Wir danken Dr. Patricia Manosalva für ihre wertvolle Einblicke in das Manuskript. Die Finanzierung wurde von der National Science Foundation Pflanzengenomforschung Programm, Vergabe-Nummer DBI-0605059 zur Verfügung gestellt.

Teil 1: Pflanzenmaterial N. benthamiana Pflanzen zum Schweigen verwendet werden, sollten ca. 2 ½ Wochen alt, die ist die Zeit, wenn die Keimblätter und die ersten 2 bis 4 Laubblätter sind entstanden. 8 Tage Nachauflauf, wenn die Laubblätter noch nicht erschienen - Tomaten (Solanum lycopersicum) Anlagen sind 7 verwendet wird. Teil 2: VIGS TAG 1 <ol><li> Für jeden Versuch sind Agrobacterium tumefaciens beherbergen pTRV1, pTRV2, pTRV2-PDS und pTRV2-host Zielgen auf LB-Agarplatten mit 50 pg / ml Kanamycin und 100 ug / mL Rifampicin ergänzt gewachsen. Die Kanamycin wählt für die pTRV Plasmid während der Rifampicin nicht so für die Agrobacterium. Die Platte bei 30 ° C für 2 Tage. Silencing der PDS wird die Pflanzen photobleach Ursache und ist als Schweigen Erfolgskontrolle eingesetzt. Außerdem müssen die Gene herunterreguliert werden in eine pTRV2 Vektor kloniert werden. Es ist ein Gateway kompatibel pTRV2 Vektor, der die Klonierung zu erleichtern kann und wird von Liu et al. (2002). </li></ol> TAG 3 <ol><li> Impfen ein 2 bis 3 ml Flüssigkeit Kultur der LB mit den oben genannten Antibiotika für jeden der Stämme. Inkubieren durch Schütteln bei 30 ° C für 16 bis 18 Stunden und 200 rpm </li></ol> TAG 4 <ol><li> Impfen eine 1: 25 Verdünnung des primären Kultur in eine sekundäre Flüssigkeit Induction Media (IM) IM Kultur mit Kanamycin, Rifampicin und 200 uM Acetosyringon (Tabellen 1, 2 und 3). Die Acetosyringon ist als Induktor der vir-Gene von Agrobacterium, die für T-DNA-Transfer in der Anlage 9 erforderlich sind, während die IM ahmt die Umwelt dieses Erregers Begegnungen in den Host-Apoplasten verwendet. Inkubieren durch Schütteln bei 30 ° C für 20 bis 24 Stunden und 200 rpm </li></ol> TAG 5 <ol><li> Ernten Sie die Zellen durch Zentrifugieren für 10 Minuten bei 3000 x g. Resuspendieren in das gleiche Volumen, dass die ursprüngliche Kultur mit 10 mM MgCl 2, 10 mM MES pH 5,5 hatte. Zellen können sanft gevortext werden, um sie zu resuspendieren. </li><li> Centrifugue die Zellen wieder für 10 Minuten bei 3000 x g. Resuspendieren in die Hälfte des Volumens der ursprünglichen Kultur mit 10 mM MgCl2, 10 mM MES pH 5,5. </li><li> Bereiten Sie eine Bakteriensuspension mit einer OD 600 von 0,3 für jede Bakterienkultur. Add Acetosyringon bis zu einer Endkonzentration von 400 nM auf die pTRV1 Kultur. </li><li> Mix der Kulturen mit dem pTRV1 und die pTRV2 (oder die pTRV2 enthält das Gen von Interesse) in einer 1 bis 1 Verhältnis. Auch eine pTRV2-PDS-Steuerung. Bitte beachten Sie, dass die endgültige Acetosyringon Konzentration ist jetzt 200 uM und dass jede Kultur bei einer OD 600 von 0,15. </li><li> Beschriften Sie die Keimlinge mit dem Gen zum Schweigen gebracht werden und das Datum des Experiments infiltriert werden. </li><li> Poke ein Loch in jedes Blatt mit einer Nadel infiltriert werden. Verwenden Sie einen 1 mL unnötige Spritze auf die Bakteriensuspension in die Sämlinge zu infiltrieren. Für Tomaten, infiltrieren beide Cotyledonen während für N. benthamiana, infiltrieren die beiden größten Laubblätter. Fünf ml jeder bakteriellen Mischung sollte ausreichen, um 15 N. infiltrieren benthamiana und 25 Tomatenjungpflanzen. Vermeiden Sie Kreuzkontaminationen, indem Handschuhe zwischen Infiltrationen und nicht die Bewässerung der Pflanzen bis zum nächsten Tag nach der Impfung. </li><li> Die Pflanzen werden bei 20 gehalten - 22 ° C in einer Klimakammer mit einer 16-Stunden-Tag Länge und 50% RH für mindestens 3 ½ Wochen, bevor sie für Tests verwendet werden können. </li></ol> Teil 3: Repräsentative Ergebnisse Abbildung 1 zeigt ein repräsentatives Experiment mit N. benthamiana-und Tomatenpflanzen für PDS zum Schweigen gebracht. Pflanzen zeigen die charakteristischen photobleaching Phänotyp in Pflanzen mit verminderter Mengen an Carotinoiden beobachtet. Für die PDS-Schweigen Kontrollpflanzen, Bleichen beginnt, sobald 1 ersichtlich ½ Wochen nach Infiltration. <img src="/files/ftp_upload/1292/Figure 1A 1292.jpg" alt="Abbildung 1a" /><img src="/files/ftp_upload/1292/Figure 1B 1292.jpg" alt="Abbildung 1b" /> Abbildung 1. Silencing der PDS Kontroll-Gen verursacht Bleichen in N. benthamiana (A) und Tomaten (B)-Anlagen. Fotos wurden 3 ½ Wochen nach Schweigen aufgenommen. Tabelle 1. Vorbereitung der Induction Medium (IM). <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 400 mL </td><td> destilliertem H 2 O </td></tr><tr><td> 4,88 g </td><td> MES (2 - (4 Morpholino)-ethansulfonsäure) </td></tr><tr><td> 2,5 g </td><td> Glucose </td></tr><tr><td> 0,12 g </td><td> NaH 2 PO 4 </td></tr></tbody></table> Bringen Sie zu einem Endvolumen von 475 ml mit dH 2 O und den pH-Wert auf 5,6. Autoclave. Nachdem das Medium abgekühlt, 25 ml 20x AB-Salze. Tabelle 2. Vorbereitung der AB-Salze. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 20 g </td><td> NH 4 Cl </td></tr><tr><td> 6 g </td><td> MgSO 4 · 7H 2 O </td></tr><tr><td> 3 g </td><td> KCl </td></tr><tr><td> 0,2 g </td><td> CaCl 2 </td></tr><tr><td> 0,05 g </td><td> FeSO 4 · 7H 2 O </td></tr></tbody></table> Bringen Sie zu einem Endvolumen von 1 Liter mit destilliertem Wasser. Autoclave. Seien Sie sich bewusst, dass AB Salze als orangefarbenes Pulver Niederschlag. Nur mischen sie gut durch Verwirbelung vor ihrer Verwendung. Tabelle 3. Vorbereitung der 200 mM Acetosyringon. Bitte beachten Sie, dass Acetosyringon der Tag sie verwendet werden sollten darauf vorbereitet sein. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 19,6 mg </td><td> Acetosyringon (3 &#39;, 5&#39;-Dimethoxy-4&#39;-Hydroxyacetophenon) </td></tr><tr><td> 500 ul </td><td> DMSO (Dimethylsulfoxid) </td></tr></tbody></table>

<ol>
	<li>Voinnet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+as+a+plant+immune+system+against+viruses.">RNA silencing as a plant immune system against viruses.</a> Trends Genet. 17, 449-459 (2001).</li><li>Carrington, J. C., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microRNAs+in+plant+and+animal+development.">Role of microRNAs in plant and animal development.</a> Science. 301, 336-338 (2003).</li><li>Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microRNA+as+a+translational+repressor+of+APETALA2+in+Arabidopsis+flower+development.">A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development.</a> Science. 303, 2022-2025 (2004).</li><li>Palatnik, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+leaf+morphogenesis+by+microRNAs.">Control of leaf morphogenesis by microRNAs.</a> Nature. 425, 257-263 (2003).</li><li>Brigneti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+pathogenicity+determinants+are+suppressors+of+transgene+silencing+in+Nicotiana+benthamiana.">Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.</a> EMBO J. 17, 6739-6746 (1998).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant Journal. 31, 777-786 (2002).</li><li>MacFarlane, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+the+tobraviruses.">Molecular biology of the tobraviruses.</a> J. Gen. Virol. 80, 2799-2807 (1999).</li><li>Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+study+to+determine+the+extent+of+gene+silencing+in+Nicotiana+benthamiana+and+other+Solanaceae+species+when+heterologous+gene+sequences+are+used+for+virus-induced+gene+silencing.">A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing.</a> New Phytol. 176, 782-791 (2007).</li><li>McCullen, C. A., Binns, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+tumefaciens+and+plant+cell+interactions+and+activities+required+for+interkingdom+macromolecular+transfer.">Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer.</a> Annu. Rev. Cell Dev. Biol. 22, 101-127 (2006).</li><li>Rosebrock, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bacterial+E3+ubiquitin+ligase+targets+a+host+protein+kinase+to+disrupt+plant+immunity.">A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity.</a> Nature. 448, 370-374 (2007).</li><li>Miki, D., Itoh, R., Shimamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+of+single+and+multiple+members+in+a+gene+family+of+rice.">RNA silencing of single and multiple members in a gene family of rice.</a> Plant Physiol. 138, 1903-1913 (2005).</li><li>Voinnet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use,+tolerance+and+avoidance+of+amplified+RNA+silencing+by+plants.">Use, tolerance and avoidance of amplified RNA silencing by plants.</a> Trends Plant Sci. 13, 317-328 (2008).</li><li>Ekengren, S. K., Liu, Y., Schiff, M., Kumar-Dinesh, S. P., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+MAPK+cascades,+NPR1,+and+TGA+transcription+factors+play+a+role+in+Pto-mediated+disease+resistance+in+tomato.">Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato.</a> Plant J. 36, 905-917 (2003).</li><li>Liu, E., Page, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+cDNA+libraries+for+virus-induced+gene+silencing+(VIGS)+using+tobacco+rattle+virus.">Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus.</a> Plant. Methods. 4, 5-5 (2008).</li></ol>

Virus-induzierte Gen-Silencing ist eine Methode, die eine schnelle reverse genetische Screens ermöglicht. Es vermeidet die Erzeugung von T-DNA oder Transposon-vermittelte Gen-Knock-outs, die nur in bestimmten Pflanzen wie Arabidopsis und Mais zur Verfügung. Zudem umgeht das zeitraubende Prozess der Transformation von Pflanzen und erlaubt das gezielte Vorgehen gegen mehrere Gene gleichzeitig, sofern sie eine ausreichende Homologie 10 oder dass unterschiedliche Host-Target-Sequenzen sind im Tandem in...

virus-induced gene silencing (vigs) in nicotiana benthamiana and tomato

RNA-Interferenz (RNAi) ist ein hoch spezifisches Gen-Silencing Phänomen durch dsRNA 1 ausgelöst. Das Schweigen Mechanismus nutzt zwei große Klassen von RNA Regulatoren: microRNAs, die aus Nicht-Protein-kodierende Gene und short interfering RNAs (siRNAs) hergestellt werden. Pflanzen nutzen RNAi auf Transposons zu kontrollieren und eine strenge Kontrolle über Entwicklungsprozesse wie Blume Organbildung und Blattentwicklung 2,3,4 auszuüben. Auch Pflanzen nutzen RNAi, um sich vor Ansteckung zu schützen, die durch Viren. Folglich haben viele Viren Suppressoren von Gen-Silencing, um ihre erfolgreiche Kolonisation des Aufnahmelandes 5 erlauben entwickelt. Virus-induzierte Gen-Silencing (VIGS) ist eine Methode, die die Vorteile der Anlage RNAi-vermittelten antiviralen Abwehrmechanismus. In Anlagen mit unveränderten Viren infiziert ist der Mechanismus der spezifisch gegen das virale Genom gezielt. Doch mit Virus-Vektoren tragen Sequenzen aus Wirtsgenen abgeleitet, kann das Verfahren zusätzlich mit den entsprechenden Host-mRNAs ausgerichtet sein. VIGS für High-Throughput-funktionelle Genomik in Pflanzen wurde mit Hilfe des Pflanzenpathogen Agrobacterium tumefaciens zu liefern angepasst, über dessen Ti-Plasmid, ein rekombinantes Virus, das die gesamte oder einen Teil der Gensequenz zum Schweigen ausgerichtet. Systemische Virus zu verbreiten und die endogenen RNAi-Maschinerie kümmern uns um den Rest. dsRNAs entsprechend dem Ziel-Gen hergestellt und dann gespalten durch die Ribonuklease Dicer zu siRNAs von 21 bis 24 Nukleotiden Länge. Diese siRNAs letztlich führen den RNA-induced silencing complex (RISC), um die Ziel-Transkript 2 degradieren. Verschiedene Vektoren wurden in VIGS eingesetzt und einer der am häufigsten verwendet wird, auf Tabak Rassel-Virus (TRV) basiert. TRV ist eine zweiteilige Antivirus und als solcher zwei verschiedene A. tumefaciens-Stämme sind für VIGS verwendet. Einer trägt pTRV1, die die Replikation und Bewegung viralen Funktionen kodiert, während das andere, pTRV2, birgt das Hüllprotein und die Sequenz für VIGS 6,7 verwendet. Inokulation von Nicotiana benthamiana und Tomatenjungpflanzen mit einer Mischung aus beiden Stämmen Ergebnisse in Gen-Silencing. Silencing des endogenen Phytoendesaturase (PDS)-Gen, das Bleichen verursacht, wird als Kontrolle für VIGS Effizienz eingesetzt. Es sollte angemerkt werden, ist jedoch, dass Schweigen in Tomaten in der Regel weniger effizient als in N. benthamiana. RNA-Transkript Fülle des Gens von Interesse sollte immer gemessen werden, um sicherzustellen, dass das Ziel-Gen effizient worden herunter reguliert werden. Dennoch heterologen Gensequenzen von N. benthamiana kann verwendet werden, um ihre jeweiligen Orthologe in Tomaten und umgekehrt 8 zum Schweigen zu bringen.

Watch this Scientific Journal Video about Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato at JoVE.com

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Beschreibung eines Virus-induzierte Gen-Silencing (VIGS)-Methode für knock-down der Genexpression in<em&gt; Nicotiana benthamiana</em&gt; Und Tomaten.

Virus-induzierten Gene Silencing (VIGS) in Nicotiana benthamiana Und Tomaten

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

virus-induced-gene-silencing-vigs-in-nicotiana-benthamiana-and-tomato

Research

JoVE Journal

Biology

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

52.4K Aufrufe.  Cornell University. Beschreibung eines Virus-induzierte Gen-Silencing (VIGS)-Methode für knock-down der Genexpression in Nicotiana benthamiana Und Tomaten.

Serie: Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Live Cell Imaging mit Calcium siRNA vermittelte Gene Silencing Combined Identifiziert Ca 2 + Leck-Kanäle in der ER-Membran und deren Regulationsmechanismen

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Forschung

Biologie

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Induktion und Prüfung von Hypoxie in der Zellkultur

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Agrobacterium-vermittelte Virus-induced gene silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Mit SECM Arrest Sequenz als Werkzeug zum Ribosom gebunden Polypeptide isolieren

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration und PVX Agroinfektion in Kartoffel und Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Optimierung und Nutzung von<em&gt; Agrobacterium</em&gt; Transient-vermittelte Proteinproduktion in<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Chitosan / interfering RNA Nanopartikel vermittelten Gen-Stummschaltung in Disease Vector Mückenlarven

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotischen Spindel Assessment in Maus-Oozyten von siRNA-vermittelte Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase Komplementierung Imaging Assay in Nicotiana Benthamiana Blätter für Transient Bestimmung Proteinprotein Interaktion Dynamik

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...