This research was conducted under USDA-ARS CRIS Project 5325-42000-037-00D and with results from CRADA 58-3K95-7-1198 and TFCA 58-5325-8-419. The authors gratefully acknowledge Suterra for the gift of the (Z,Z)-11,13-hexadecadienal, B. Higbee for productive discussions, and J. Baker for technical assistance.

1. Preparation of Volatiles Detected from the Host Plant for EAG Screening
<ol>
 <li>After appropriate identification and authentication of all volatiles via GC-MS, perform EAG puff analysis of each available volatile. Initial screening can be a low replicate number of antennal responses (N=3-5) for each sex in order to achieve an indication of relative chemoreceptivity in a short amount of time (Table 1).</li>
 <li>Prepare a solution of each volatile at a 5 mg/mL concentration in pentane. Tightly seal and refrigerate the sample until ready for immediate use (e.g., acetophenone, density = 1.03 g/mL, volume = (0.005 g/1.03 g/mL) &times; 1,000 = 4.85 &mu;L of acetophenone into a 1.0 mL volumetric flask and dilute to 1.0 mL with pentane).</li>
 <li>Just prior to EAG analysis, remove vials containing the 5 mg/mL concentration of volatiles to be tested and allow to warm to room temperature. Meanwhile, label the proper number of Pasteur pipettes to correlate with each volatile to be tested, and wrap a small piece of parafilm (ca. 15 &times; 15 mm) over the tip of the pipette. For the initial screening 10 stimulus pipettes (N=4 for each male and female) are loaded in the event of a bad prep or other unforeseen obstacle.</li>
 <li>Using a pair of tweezers, gently fold the bioassay disc in half to facilitate placement into the pipette, then partially place the folded disc into the large end of the pipette so that ca. 2-3 mm of the disc are exposed. Align the labeled and prepped pipettes in a rack holder.</li>
 <li>Using a pipettor or syringe, load 10 &mu;L (50 &mu;g) of volatile solution onto each disc and allow 2 minutes to pass before fully inserting discs into the pipette. Once loaded, immediately seal the end of the pipette with parafilm (ca. 15 &times; 30 mm). The amount of volatile stimulus to be loaded and puffed will most likely vary with insects species.</li>
 <li>Following the same protocol noted above prepare controls to include in each analysis. For the negative control load 10 &mu;L of just pentane onto each disc, wait 2 minutes, load into the labeled pipette, and seal. For the positive control, load 10 &mu;L (50 &mu;g) onto the disc, wait 2 minutes, load into the labeled pipette, and seal with parafilm.</li>
 <li>The viability of the insect antennal prep will most likely vary with species, but we have found that Amyelois transitella antennae typically stay active and consistent for &gt; 30 minutes after excision using the described protocol. This amount of time will generally allow for up to 4-10 volatile samples to be evaluated. Table 2 provides an example of times and sequences.</li>
</ol>
2. Preparation of Insect Antennae for EAG Bioassay
<ol>
 <li>The focus of this experiment is EAG analysis; thus, the assumption that each investigator will have access to appropriately reared insects.</li>
 <li>For this experiment we will be studying 3-4 day old, mated male and female moths. Individual moths are transferred into a small, lidded, plastic container the day of analysis. Immediately prior to the bioassay, the moth to be tested is transferred head-first into a holding apparatus (i.e., made from various plastic pipettor tips) and secured from behind. Manipulation of the insect can be viewed under a low-power stereo-microscope to facilitate excision.</li>
 <li>Antennae are teased out using a wire-tipped tool. The fork holder, with a small film of electrode gel, is placed in close proximity for quick transfer of antennae. The first antenna is excised using micro scissors and placed on the fork, ensuring the base of the antenna is placed on the non-red portion of the fork (the indifferent ground electrode). A timer set for 10 minutes is started and the second antenna is excised and placed next to the first antenna with both bases on the same side of the fork.</li>
 <li>Alternatively, the antennae may be left secured within the holding tube, excised, then gently removed from between the tube and the insect using a wire-tipped tool with a dab of gel on the end.</li>
 <li>Antennae are checked to ensure the base and tip are immersed in electrode gel and no bubbles are present in the gel. The ends/tips can be trimmed to guarantee full exposure to the electrode gel. Care should be used to ensure that the remainder of each antenna is not covered with gel, thus guaranteeing maximum antennal surface area is exposed to the puff.</li>
 <li>The prepped fork is then inserted into the probe pre-amplifier and kept under a constant stream of humidified air at 200 mL/min for the remaining time of the 10 minute waiting period. The excise time and EAG initiation times are noted (Table 2) and 30 seconds prior to the first stimulus puff, the pipette containing the positive control is unsealed and placed in the holder which is adjusted to direct air/puff flow 2-3 mm from the prepped antennae.</li>
 <li>After each experiment, the test moths are euthanized in a dry ice environment and properly disposed.</li>
 <li>Female moths are dissected to check mating status. Cohabitating males are assumed to have mated.</li>
</ol>
3. EAG Protocol for Individual Components
<ol>
 <li>Antennal responses are recorded via the AutoSpike software included with the Syntech EAG instrument. The configuration in the AutoSpike Properties tab for the channel with the EAG probe is set at a sampling rate of 106.7, NO to rectify, and a filter of 0-42 Hz. For the Filter tab, the EAG filter is ON and the low-cutoff is set to 0.1 Hz.</li>
 <li>Stimulus puffs are 1-2 seconds in duration. A timer set for 1 minute is started after each puff.</li>
 <li>The next test volatile pipette is unsealed and placed in the holder. The sequence of test volatiles, randomized between runs to ensure no test compounds consistently follow another test volatile, is then followed (e.g., Table 2) carefully allowing 1 minute between puffs.</li>
 <li>To facilitate ease of reading EAG responses, portions of rulers are placed on the computer screen and the baseline level to the apex of the downward deflection signal is measured in mm and logged on a data sheet (i.e., for the 5 mV setting, 33 mm = 2.5 mV). The software has the capability of playback of the saved recordings for future analysis. Conversion of mm to either mV or &mu;V amplitude is performed in later data analysis.</li>
 <li>After the final positive control puff (e.g., record #12) is administered, the antennae are removed, the fork is cleaned with an ethanol-saturated wipe and allowed to dry before subsequent use.</li>
 <li>To increase the throughput of assays, the excision of the next set of antennae can be started by a second laboratory personnel approximately 10 minutes before the end of current experiment - after the fourth puff; the second test volatile of the current experiment, as per Table 2. This will allow for the start of the next experiment directly after the current experiment ends.</li>
 <li>Higher number of replications (on different antennae) can be performed on compounds of interest to provide further statistical validation.</li>
</ol>
4. An Example of EAG Analysis of Blends or Other Matrices (Table 3)
<ol>
 <li>Next, the ratios and volumes for two tertiary blends (3-component mixtures) are calculated to provide an example of combining volatiles eliciting high EAG responses (Table 4). The calculations are for some basic ratios, a 1:1:1 molar ratio of &alpha;-humulene : 2-undecanone : 2-phenylethanol and then comparing to a second blend ratio of 1:2:4.</li>
 <li>Using similar protocols described earlier, the mixtures are prepared and labeled Pasteur pipettes are loaded along with the necessary positive and negative controls.</li>
 <li>The volatile samples are puffed across male and female antennae and the responses are measured and logged onto a data sheet (Table 3).</li>
</ol>
5. Representative Results
For female navel orangeworm the following settings are used: 2 second puffs, 10 second recording times, 10 second window, and 5 mV scale. A negative deflection is the typical response, yet the absolute value is recorded (e.g., -3,400 &mu;V deflection is recorded as 3,400 &mu;V). A relatively weak response of the prep to the positive control is discarded. Figure 1 provides a graphical representation of a poor response to the positive control by navel orangeworm.
For example of a poor control result, the average female antennal response to acetophenone is typically ca. 2,600 &mu;V (Figure 2), if the prep only gave a response of ca. 1,300 &mu;V it would be discarded and another pair of antennae prepped. Similarly, the average male response to (Z,Z)-11,13-hexadecadienal was typically 3,000 &mu;V; thus, any response less than 1,500 &mu;V was typically discarded.
The positive control at the start and end of each experiment also provides information regarding the condition of the antennae. A rule of thumb we follow for rapid screening is if the antennal response to the puff of the post-control (record #12, Table 2) is either less than 75% of the 1st puff of the pre-control (record #1, Table 2) or less than the 2nd puff of the pre-control (record #2, Table 2) then the experiment is not used in the data analysis due to possible degradation of the prep (Figure 3). An example of the first rule of thumb would be record #1 = 2,730 &mu;V and record #12 = 1,680 &mu;V; and the second rule of thumb if record #2 = 2,350 &mu;V and record #12 = 1,680 &mu;V, In each of these cases, the prep and experiment's results would be discarded.
A representative example of correcting the EAG response values as measured to the positive control would be as follows.
<table border="1">
 <tbody>
 <tr>
 <td>Run #</td>
 <td>EAG (&mu;V)</td>
 <td>Run #2</td>
 <td>EAG (&mu;V)</td>
 <td>Run #3</td>
 <td>EAG (&mu;V)</td>
 </tr>
 <tr>
 <td>(+) Ctrl</td>
 <td>2800</td>
 <td>(+) Ctrl</td>
 <td>2420</td>
 <td>(+) Ctrl</td>
 <td>3030</td>
 </tr>
 <tr>
 <td>Cmpnd A</td>
 <td>3000</td>
 <td>Cmpnd A</td>
 <td>2500</td>
 <td>Cmpnd A</td>
 <td>3440</td>
 </tr>
 <tr>
 <td>(-) Ctrl</td>
 <td>530</td>
 <td>(-) Ctrl</td>
 <td>755</td>
 <td>(-) Ctrl</td>
 <td>910</td>
 </tr>
 <tr>
 <td>Cmpnd B</td>
 <td>2400</td>
 <td>Cmpnd B</td>
 <td>2000</td>
 <td>Cmpnd B</td>
 <td>2560</td>
 </tr>
 <tr>
 <td>(+) Ctrl</td>
 <td>2770</td>
 <td>(+) Ctrl</td>
 <td>2400</td>
 <td>(+) Ctrl</td>
 <td>3020</td>
 </tr>
 </tbody>
</table>
Using the values above for an N=3 experiment, the negative control response is subtracted from every value within each experiment under the assumption the negative control is the baseline antennal response to the mechanical puff and residual solvent.
<table border="1">
 <tbody>
 <tr>
 <td>Run #1</td>
 <td>EAG (&mu;V)</td>
 <td>Run #2</td>
 <td>EAG (&mu;V)</td>
 <td>Run #3</td>
 <td>EAG (&mu;V)</td>
 </tr>
 <tr>
 <td>(+) Ctrl</td>
 <td>2270</td>
 <td>(+) Ctrl</td>
 <td>1665</td>
 <td>(+) Ctrl</td>
 <td>2120</td>
 </tr>
 <tr>
 <td>Cmpnd A</td>
 <td>2470</td>
 <td>Cmpnd A</td>
 <td>1745</td>
 <td>Cmpnd A</td>
 <td>2530</td>
 </tr>
 <tr>
 <td>(-) Ctrl</td>
 <td>0</td>
 <td>(-) Ctrl</td>
 <td>0</td>
 <td>(-) Ctrl</td>
 <td>0</td>
 </tr>
 <tr>
 <td>Cmpnd B</td>
 <td>1870</td>
 <td>Cmpnd B</td>
 <td>1245</td>
 <td>Cmpnd B</td>
 <td>1650</td>
 </tr>
 <tr>
 <td>(+) Ctrl</td>
 <td>2240</td>
 <td>(+) Ctrl</td>
 <td>1645</td>
 <td>(+) Ctrl</td>
 <td>2110</td>
 </tr>
 </tbody>
</table>
The positive controls for each experiment would then be averaged and corrected to 1,000 &mu;V, noting the ratio for correction to 1,000 &mu;V. A data sheet (e.g., Excel) can easily be manipulated to convert responses to usable data.
<table border="1">
 <tbody>
 <tr>
 <td>Run #1</td>
 <td>Avg (&mu;V)</td>
 <td>Run #2</td>
 <td>Avg (&mu;V)</td>
 <td>Run #3</td>
 <td>Avg (&mu;V)</td>
 </tr>
 <tr>
 <td>(+) Ctrl</td>
 <td>2255</td>
 <td>(+) Ctrl</td>
 <td>1655</td>
 <td>(+) Ctrl</td>
 <td>2115</td>
 </tr>
 <tr>
 <td>(+) Ctrl adj.</td>
 <td>1000 (0.443)</td>
 <td>(+) Ctrl adj.</td>
 <td>1000 (0.604)</td>
 <td>(+) Ctrl adj.</td>
 <td>1000 (0.473)</td>
 </tr>
 </tbody>
</table>
Multiplying by the correction ratio within each experiment, the values for compound A and compound B are then adjusted.
<table border="1">
 <tbody>
 <tr>
 <td>Run #1</td>
 <td>Adj. EAG(&mu;V)</td>
 <td>Run #2</td>
 <td>Adj. EAG(&mu;V)</td>
 <td>Run #3</td>
 <td>Adj. EAG(&mu;V)</td>
 </tr>
 <tr>
 <td>Cmpnd A</td>
 <td>1094</td>
 <td>Cmpnd A</td>
 <td>1054</td>
 <td>Cmpnd A</td>
 <td>1197</td>
 </tr>
 <tr>
 <td>Cmpnd B</td>
 <td>828</td>
 <td>Cmpnd B</td>
 <td>752</td>
 <td>Cmpnd B</td>
 <td>780</td>
 </tr>
 </tbody>
</table>
The averages (means) for each compound are then determined along with other relevant statistical data and the EAG responses for the compounds tested can then be evaluated for candidacy for further investigation.
<table border="1">
 <tbody>
 <tr>
 <td>Compound</td>
 <td>EAG (&mu;V)</td>
 <td>No. Runs, N=</td>
 </tr>
 <tr>
 <td>A</td>
 <td>1115</td>
 <td>3</td>
 </tr>
 <tr>
 <td>B</td>
 <td>787</td>
 <td>3</td>
 </tr>
 </tbody>
</table>
<img alt="Figure 1" src="/files/ftp_upload/3931/3931fig1.jpg" /> 
Figure 1. Representative EAG for male antennal response (1,180 &mu;V) to the pre-control (1st positive control puff) that would be discarded due to poor antennal response after ensuring the antennae have good contact with the gel. Blue bars in bottom windows represent the two second puff of volatile. <a href="https://www.jove.com/files/ftp_upload/3931/3931fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/3931/3931fig2.jpg" /> 
Figure 2. Representative EAG for the female antennal response (3,400 &mu;V) to the 1st puff pre-control that would be considered appropriate. <a href="https://www.jove.com/files/ftp_upload/3931/3931fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/3931/3931fig3.jpg" /> 
Figure 3. Representative EAG for the female antennal response (1,680 &mu;V) to the post-control (last positive control for each experiment) that would be considered poor, and suggestive of antennae degradation (&lt;75% of 1st pre-control or &lt; 2nd puff value of pre-control). For this example the 1st pre-control puff was 2,730 &mu;V and 2nd pre-control puff was 2,350 &mu;V. <a href="https://www.jove.com/files/ftp_upload/3931/3931fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/3931/3931fig4.jpg" /> 
Figure 4. Representative EAG for the female antennal response (3,800 &mu;V) to the puff of a candidate volatile blend and the subsequent measurements of the maximum initial deflection (3,800 &mu;V), the initial slope during the puff duration (0.3 s/1.2 mV = 0.25 s/mV), and the slope for the remaining puff duration (1.6 s/1.9 mV = 0.84 s/mV). <a href="https://www.jove.com/files/ftp_upload/3931/3931fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" src="/files/ftp_upload/3931/3931fig5.jpg" /> 
Figure 5. Small modified vessel containing a sample matrix and associated volatiles to be puffed across A. transitella antennae.
<img alt="Table 1" src="/files/ftp_upload/3931/3931table1.jpg" /> 
Table 1. In situ volatile emission of Nonpareil almonds (2007) and EAG responses determined by a different and less sensitive configuration in the Autospike program.
<img alt="Table 2" src="/files/ftp_upload/3931/3931table2.jpg" /> 
Table 2. Example of a form for recording male and female antennal responses to individual volatile components.
<img alt="Table 3" src="/files/ftp_upload/3931/3931table3.jpg" /> 
Table 3. Example of a form for recording male and female antennal responses to volatile blends and/or bouquets.
<img alt="Table 4" src="/files/ftp_upload/3931/3931table4.jpg" /> 
Table 4. Examples of preparation of 10 mL of a 5 mg/mL solution for two different ratios of blends.
<img alt="Table 5" src="/files/ftp_upload/3931/3931table5.jpg" /> 
Table 5. Example of form for recording two consecutive puffs of single volatile components across male and female antennae.

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The author has an existing cooperative research and development agreement with Paramount Farming Company, a company with ties to Suterra LLC.

Use of electroantennogram recordings as a bioassay to determine chemoreception responses of a target insect is fairly common and numerous studies utilizing EAG as a detector for effluent from a gas chromatogram (GC-EAD) can be found in the literature.9,10 The method demonstrated will provide a rapid screening of equivalent amounts of volatile components with high replications for confident assignment of the relative responsiveness. The AutoSpike program in the Syntech software is a good program for screening v...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Acetophenone</td>
			<td>Alfa-Aesar</td>
			<td>A12727</td>
			<td>Female positive control</td>
		</tr>
		<tr>
			<td>(Z,Z)-11,13-Hexadecadienal</td>
			<td>Suterra</td>
			<td>&nbsp;</td>
			<td>Male positive control</td>
		</tr>
		<tr>
			<td>&alpha;-Humulene</td>
			<td>Aldrich</td>
			<td>53675</td>
			<td>Sesquiterpene</td>
		</tr>
		<tr>
			<td>2-Undecanone</td>
			<td>Aldrich</td>
			<td>U1303</td>
			<td>Fatty acid derivative</td>
		</tr>
		<tr>
			<td>2-Phenylethanol</td>
			<td>Aldrich</td>
			<td>77861</td>
			<td>Benzenoid</td>
		</tr>
		<tr>
			<td>Pentane</td>
			<td>EMD</td>
			<td>PX0167-1</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>4-Channel acquisition controller</td>
			<td>Syntech</td>
			<td>IDAC-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EAG probe, pre-amplifier</td>
			<td>Syntech</td>
			<td>Type PRG-2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Antenna holder</td>
			<td>Syntech</td>
			<td>For PRG-2</td>
			<td>Fork electrode</td>
		</tr>
		<tr>
			<td>Stimulus controller</td>
			<td>Syntech</td>
			<td>CS-55</td>
			<td>Air flow and puffs</td>
		</tr>
		<tr>
			<td>Spectra Electrode Gel</td>
			<td>Parker</td>
			<td>12-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bioassay discs</td>
			<td>Whatman</td>
			<td>2017-006</td>
			<td>6 mm</td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td>5 &frac34;" (14.6 cm)</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM-992</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

electroantennographic bioassay as a screening tool for host plant volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.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

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

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

electroantennographic-bioassay-as-screening-tool-for-host-plant

Research

JoVE Journal

Biology

13.6K Views.  Agricultural Research Service. A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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