מחקר זה נערך על פי USDA-ARS קריס Project 5325-42000-037-00D ועם תוצאות CRADA 58-3K95-7-1198 ו TFCA 58-5325-8-419. המחברים מודים Suterra בתודה על המתנה של (Z, Z) -11,13-hexadecadienal, ב Higbee לדיונים פוריים, וג&#39; בייקר לקבלת סיוע טכני.

<ol>
	<li>Bruce, T. J. A., Wadhams, L. J., Woodcock, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+host+location:+a+volatile+situation.">Insect host location: a volatile situation.</a> Trends in Plant Sci. 10, 1360-1385 (2005).</li><li>Unsicker, S. B., Kunert, G., Gershenzon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+perfumes:+the+role+of+vegetative+volatiles+in+plant+defense+against+herbivores.">Protective perfumes: the role of vegetative volatiles in plant defense against herbivores.</a> Curr. Opin. Plant Biol. 12, 479-485 (2009).</li><li>Norin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiochemicals+for+insect+pest+management.">Semiochemicals for insect pest management.</a> Pure Appl. Chem. 79, 2129-2136 (2007).</li><li>Beck, J. J., Merrill, G. B., Higbee, B. S., Light, D. M., Gee, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+seasonal+study+of+the+volatile+production+of+almonds+(Prunus+dulcis)+var.+'nonpareil'+and+relationship+to+navel+orangeworm.">In situ seasonal study of the volatile production of almonds (Prunus dulcis) var. 'nonpareil' and relationship to navel orangeworm.</a> J. Agric. Food Chem. 57, 3749-3753 (2009).</li><li>Beck, J. J., Higbee, B. S., Gee, W. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+pheromone+chemistry+in+the+navel+orangeworm:+novel+sex+attractants+and+a+behavioral+antagonist.">Unusual pheromone chemistry in the navel orangeworm: novel sex attractants and a behavioral antagonist.</a> Naturwissenschaften. 92, 139-146 (2005).</li><li>Kanno, H., Kuenen, L. P. S., Klingler, K. A., Millar, J. G., Carde, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attractiveness+of+a+four-component+pheromone+blend+to+male+navel+orangeworm+moths.">Attractiveness of a four-component pheromone blend to male navel orangeworm moths.</a> J. Chem. Ecol. 36, 584-591 (2010).</li><li>Takacs, S., Gries, G., Gries, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiochemical-mediated+location+of+host+habitat+by+Apanteles+carpatus+(Say)+(Hymenoptera:+Braconidae),+a+parasitoid+of+cloths+moth+larvae.">Semiochemical-mediated location of host habitat by Apanteles carpatus (Say) (Hymenoptera: Braconidae), a parasitoid of cloths moth larvae.</a> J. Chem. Ecol. 23, 459-472 (1997).</li><li>Karimifar, N., Gries, R., Khaskin, G., Gries, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+food+semiochemicals+attract+omnivorous+German+cockroaches,+Blattella+germanica.">General food semiochemicals attract omnivorous German cockroaches, Blattella germanica.</a> J. Agric. Food Chem. 59, 1330-1337 (2011).</li><li>Molyneux, R. J., Schieberle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound+identification:+a+Journal+of+Agricultural+and+Food+Chemistry+perspective.">Compound identification: a Journal of Agricultural and Food Chemistry perspective.</a> J. Agric. Food Chem. 55, 4625-4629 (2007).</li><li>Marion-Poll, F., Thiery, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+EAG+responses+to+host-plant+volatiles+delivered+by+a+gas+chromatograph.">Dynamics of EAG responses to host-plant volatiles delivered by a gas chromatograph.</a> Entomol. Exp. Appl. 80, 120-123 (1996).</li><li>Beck, J. J., Higbee, B. S., Merrill, G. B., Roitman, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+volatile+emissions+from+undamaged+and+mechanically+damaged+almonds.">Comparison of volatile emissions from undamaged and mechanically damaged almonds.</a> J. Sci. Food Agric. 88, 1363-1368 (2008).</li><li>Lucas, P., Renou, M., Tellier, F., Hammoud, A., Audemard, H., Descoins, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+and+field+activity+of+halogenated+analogs+of+(E,E)-8-10-dodecadien-1-ol,+the+main+pheromone+component+in+codling+moth+(Cydia+pomonella+L.).">Electrophysiology and field activity of halogenated analogs of (E,E)-8-10-dodecadien-1-ol, the main pheromone component in codling moth (Cydia pomonella L.).</a> J. Chem. Ecol. 20, 489-503 (1994).</li><li>Rodriguez-Saona, C., Poland, T. M., Miller, J. R., Stelinski, L. L., Grant, G. G., de Groot, P., Buchan, L., MacDonald, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+electrophysiological+responses+of+the+emerald+ash+borer,+Agrilus+planipennis,+to+induced+volatiles+of+Manchurian+ash,+Fraxinus+mandshurica.">Behavioral and electrophysiological responses of the emerald ash borer, Agrilus planipennis, to induced volatiles of Manchurian ash, Fraxinus mandshurica.</a> Chemoecology. 16, 75-86 (2006).</li><li>Burks, C. S., Brandl, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+abundance+of+navel+orangeworm+(Leipidoptera:+Pyralidae)+in+figs+and+effect+of+peripheral+aerosol+dispensers+on+sexual+communication.">Seasonal abundance of navel orangeworm (Leipidoptera: Pyralidae) in figs and effect of peripheral aerosol dispensers on sexual communication.</a> J. Insect Sci. 4, 1-8 (2004).</li></ol>

מחבר המחקר יש שיתוף פעולה קיים הסכם פיתוח עם חברת פרמאונט חקלאות, חברה עם קשרים Suterra LLC.

השימוש בהקלטות electroantennogram כמו המבדק לקבוע התגובות chemoreception של חרק היעד נפוץ למדי, מחקרים רבים ניצול EAG כגלאי של שפכים מן הכרומתוגרמה גז (GC-EAD) ניתן למצוא בספרות. 9,10 שיטת הדגימה תספק סינון מהיר של כמויות מקבילות של מרכיבים נדיפים עם משוכפלים גבוהות המשימה בטוחים הי?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> Acetophenone </td><td> אלפא-Aesar </td><td> A12727 </td><td> בקרה חיובית נקבה </td></tr><tr><td> (Z, Z) -11,13-Hexadecadienal </td><td> Suterra </td><td></td><td> בקרה חיובית זכר </td></tr><tr><td> α-Humulene </td><td> אולדריץ </td><td> 53675 </td><td> Sesquiterpene </td></tr><tr><td> 2-Undecanone </td><td> אולדריץ </td><td> U1303 </td><td> חומצת שומן הנגזרת </td></tr><tr><td> 2-Phenylethanol </td><td> אולדריץ </td><td> 77861 </td><td> Benzenoid </td></tr><tr><td> פנטן </td><td> EMD </td><td> PX0167-1 </td><td> ממס </td></tr><tr><td> 4-Channel רכישת בקר </td><td> Syntech </td><td> אניDAC-4 </td><td></td></tr><tr><td> EAG בדיקה, קדם מגבר </td><td> Syntech </td><td> סוג PRG-2 </td><td></td></tr><tr><td> בעל אנטנה </td><td> Syntech </td><td> עבור PRG-2 </td><td> מזלג האלקטרודה </td></tr><tr><td> גירוי בקר </td><td> Syntech </td><td> CS-55 </td><td> זרימת האוויר שאיפות </td></tr><tr><td> אלקטרודה ספקטרה ג&#39;ל </td><td> פרקר </td><td> 12-02 </td><td></td></tr><tr><td> המבדק דיסקים </td><td> Whatman </td><td> 2017-006 </td><td> 6 מ&quot;מ </td></tr><tr><td> פסטר pipets </td><td> VWR </td><td> 14673-010 </td><td> 5 ¾ &quot;(14.6 ס&quot;מ) </td></tr><tr><td> Parafilm M </td><td> Bemis </td><td> PM-992 </td><td></td></tr></tbody></table>

electroantennographic bioassay as a screening tool for host plant volatiles

נדיפים צמחים תפקיד חשוב במפעל, חרקים אינטראקציות. חרקים אוכלי עשב להשתמש נדיפים צמחים, המכונה kairomones, לאתר את הצמח הפונדקאי שלהם. 1,2 כאשר הצמח הפונדקאי היא האכלה חשוב אגרונומית מצרך נזק על ידי חרקים מזיקים יכולים לגרום הפסדים כלכליים קשים על המגדלים. בהתאם לכך, kairomones יכול לשמש משיכה לפתות או לבלבל את החרקים, ולכן להציע חלופה ידידותית לסביבה חומרי הדברה עבור חרקים. 3 למרבה הצער, צמחים יכולים לפלוט כמה נדיפים מספר עצום עם יצירות ויחסים שונים של פליטות תלויים בפנולוגיה של הנכס או השעה ביום. זה הופך את זיהוי המרכיבים בעלי הפעילות הביולוגית או תערובות של מרכיבים נדיפים תהליך מפרך. כדי לזהות את המרכיבים של הצמח ביו פליטות המארחות נדיפים נשתמש במעבדה מבוסס המבדק ההקרנה electroantennography (EAG). EAG הוא כלי יעיל להערכת recorד electrophysiologically התגובות חוש הריח של החרק דרך קולטנים מחושים שלהם. תהליך המיון EAG יכול לעזור להפחית את מספר נדיפים שנבדקו לזהות רכיבים ביו מבטיחות. עם זאת, bioassays EAG רק לספק מידע אודות הפעלת הקולטנים. זה אינו מספק מידע על סוג של התנהגות חרקים המתחם מעורר, שיכול להיות סוג, מושך ודוחה או אחר של תגובה התנהגותית. נדיפים לעורר תגובה משמעותית על ידי EAG, ביחס לשליטה חיובית מתאימה, נלקחים בדרך כלל על בדיקות נוספות של תגובות התנהגותיות של חרקים מזיקים. מערך הניסוי הציג יפרט את המתודולוגיה מועסקים למסך שקדים מבוססי צמחים מארחים נדיפים 4.5 ידי מדידת התגובות מחושים אלקטרו של הדברה מבוגר חרק הטבור orangeworm (Amyelois transitella) לרכיבים בודדים תערובות פשוטות של רכיבים באמצעות EAG המבדק. השיטה משתמשת 2 לשעבראנטנות cised ממוקם על פני בעל &quot;מזלג&quot; האלקטרודה. פרוטוקול הפגינו כאן מציג מהירה, תפוקה גבוהה שיטה סטנדרטית נדיפים ההקרנה. כל תנודתי הוא בסכום קבוע מראש, קבוע על מנת לתקנן את רמת הגירוי ובכך לאפשר תגובות מחושים כדי להצביע על chemoreceptivity יחסית. הביקורת השלילית עוזר לחסל את התגובה אלקטרו לכוח הן ממס מכני שיורית של עלים. הביקורת החיובית (ב acetophenone זה למשל) הוא תרכובת אחת אשר עוררה תגובה עקבית זכר ונקבה עש orangeworm (NOW) הטבור. תקן semiochemical נוסף המספק מענה עקבי משמש ללימודי המבדק עם עש עכשיו הוא זכר (Z, Z) -11,13-hexdecadienal, מרכיב אלדהיד מ פרומון הנקבה בייצור מין. 6-8

Watch this Scientific Journal Video about Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles at JoVE.com

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

השיטה במהירות המארחות נדיפים מסך צמחים ידי מדידת התגובה של אלקטרו orangeworm הטבור מבוגר (<em&gt; Amyelois transitella</em&gt;) אנטנות לרכיבים בודדים תערובות באמצעות ניתוח electroantennographic באה לידי ביטוי.

המבדק Electroantennographic ככלי מיון עבור נדיפים צמחים מארח

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

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13.6K Views.  Agricultural Research Service. השיטה במהירות המארחות נדיפים מסך צמחים ידי מדידת התגובה של אלקטרו orangeworm הטבור מבוגר ( Amyelois transitella) אנטנות לרכיבים בודדים תערובות באמצעות ניתוח electroantennographic באה לידי ביטוי.

Video: Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

Β-glucuronidase (גאס) נייד מבוסס מוות Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

אוכלי עשב הנגרמת נדיפים אוכמניות צמח תוך איתות

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

פלואורסצנטי, מיקרוסקופ הקרנת ואת הדור הבא הרצף: כלים שימושיים לזיהוי גנים המעורבים אברון Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter 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.

באמצעות רצף מעצר SecM ככלי לבודד פוליפפטידים כרוכים הריבוזום

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

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA Catalyst ככתב להקרנת סמים נגד RNA עריכה בtrypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

מבדק פשוט עבור הערכת גורמי הגדילה של אנדותל כלי דם

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

Microdissection לייזר בסיוע (LAM) ככלי פרופיל תעתיק של סוגי תאים בודדים

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

Assay תחרות צלחת כמו הערכה ראשונית מהירה של דיכוי מחלות

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify&#160;molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts.
Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is&#160;carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

This is an adapted method for identifying candidate insect colonization factors in a Burkholderia beneficial symbiont. The beetle host is infected with a random mutant library generated via transposon mutagenesis, and library complexity after colonization is compared to a control grown in vitro.

רצף הכנסה טרנספוסון ככלי להבהב גורמי קולוניזציה חיידקיים בבורקהולדריה גלדיולי סימביונט של לגריה וילוסה בילס

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological ...

Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran insects that have piercing-sucking mouthparts. During viral transmission, the insect saliva bridges the virus-vector-host because the saliva vectors viruses, and the insect proteins, trigger or suppress the immune response of plants from insects into plant hosts. The identification and functional analyses of salivary proteins are becoming a new area of focus in the research field of arbovirus-host interactions. This protocol provides a system to detect proteins in the saliva of leafhoppers using the plant host. The leafhopper vector Nephotettix cincticeps infected with rice dwarf virus (RDV) serves as an example. The vitellogenin and major outer capsid protein P8 of RDV vectored by the saliva of N. cincticeps can be detected simultaneously in the rice plant that N. cincticeps feeds on. This method is applicable for testing the salivary proteins that are transiently retained in the plant host after insect feeding. It is believed that this system of detection will benefit the study of hemipteran-virus-plant or hemipteran-plant interactions.

This protocol demonstrates how to use the plant host to detect salivary proteins of leafhopper and plant viral proteins released by leafhopper vectors.

איתור חלבוני וירוס ורוק של וקטור עלה בצמח המארח

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran ...