavior\| PubMed ID: 20890133	2010
Involvement of KDM1C histone demethylase-OTLD1 otubain-like histone deubiquitinase complexes in plant gene repression. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21690391	2011
Expression of complete metabolic pathways in transgenic plants. Biotechnology & genetic engineering reviews\| PubMed ID: 22616478	2012
Disassembly of synthetic Agrobacterium T-DNA-protein complexes via the host SCF(VBF) ubiquitin-ligase complex pathway. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23248273	2013

Adi Zaltsman

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