Name: screening of tobacco genotypes for phytophthora nicotianae resistance
Uploaded: 2022-04-15T12:30:00
Description: Watch this Scientific Journal Video about Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance at JoVE.com

This research was funded by the National Natural Science Foundation of China (31571738) and the Agricultural Science and Technology Innovation Program of China (ASTIP-TRIC01).

1. Materials
<ol>
	<li>Obtain tobacco varieties. 
	NOTE: For this experiment &#34;Beinhart1000-1&#34; (a selection of Beinhart 1000) (BH) and &#34;Xiaohuangjin1025&#34; (XHJ) were obtained from the National Medium-term Genbank of the Tobacco Germplasm Resource of China. BH is resistant, whereas XHJ is susceptible to P. nicotianae infection16. A field isolate of P. nicotianae race 0, which was preserved in the Tobacco Research Institute of the Chinese Acade...

<ol>
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Multiple resistance sources have been used to improve P. nicotianae resistance in cultivated tobacco. Single dominant R genes, Php and Phl, have been introgressed from Nicotiana plumbaginifolia and Nicotiana longiflora, respectively10. The cigar tobacco variety Beinhart 1000 has the highest reported level of quantitative resistance to P. nicotianae13. Multiple interval mapping experiments have suggested that at least six...

P. nicotianae is destructive to many solanaceous crops. It can cause tobacco &#34;black shank&#34;1, potato foliar and tuber rot2, tomato and sweet pepper crown and root rot3, and Goji collar and root rot4. P. nicotianae can attack all parts of tobacco plants, including the roots, stems, and leaves at any growing stage5. The most common symptom of the disease is the black base of the stalk. The roots are initially visible as water-soaked and then become necrotic, and the leaves show large circular lesions5. This disease can be devastating to a tobacco plant in the greenhouse, as well as in the field6. The most practical and economical method for controlling P. nicotianae is the use of resistant varieties7. However, an effective screening protocol is required for the identification of P. nicotianae-resistant accessions from tobacco germplasm collections.
Various identification methods have been described to assess P. nicotianae resistance in tobacco7,8,9,10,11,12,13,14,15,16. In general, three major approaches have been used for the identification of P. nicotianae-resistant tobacco genotypes. The first includes mixing mycelia with agar medium on Petri plates containing P. nicotianae. The mycelia are then cultivated in the dark at room temperature for 2 weeks. 1 L of deionized water is added to the mycelia and homogenized for 30 s. The inoculum is kept on ice until needed. Two holes (1 cm in diameter and 4-5 cm deep) are made on each side of the plant, and 10 mL of the inoculum is poured into each hole. The holes are then filled with the surrounding soil, and disease development is monitored daily for 2 weeks8,10.
In the second method, the plants are inoculated with pathogen-infested toothpicks. For this approach, the plants should be used approximately 6 weeks after transplanting and should have a minimum height of 30 cm. Autoclaved toothpicks are placed on the surface of cultures containing P. nicotianae mycelia. The culture dishes are then stored under the light at room temperature for 7 days. Then, colonized toothpicks are used to inoculate the plants. Toothpicks are inserted into the tobacco stems between the fourth and fifth nodes. The plants are monitored daily for 5 days9,15. This method is not applicable for small seedlings. As the inoculum is pathogen-infested toothpicks, the inoculation intensity cannot be precisely controlled.
The most frequently used approach involves oat grains for inoculation. In this case, oat grains are prepared by autoclaving 500 mL of oats and 300 mL of deionized water at 121 &#176;C for 1 h once per day for 3 days. Then, oat grains are added to the pathogen-colonized culture medium. The dishes are sealed with paraffin film and incubated at 25 &#176;C in light for 7-12 days. Four separate 5 cm deep holes are madeon the potting soil, 4 cm from each plant, and one pathogen-infested oat grain is placed into each hole. The incubation period is determined based on when the first aboveground symptom occurrs7,11,12,13,14,15,16. This method is efficient and applicable for large-scale resistance screening. However, one limitation of this approach is that the inoculum is pathogen-infested oat grains, hence the inoculation intensity cannot be precisely controlled.
However, presented here is a more accurate method that is applicable to growth chamber resistance evaluation. Compared to the other approaches, the inoculum is zoospore suspension, hence the inoculation intensity is controllable and adjustable. As the tobacco plants in this study are cultivated without soil, the results are easier to observe. Moreover, sampling plant roots from soil always causes damage to the roots, which induces a series of physiological responses17. In this method, as plants are cultivated without soil, the interference in root damage can be eliminated. In conclusion, this method is more practical for molecular mechanism research and precision breeding. Using this protocol, data are typically obtained within 5 days, with more than 200 plants evaluated in a single experiment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sinopharm</td>
			<td>10002917</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>(NH4)6 Mo7O24&#8226;2 H2O</td>
			<td>Sinopharm</td>
			<td>XW131067681</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>1.5 ml Safe-lock Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>Used for Sample Extarction</td>
		</tr>
		<tr>
			<td>2 ml Safe-lock Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td>Used for Sample Extarction</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>MDBio, Inc</td>
			<td>9002-18-0</td>
			<td>Materials of Culture Medium</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>AOHAOSI</td>
			<td>AX2202ZH</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Yamatuo</td>
			<td>SQ510C</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>YAMATUO</td>
			<td>SQ510C</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>Bio Best</td>
			<td>DHSB-2L</td>
			<td>Materials of Culture Medium</td>
		</tr>
		<tr>
			<td>Biological Incubator</td>
			<td>JINGHONG</td>
			<td>SHP-250</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Ca(NO3)2&#8226;4 H2O</td>
			<td>Sinopharm</td>
			<td>80029062</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sinopharm</td>
			<td>10005817</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>CuSO4&#8226;5 H2O</td>
			<td>Sinopharm</td>
			<td>10008218</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>Electromagnetic Oven</td>
			<td>Bio Best</td>
			<td>DHDCL</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>FeSO4&#8226;7 H2O</td>
			<td>Sinopharm</td>
			<td>10002918</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>Filter Paper</td>
			<td>Bio Best</td>
			<td>DHLZ-9CM</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Fluorescence Ration PCR Instrument</td>
			<td>Roche</td>
			<td>LightCycler96</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Bio Best</td>
			<td>17071202</td>
			<td>Materials of Culture Medium</td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>Phytotechnology</td>
			<td>B210-500G</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Solarbio</td>
			<td>17072801</td>
			<td>Material for disease-resistant&#160; identification</td>
		</tr>
		<tr>
			<td>K2SO4</td>
			<td>Sinopharm</td>
			<td>10017918</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Sinopharm</td>
			<td>10017218</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>KT Foam Sheet</td>
			<td>Bio Best</td>
			<td>DHKTB</td>
			<td>Material for Seedling</td>
		</tr>
		<tr>
			<td>Low Constant Incubator</td>
			<td>Jinghong</td>
			<td>SHP-250</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Measuring Cylinder</td>
			<td>Bio Best</td>
			<td>DHBLLT-1000ML</td>
			<td>Materials of Culture Medium</td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7 H2O</td>
			<td>Sinopharm</td>
			<td>10013080</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ECHO</td>
			<td>RVL-100-G</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>MnCl2&#8226;4 H2O</td>
			<td>Sinopharm</td>
			<td>G5468154</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>Na2-EDTA</td>
			<td>Sinopharm</td>
			<td>G21410-250</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;2 H2O</td>
			<td>Sinopharm</td>
			<td>20040717</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>NH4NO3</td>
			<td>Sinopharm</td>
			<td>B64586-100g</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>Oatmeal</td>
			<td>Bio Best</td>
			<td>DHYMP-1.5KG</td>
			<td>Materials of Culture Medium</td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Bio Best</td>
			<td>DHPYM-9CM</td>
			<td>Material for disease-resistant&#160; identification</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>THERMO</td>
			<td>S1</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Potting</td>
			<td>Bio Best</td>
			<td>DHYCXHP-12CM</td>
			<td>Material for Seedling</td>
		</tr>
		<tr>
			<td>Potting Soil</td>
			<td>Bio Best</td>
			<td>DHYMJZ-50L</td>
			<td>Seedling Material</td>
		</tr>
		<tr>
			<td>Punch</td>
			<td>Bio Best</td>
			<td>DHDKW</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>qRT-PCR Plate</td>
			<td>Monad</td>
			<td>MQ50401S</td>
			<td>qRT-PCR Plate</td>
		</tr>
		<tr>
			<td>SYBR&#160;Green Premix&#160;Pro Taq&#160;HS qPCR Kit</td>
			<td>Accurate Biology</td>
			<td>AG11718</td>
			<td>PCR Reagent</td>
		</tr>
		<tr>
			<td>Toothpick</td>
			<td>Bio Best</td>
			<td>DHYQ-900</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Total RNA Kit II</td>
			<td>Omega</td>
			<td>R6934-01</td>
			<td>PCR Reagent</td>
		</tr>
		<tr>
			<td>TransScript&#174;&#160;II One-Step gDNA Removal and cDNA Synthesis SuperMix</td>
			<td>Transgen</td>
			<td>AH311-02</td>
			<td>PCR Reagent</td>
		</tr>
		<tr>
			<td>Trays</td>
			<td>Bio Best</td>
			<td>DHYMTP-90G</td>
			<td>Material for Seedling</td>
		</tr>
		<tr>
			<td>Vermiculite</td>
			<td>Bio Best</td>
			<td>DHZS</td>
			<td>Seedling Material</td>
		</tr>
		<tr>
			<td>Water Purification System</td>
			<td>HEAL FORCE</td>
			<td>HSE68-2</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>ZnSO4&#8226;7 H2O</td>
			<td>Sinopharm</td>
			<td>10024018</td>
			<td>Analytical Reagent</td>
		</tr>
	</tbody>
</table>

screening of tobacco genotypes for phytophthora nicotianae resistance

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. P. nicotianae is well adapted to high temperatures; therefore, research on this pathogen is gaining importance in agriculture worldwide because of global warming. P. nicotianae-resistant varieties of tobacco plants are commonly screened by inoculation with oat grains colonized by P. nicotianae and monitoring for the disease symptoms. However, it is difficult to quantify the inoculation intensity since accurate inoculation is crucial in this case. This study aimed to develop an efficient and reliable method for evaluating the resistance of tobacco to infection with P. nicotianae. This method has been successfully used to identify resistant varieties, and the inoculation efficiency was confirmed by real-time PCR. The resistance evaluation method presented in this study is efficient and practical for precision breeding, as well as molecular mechanism research.

4-week-old plants of the resistant variety BH and susceptible variety XHJ were challenged with P. nicotianae using the method presented in this article. The experiment was designed with three replicates, each with 8 plants per group. P. nicotianae infection of the two tobacco varieties, BH and XHJ, is presented in Figure 2. At 3 days post inoculation, for XHJ, stem lesions covered approximately one-half of the stem girth, and one-half of the leaves were slightly wilted; in ...

Watch this Scientific Journal Video about Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance at JoVE.com

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. This is a practical approach for precision breeding, as well as molecular mechanism research.

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. ...

Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. It is practical for precision breeding, as well as molecular mechanism research.Materials. Obtain tobacco varieties Beinhart1000-1, a selection of Beinhart1000 BH, and Xiaohuangjin1025 XHJ from the National Medium-term Geneback of the Tobacco Germplasm Resource of China.BH is resistant, whereas XHJ is susceptible to Phytophthora nicotianae infection. Planting tobacco genotypes for Phytophthora nicotianae resistance evaluation. Mix tobacco seeds with vermiculite and broadcast the seeds gently on the sterilized potting soil.Place the pots in the growth chamber. Maintain a constant temperature of 25 degrees Celsius under a 16-hour light/eight-hour dark photo period. Prepare hydroponic devices with trays and foam sheets.After seeds germinate, prick seedlings out from the potting soil. Wash the roots gently with sterile deionized water and transplant them into the hydroponic devices. Place the devices in climate chambers at 25 degrees Celsius under a six-hour light/eight-hour dark photo period for 24 hours.Prepare Hoagland nutrient solution beforehand. Transplant the seedlings to hydroponic devices with Hoagland nutrient solution. Place the devices in climate chamber at 25 degrees Celsius under 16-hour light/eight-hour dark photo period for two weeks.Preparation of Phytophthora nicotianae zoospore suspension. Preparation of oatmeal agar medium. Weigh 33 grams of oatmeal into a hollowware and add 1, 000 milliliters of sterile water.Boil on an electromagnetic oven. After the oatmeal becomes sticky, strain the liquid through a piece of sterile gauze. Pour the liquid into a 1, 000 milliliter graduated cylinder and adjust the volume to 1, 000 milliliter with sterile water.Pour the liquid into a glass reagent bottle and add 18 grams of agar. Shake well and autoclave the mixture oatmeal agar medium at 121 degrees Celsius for 15 minutes. Leave it at room temperature for 30 minutes.Pour around 20 milliliters of sterilized oatmeal agar medium to each Petri dish. Leave the Petri dishes at room temperature to cool thoroughly before mycelial cultivation. Mycelial cultivation.Prepare one centimeter diameter punches and toothpicks beforehand by autoclaving them in an autoclave at 121 degrees Celsius for 15 minutes. Punch holes in Phytophthora nicotianae mycelial agar cultures to make round mycelial mats. Pick out mycelial mats, place the mycelial side down on oatmeal agar medium and incubate the mycelium at 25 degrees Celsius in the dark for 14 days.Preparation of Phytophthora nicotianae zoospore suspension. Add 0.1%potassium nitrate solution to each mycelial cultivation 15 milliliters per dish, followed by culturing at four degrees Celsius for 20 minutes to induce sporangium. Keep the dishes at 25 degrees Celsius for 25 minutes to release zoospores.Collect the zoospore suspension to a beaker and measure the zoospore concentration by microscope and hemocytometer. Adjust the zoospore concentration to one times 10 to the fourth zoospore per milliliter with sterile water. Identification of disease-resistant tobacco varieties.Take seedlings from Hoagland nutrient solution and inoculate them by immersing the roots in 20 milliliters of Phytophthora nicotianae zoospore suspension in a Petri dish 90 millimeters at 25 degrees Celsius for three hours in the dark. After inoculation, put tobacco seedlings to new Petri dishes with 10 milliliters of sterile water, immersing the roots. Keep the roots moist by covering with two pieces of filter paper.Keep the dishes in 25 degrees Celsius 16-hour light/eight-hour dark photo period. After two to three days, observe the disease severity. For the control treatment, put the tobacco seedlings directly in the Petri dishes with 10 milliliter sterile water, immersing the roots and cover the roots by two pieces of filter paper.Evaluation of Phytophthora nicotianae infection. Evaluate disease severity four to five days after inoculation. Based on the Chinese national standard, score individual plant disease severity on a scale from zero to nine.Grade 0 means no symptoms on the whole plant. Grade 1 means stem lesions less than 1/3 of stem girth or 1/3 of leaves wilting. Grade 3 means stem lesions between 1/3 and 1/2 of stem girth or between 1/3 and 1/2 of leaves slightly wilting.Grade 5 means stem lesions greater than 1/2 of stem girth, but not completely around the girth or between 1/2 and 2/3 of leaves wilting. Grade 7 means stem lesions around whole stem girth or greater than 2/3 of leaves wilting. Grade 9 means plants look dead.Calculate the disease index using the following formula. Disease severity was divided into six grades. Representative results.Four-week-old plants of the resistant variety BH and susceptible variety XHJ were challenged with Phytophthora nicotianae using the method presented in this article. At three days post inoculation, in XHJ, stem lesions covered approximately 1/2 of the stem girth and 1/2 of the leaves were slightly wilted. In the resistant variety BH, no symptoms were observed.At four days post inoculation, leaf wilting and severe stem lesions occurred in XHJ whereas these symptoms did not appear in BH.At five days post inoculation, individual plant disease severity was recorded and calculated based on the Chinese national standard for the grade and investigation method of tobacco diseases and insect pests. The mean disease index of BH was 6.48, which shows resistance R according to the standard, and the mean disease index of XHJ was 76.85, which shows susceptibility S according to the standard. To confirm the inoculation efficiency, relative pathogen biomass was quantified by realtime PCR.The result of the realtime PCR confirms the phenotypic observations. Five progenies from the BC4 F2 population of a cross between BH and XHJ, two intermediate resistance varieties, K326 and Yunyan87, were evaluated by zoospore suspension inoculation method and oat grains inoculation method. Zoospore suspension method was performed on small seedlings and oat grains inoculation method was performed on adult plants.For the five progenies from the BC4 F2 population, the disease index ranged from 16.49 to 77.60 by zoospore suspension method and ranged from 10.33 to 83.08 by oat grains method. The resistance classifications between two infection methods are mostly consistent with each other. With the two intermediate resistance varieties K326 and Yunyan87, the evaluation showed resistant in both methods.These data illustrate the correlation between two inoculation method despite they were performed on tobacco in different growth period. The protocol described here is an efficient and reliable method to evaluate the resistance of tobacco to infection by P.nicotianae seedling stage. This protocol is practical for breeding as well as for molecular mechanism research as the oat grain method is applicable for large-scale resistance screening of adult plants.This two methods can use complementary. Thanks for watching.

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Research

JoVE Journal

Biology

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

A Finite Element Approach for Locating the Center of Resistance of Maxillary Teeth

The center of resistance (CRES) is regarded as the fundamental reference point for predictable tooth movement. The methods used to estimate the CRES of teeth range from traditional radiographic and physical measurements to in vitro analysis on models or cadaver specimens. Techniques involving finite element analysis of high-dose micro-CT scans of models and single teeth have shown a lot of promise, but little has been done with newer, low-dose, and low resolution cone beam computed tomography (CBCT) images. Also, the CRES for only a few select teeth (i.e., maxillary central incisor, canine, and first molar) have been described; the rest have been largely ignored. There is also a need to describe the methodology of determining the CRES in detail, so that it becomes easy to replicate and build upon.
This study used routine CBCT patient images for developing tools and a workflow to obtain finite element models for locating the CRES of maxillary teeth. The CBCT volume images were manipulated to extract three-dimensional (3D) biological structures relevant in determining the CRES of the maxillary teeth by segmentation. The segmented objects were cleaned and converted into a virtual mesh made up tetrahedral (tet4) triangles having a maximum edge length of 1 mm with 3matic software. The models were further converted into a solid volumetric mesh of tetrahedrons with a maximum edge length of 1 mm for use in finite element analysis. The engineering software, Abaqus, was used to preprocess the models to create an assembly and set material properties, interaction conditions, boundary conditions, and load applications. The loads, when analyzed, simulated the stresses and strains on the system, aiding in locating the CRES. This study is the first step in accurate prediction of tooth movement.

This study outlines the necessary tools for utilizing low-dose three-dimensional cone beam-based patient images of the maxilla and maxillary teeth to obtain finite element models. These patient models are then used to accurately locate the CRES of all the maxillary teeth.

The center of resistance (CRES) is regarded as the fundamental reference point for predictable tooth movement. The methods used to estimate the CRES of ...