Projects at the M.S.K lab are supported by the National Institute of Plant Genome Research core funding. VI acknowledges DBT- JRF (DBT/2015/NIPGR/430). We thank trainee students, Miss. Rishika, Mr. Jayachendrayan, and Miss. Durgadevi for technical help during video shooting and Mr. Sandeep Dixit, Miss. Anjali and Dr. Avanish Rai for critically assessing raw data and the manuscript files. We thank Mr. Rahim H Tarafdar and Mr. Sunder Solanki for their help in the laboratory. We acknowledge DBT-eLibrary Consortium (DeLCON) and NIPGR Library for providing access to e-resources and NIPGR Plant Growth Facility for plant growth support/space.

1. Isolation of R. bataticola and storage
<ol>
	<li>Details of the chickpea genotype and DRR symptoms
	<ol>
		<li>Use chickpea plants (genotype, JG 62) that generally show typical DRR symptoms, such as dry, brittle primary root with no lateral roots and microsclerotia beneath the bark and inside the pith1,3.</li>
	</ol>
	</li>
	<li>Collection and washing
	<ol>
		<li>Uproot plants showing symptoms such as dry straw-colored foliar and brittle primary root with microsclerotia under the epidermis layer. While uprooting, a major part of the roots will remain inside the soil as the infected roots are brittle. Remove the coarse soil debris attached to the root. Cut the roots separately and collect them in paper envelopes (27.5 cm x 12 cm) with the proper label and place them in a sample collection box.</li>
		<li>After transporting samples to the laboratory, place the roots in a 200 mL beaker and cover with mesh (Nylon mesh with the pore size of 3 mm diameter), and wash the roots thoroughly with running tap water to remove adhering soil particles.</li>
		<li>Use reverse osmosis (RO) water to rinse once at the end.</li>
	</ol>
	</li>
	<li>Surface sterilization
	<ol>
		<li>Break the roots into four pieces with 2 cm length each with a scalpel blade and put them in a clean 200 mL beaker.</li>
		<li>Assemble autoclaved RO water, 2% NaOCl, discarding jar, and autoclaved blotting paper (5 cm2) inside the laminar flow chamber.</li>
		<li>Wash the roots with 50 mL of autoclaved RO water thrice and then with 50 mL of 2% NaOCl for 10 min. Wash the roots with 50 mL of RO water three times again to remove the NaOCl.</li>
		<li>Blot dry the roots by placing the roots on autoclaved blotting paper and leave until the roots are dried.</li>
	</ol>
	</li>
	<li>Media and incubation
	<ol>
		<li>Remove the edges of the roots with a sterilized scalpel blade. Split the roots using the blade and use the sterilized forceps to place them on a potato dextrose agar (PDA) media Petri plate containing streptomycin sulfate (50 mg L-1) and ampicillin (50 mg L-1).</li>
		<li>Close the plate, seal with parafilm, and incubate in an incubator at 28 &#176;C for two days in the dark.</li>
	</ol>
	</li>
	<li>Hyphal tip method
	<ol>
		<li>After two days of incubation, bring the plates into the laminar flow chamber. Cut the tip of the hyphal growth8 under a stereomicroscope (Leica EZ4 educational stereomicroscope) and transfer it into a fresh PDA plate with streptomycin sulfate and ampicillin.</li>
		<li>Incubate the plates in the incubator at 28 &#176;C for ten days in the dark.</li>
	</ol>
	</li>
	<li>Storage and maintenance of R. bataticola fungal inoculum
	<ol>
		<li>Make PDA slants in test tubes and transfer a fungal agar plug from a ten-days-old culture plate using the flame sterilized inoculation loop. Seal the test tube cap with parafilm.</li>
		<li>Incubate the slants at 28 &#176;C for ten days in the dark.</li>
		<li>Seal the cap with parafilm again and store it at 4 &#176;C in the refrigerator for the future use.</li>
		<li>Subculture every six months to maintain the fungus.</li>
	</ol>
	</li>
	<li>Maintenance of virulence
	<ol>
		<li>Infect the plants with pure fungal inoculum prepared as mentioned in the sick pot technique3. Isolate the same fungus from the infected plants (Koch&#8217;s postulates)9 and use for further experiments. 
		NOTE: In this study, a field isolated strain of the fungus was used (GenBank: MH509971.1 https://www.ncbi.nlm.nih.gov/nuccore/MH509971)</li>
	</ol>
	</li>
</ol>
2. Blotting paper technique
NOTE: The blotting paper technique entails the preparation of liquid fungal inoculum, seedling preparation, and disease assessment.
<ol>
	<li>Preparation of liquid R. bataticola inoculum 
	NOTE: Liquid R. bataticola fungal inoculum contains both mycelia and microsclerotia. Both these structures act as primary inoculum.
	<ol>
		<li>Prepare 500 mL of PDB media in a 1,000 mL flask and autoclave it at 121 lbs for 15 min.</li>
		<li>After the media is cooled down, inoculate the broth with a loopful of fungal agar plug from a fungal slant culture and incubate at 28 &#176;C for five days in a shaker at 180 rpm in the dark.</li>
		<li>Assemble an autoclaved glass or plastic funnel (10 cm), one-liter conical flask, and two layers of mesh (10 cm2 size) (Nylon mesh with the pore size of 3 mm diameter) on the table. Filter out the fungal mycelia and microsclerotia using the mesh. Blot dry the fungus in autoclaved blotting paper.</li>
		<li>Weigh 100 g mycelia for 50% inoculum and keep at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of chickpea plant
	<ol>
		<li>Select 50 healthy seeds (in this study, the DRR-susceptible genotype JG 62 was used), place them in a 100 mL beaker, and cover with a mesh.</li>
		<li>Wash the seeds with tap water first to remove soil debris.</li>
		<li>Take the beaker with seeds into the laminar flow chamber and wash them with sterilized 50 mL of RO water thrice for 1 min per wash.</li>
		<li>Sterilize the seeds with 50 mL of 2% aqueous NaOCl for 2 min with continuous shaking and wash the seeds with 50 mL of sterilized water five times for 1 min each.</li>
		<li>Fill a polythene bag (47.5 cm x 25 cm) with Soilrite (a mixture of horticulture grade expanded perlite, Irish Peat moss, and exfoliated vermiculite in equal ratio i.e., 1/3:1/3:1/3), sow the surface-sterilized 50 seeds two cm deep, and keep in a growth chamber/room with 28 &#176;C &#177; 2 &#176;C temperature, 16 h photoperiod with a light intensity of 150 &#956;mol m&#8722;2 s&#8722;1, and relative humidity of 70%. Water them with RO water and uproot the plants eight days after sowing.</li>
		<li>Wash the roots in tap water to remove the Soilrite particles. Rinse the roots with sterilized RO water, and keep them in the RO water in a 1000 mL beaker.</li>
	</ol>
	</li>
	<li>Preparation of blotting paper in trays
	<ol>
		<li>Take a blotting paper and cut them into pieces (30 cm &#215; 23 cm) in sufficient numbers to meet the replications.</li>
		<li>Pack the sheets in autoclavable polythene bag&#160;and autoclave them at 121 lbs for 15 minutes and keep it in a hot air oven for drying.</li>
		<li>Fold each blotting paper sheet in half.</li>
		<li>Place the paper on a clean, spirit-wiped plastic tray, as shown in the Figure 2i-iv.</li>
	</ol>
	</li>
	<li>Plant inoculation by dipping
	<ol>
		<li>Dissolve 100 g of fungal inoculum in 200 mL autoclaved RO water in a 200 mL beaker to obtain 50% inoculum.</li>
		<li>Dip the plant roots in the prepared inoculum for 1 min with intermittent up-and-down movement to ensure uniform attachment of fungal inoculum.</li>
	</ol>
	</li>
	<li>Placing the plants in the blotting paper
	<ol>
		<li>Wet the bottom side of the blotting paper placed on the tray with sterile RO water. Place the plants on the paper in a way where only the roots are covered by the paper, and shoots are left out.</li>
		<li>Close it by folding the top side of the blotting paper and wet the entire paper to provide enough water to sustain plant growth.</li>
		<li>Water the tray once a day and keep the trays at 28 &#176;C. Observe the symptoms such as necrosis, root rot, and leaf yellowing daily.</li>
	</ol>
	</li>
</ol>
3. Sick pot technique
NOTE: The sick pot technique entails the preparation of virulent inoculum and a sick pot, maintenance of moisture level, and assessment of disease symptoms.
<ol>
	<li>Preparation of substrate
	<ol>
		<li>Take one kg of any commercially available chickpea seeds. Remove infected seeds (seeds with fungal growth, infection spots, and insect damage). Place them in 5 L plastic beaker and wash with tap water thoroughly 3&#8211;4 times to remove major debris.</li>
		<li>Wash the seeds with 2 L of RO water thrice and soak the 1 kg seeds in a 5 L beaker with threefold more water for five h.</li>
		<li>Once the seeds imbibed the water, rewash them with RO water thrice to remove the seed exudates.</li>
		<li>Remove the water completely and pack the seeds in jam bottles (300 mL, 12 cm height, 6 cm diameter, and 155 g weight) to about 1/4th capacity (100 g per jam bottle) and close the bottles with caps. Pack ten jam bottles in an autoclavable plastic bag (48 cm x 30 cm) autoclave twice at 121 lbs for 15 min continuously.</li>
		<li>Dry the seeds at 40 &#176;C in an oven overnight to remove the water droplets inside the bottle.</li>
	</ol>
	</li>
	<li>Preparation of sick culture
	<ol>
		<li>Turn on the BSL-2 level laminar flow chamber. Wipe the floor thoroughly with 70% ethanol, and turn on UV for 15 min. Then, keep the seeds inside the laminar flow chamber.</li>
		<li>Take 10-day-old freshly isolated R. bataticola culture (part 1). Then, take three fungal agar plugs (4 mm diameter) using a sterile pipette tip or cork borer, and put them into jam bottles aseptically. Cover the bottles with caps and seal with parafilm.</li>
		<li>Shake the bottles to mix the fungal disc with the chickpea seeds uniformly.</li>
		<li>Incubate the bottles at 30 &#176;C for 15 days in the dark.</li>
		<li>Take jam bottles with black fungal growth (Figure 4iii) and transfer the sick culture (seeds with microsclerotia) from jam bottles to a glass Petri plate using sterile forceps. Dry them at room temperature for two days in a glass Petri plate.</li>
		<li>Powder the fungal mass using a mortar and pestle, and store the powder at 4 &#176;C.</li>
		<li>Autoclave Soilrite twice at 121 lbs for 15 min.</li>
		<li>Dry the autoclaved Soilrite mix under a sunshade.</li>
		<li>Mix the fungal powder with Soilrite at 50% w/w, fill the pots with the mixture, and keep them at room temperature for a day.</li>
		<li>Sow a surface-sterilized DRR-susceptible chickpea seed per pot (10 cm round pots) (Table of Materials) and maintain a moisture level of 80% field capacity (FC).</li>
		<li>Observe the symptoms such as necrosis and root rot by uprooting the plants after germination.</li>
	</ol>
	</li>
	<li>Assessment of sick pot efficiency 
	NOTE: Plants show yellow foliar symptoms when the roots are completely rotten.
	<ol>
		<li>Develop a disease score based on the lesion (necrotic spots) (Supplementary Figure 2C) numbers and severity of root rot. Sick pots showing 90% plant death can be used further.</li>
	</ol>
	</li>
	<li>Genotype screening
	<ol>
		<li>Prepare sick culture in large quantities and mix it either with sterilized Soilrite or field soil (5% w/w) in 30 cm-tall pots. Water the pots to wet the surface and leave them undisturbed for seven days.</li>
		<li>Sow one surface-sterilized seed per 10 cm round pot and three seeds per 30 cm round pots and water them adequately.</li>
		<li>Observe the yellow foliar and root rot symptoms.</li>
	</ol>
	</li>
	<li>Combined drought and R. bataticola infection
	<ol>
		<li>Impose drought stress by following the protocols mentioned in Sinha et al. (2019).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Sharma, M., Ghosh, R., Pande, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+root+rot+(Rhizoctonia+bataticola+(Taub.)+Butler):+an+emerging+disease+of+chickpea+-+where+do+we+stand.">Dry root rot (Rhizoctonia bataticola (Taub.) Butler): an emerging disease of chickpea - where do we stand.</a> Archives of Phytopathology and Plant Protection. 48 (13-16), 797-812 (2015).</li><li>Sinha, R., Irulappan, V., Mohan-Raju, B., Suganthi, A., Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+drought+stress+on+simultaneously+occurring+pathogen+infection+in+field-grown+chickpea.">Impact of drought stress on simultaneously occurring pathogen infection in field-grown chickpea.</a> Scientific Reports. 9 (1), (2019).</li><li>Nene, Y., Haware, M., Reddy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chickpea+diseases:+resistance+screening+techniques,+information+bulletins+No.+10.+Patancheru.">Chickpea diseases: resistance screening techniques, information bulletins No. 10. Patancheru.</a> Information Bulletin No. 10. Patancheru, A.P., India: International CroDS Research Institute for the Semi-Arid Tropics. , 1-10 (1981).</li><li>Pande, S., Krishna Kishore, G., Upadhyaya, H. D., Narayana Rao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+sources+of+multiple+disease+resistance+in+mini-core+collection+of+chickpea.">Identification of sources of multiple disease resistance in mini-core collection of chickpea.</a> Plant Disease. , (2006).</li><li>Pandey, P., Irulappan, V., Bagavathiannan, M. V., Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+combined+abiotic+and+biotic+stresses+on+plant+growth+and+avenues+for+crop+improvement+by+exploiting+physio-morphological+traits.">Impact of combined abiotic and biotic stresses on plant growth and avenues for crop improvement by exploiting physio-morphological traits.</a> Frontiers in Plant Science. 8, (2017).</li><li>Irulappan, V., Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morpho-physiological+traits+and+molecular+intricacies+associated+with+tolerance+to+combined+drought+and+pathogen+stress+in+plants.">Morpho-physiological traits and molecular intricacies associated with tolerance to combined drought and pathogen stress in plants.</a> Biotechnologies of Crop Improvement, Volume 3: Genomic Approaches. , (2018).</li><li>Jensen, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+methods+for+fungal+brood+disease+research.">Standard methods for fungal brood disease research.</a> Journal of Apicultural Research. 52 (1), 1-39 (2013).</li><li>Agrios, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Pathology: Fifth Edition. , 9780080473 (2004).</li><li>Coley-Smith, J. R., Cooke, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+germination+of+fungal+sclerotia.">Survival and germination of fungal sclerotia.</a> Annual Review of Phytopathology. , (1971).</li><li>Nagamma, G., Saifulla, M., Sab, J., Pavitra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Screening of chickpea genotypes against dry root rot caused by Macrophomina phaseolina (tassi) goid. 10 (4), 1795-1800 (2015).</li><li>Infantino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+techniques+and+sources+of+resistance+to+root+diseases+in+cool+season+food+legumes.">Screening techniques and sources of resistance to root diseases in cool season food legumes.</a> Euphytica. 147 (1-2), 201-221 (2006).</li><li>Khaliq, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+control+of+dry+root+rot+of+chickpea+caused+by+Rhizoctonia+bataticola+under+the+natural+field+condition.">Integrated control of dry root rot of chickpea caused by Rhizoctonia bataticola under the natural field condition.</a> Biotechnology Reports. 25, 00423 (2020).</li></ol>

We have nothing to disclose

The blotting paper technique provides a straightforward approach to screen chickpea genotypes under laboratory conditions. Dip inoculation enables the investigation of interaction on a temporal basis with easy control over inoculum load (Supplementary Figure 1) and facilitates in vitro screening. Furthermore, even young seedlings can be used. Five-day-old fungal culture (Figure 1B) can yield enough inoculum to infect the plants. Liquid inoculum contains both mycelia and microsclerotia (Figure 1D &#38; E). Root rot symptoms (Figure 3B) can be used to score the disease and identify resistant genotypes. DRR occurrence is majorly influenced by drought stress1,3,5. However, with this technique alone, drought stress imposition is impossible, and screening with this technique will not reflect natural responses.
The sick pot technique allows the study of the interactions among plants, pathogens, and drought stress. It provides a way to screen the genotypes under combined drought and pathogen stress to identify resistant genotypes. In a sick pot, drought stress can be imposed at any age of the plant and screen the plants. Plants show typical DRR symptoms (Figure 5C &#38; F and Supplementary Figure 2B &#38; C) in the sick pot method. Plants subjected to combined drought and pathogen infection showed severe root rot as compared to pathogen-only treatment. This implies that available chickpea germplasm needs to be screened to identify a resistant genotype to not only pathogen but also combined pathogen and drought stress. Several studies were attempted earlier to screen the genotypes but by using blotting paper technique11,12. Besides, field screening has also been conducted but without imposing drought stress11. It is crucial to impose drought stress during different stages of chickpea and assess the genotype response.
TROUBLE SHOOTING
For the blotting paper technique, the volume of the inoculum in the beaker should be at the level where the entire plant root is dipped. Additionally, excess watering will lead to wet rot of the plant roots. Do not use tap water for watering the plants because it can cause contamination.
For the sick pot technique, Desi chickpea varieties are preferable. The number of seeds can vary depending upon the researchers&#8217; needs. The volume of water used to soak seeds should be threefold more because the seeds imbibe water. Bacterial growth will occur if the washing is not done correctly. Non-autoclaved water can be used to soak the seeds. The color of the seeds after autoclaving should be black instead of brown, which indicates improper autoclaving. Over drying in a hot air oven leads to the drying of seeds; such seeds cannot be used for fungal inoculation. Inoculating the bottles outside the laminar flow may cause contamination. White fungal growth in inoculated chickpea meal is a sign of improper autoclaving. Biosafety precautions should be followed in discarding the used inoculum, blotting paper, and infected plants.

Dry root rot (DRR) is one of the economically significant diseases in chickpea1,2. It is a root-specific disease caused by Rhizoctonia bataticola (teleomorph, Macrophomina phaseolina). Infected plants lack lateral roots and possess brittle taproots and yellow foliage1,3. DRR under drought stress has been reported to be an emerging threat to chickpea cultivation1,2,3. Moreover, DRR incidence is reported to be aggravated under drought stress under field conditions1,2,3. DRR is more prevalent in rainfed areas than in irrigated fields4. The utilization of resistant varieties is the way to overcome the disease and circumvent fungicide use1,13. Because chickpea germplasm available across the globe harbors genetic variation for the trait5, the screening and identification of resistant/susceptible genotypes are critical for molecular breeding for crop improvement.
Robust, easy, and cost-effective disease assays are essential to investigate R. bataticola infection patterns in chickpea. The primary disease assay used to observe the response of chickpea genotypes to R. bataticola infection is the blotting paper technique1,4. It is a simple technique and can be executed using liquid fungal inoculum, seedlings with roots, and sterile blotting paper. However, this technique has not been utilized to its maximum because no step-by-step-protocol is available in the literature.
Meanwhile, the sick pot technique involves the preparation of a potential sick culture and the imposition of drought stress. Given that drought stress aggravates DRR disease incidence3, it is essential to study the plant-pathogen interaction under drought stress6,7. The sick pot technique provides the platform for such a simultaneous study, promoting better possibilities for germplasm screening and understanding the mechanistic basis of the interaction. Pathomorphological changes such as an increase in root length and reduction in lateral root number&#8212;inherent to DRR disease&#8212;can be addressed using the sick pot technique1,3,7.
Herein, a detailed protocol for blotting paper and sick pot techniques, which can be used to study the interaction between chickpea and R. bataticola and screen chickpea germplasm, is presented. The details of the materials used in the study are given in the&#160;Table of Materials.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fungus- Rhizoctonia bataticola</td>
			<td>Pathogen inoculum</td>
			<td>Indian Type Culture Collection No. 8365</td>
			<td>GenBank: MH509971.1, ITCC 8635 (https://www.iari.res.in/index.php?option=com_content&#38;view=article&#38; id=1251&#38;Itemid=1370)</td>
		</tr>
		<tr>
			<td>Soilrite mix</td>
			<td>Soil medium in the lab</td>
			<td>Keltech Energies Limited, Bangalore, India</td>
			<td>http://www.keltechenergies.com/</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Blotting paper to support the plant growth</td>
			<td>Himedia</td>
			<td>http://himedialabs.com/catalogue/chemical2017/index.html#374</td>
		</tr>
		<tr>
			<td>Pot</td>
			<td>Growing plants</td>
			<td>10 and 30 cm size pots</td>
			<td>Routinely used nursery pots, for example, https://dir.indiamart.com/impcat/nursery-pots.html</td>
		</tr>
		<tr>
			<td>Potato dextrose agar/broth</td>
			<td>Culture and maintain the fungus</td>
			<td>Cat# 213400, DifcoTM, MD, USA</td>
			<td>https://www.fishersci.com/shop/products/bd-difco-dehydrated-culture-media-potato-dextrose-agar-3/p-4901946</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Culture the fungus</td>
			<td>LOM-150-2, S/N AI13082601-38, MRC, incubator, and shaker</td>
			<td>http://www.mrclab.com/productDetails.aspx?pid=91131</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Growing plants in controlled condition</td>
			<td>Model No. A1000, Conviron, Canada</td>
			<td>https://www.conviron.com/products/gen1000-reach-in-plant-growth-chamber</td>
		</tr>
		<tr>
			<td>Laminar airflow</td>
			<td>Carrying out aseptic exercises</td>
			<td>Telstar, Bio II advance, Class II cabinet, EN-12469-2000</td>
			<td>https://www.telstar.com/lab-hospitals-equipment/biological-safety-cabinets/bio-ii-advance-plus/, http://www.atlantisindia.co.in/laminar-air-flow.html</td>
		</tr>
		<tr>
			<td>Mesh</td>
			<td>Filtering the fungal mycelia</td>
			<td>Nylon mosquito net</td>
			<td>Mesh with 0.6-1 mm diameter pore size</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Autoclaving media and chickpea seeds</td>
			<td>Autoclave</td>
			<td>http://www.scientificsystems.in/autoclave</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td>Visualizing the infection ang fungal mycelia</td>
			<td>SMZ25 / SMZ18, Research Stereomicroscopes, Leica EZ4 educational stereomicroscope</td>
			<td>https://www.microscope.healthcare.nikon.com/products/stereomicroscopes-macroscopes/smz25-smz18 
			https://www.leica-microsystems.com/products/stereo-microscopes-macroscopes/p/leica-ez4/ 
			https://www.microscopyu.com/museum/eclipse-80i</td>
		</tr>
		<tr>
			<td>Weighing balance</td>
			<td>Weighing fungus and chemicals</td>
			<td>Sartorius Electronic Weighing Balance, BSA 4202S-CW</td>
			<td>https://www.sartorius.com/en/products/weighing/laboratory-balances</td>
		</tr>
		<tr>
			<td>WGA-FITC</td>
			<td>Fungus staining</td>
			<td>Sigma</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/l4895?lang=en&#38;region=IN</td>
		</tr>
		<tr>
			<td>Aniline blue</td>
			<td>Fungus staining</td>
			<td>Himedia</td>
			<td>http://www.himedialabs.com/intl/en/products/Chemicals/Dyes-Indicators-and-Stains/Aniline-blue-Water-soluble-Practical-grade-GRM901</td>
		</tr>
	</tbody>
</table>

dry root rot disease assays in chickpea: a detailed methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study aimed to demonstrate techniques such as blotting paper and sick pot techniques to facilitate pathomorphological and molecular understanding of plant-pathogen interaction under drought stress. To accomplish this, plants exhibiting DRR symptoms1,3,4 were collected from a chickpea field, and the fungus was isolated using the hyphal tip method8. R. bataticola fungal culture appears dark gray on the PDA plate and slant on four days after incubation (Figure 1A &#38; C) and darker in grey color in the PDB medium on five days after incubation (Figure 1B). R. bataticola has septate mycelia (Figure 1D), and it produces microsclerotia (Figure 1E), which act as primary inoculum in the soil10.
The steps given in Figure 2 were followed to execute the blotting paper technique. Eight-day-old plants were infected with liquid inoculum (50%), and eight days after infection, plants were observed for symptoms. Plants showed root rot because of extensive necrosis, as well as leaf yellowing, which is the typical root and foliar symptoms of DRR disease (Figure 3B and Supplementary Figure 1A).
The sick pot technique was carried out using the steps shown in Figure 4. The concentrations of fungal inoculum in Soilrite and field soil was 10% and 5%, respectively. DRR-susceptible seeds show the typical DRR symptoms such as root rot, lack of lateral roots, leaf yellowing, and premature death, as compared to control plants (Figure 5A and B). Plants subjected to infection in the sick pot made with Soilrite died and showed root rot seven days after sowing (Figure 5C and Supplementary Figure 2C). Meanwhile, plants growing in the sick pot made with field soil showed typical foliar symptoms, i.e., straw-colored foliage 48 days after sowing (Figure 5E).
The influence of drought on DRR disease was also studied in the sick pot under laboratory conditions. Drought stress was imposed by withholding water3. The plants under drought stress (30% FC) showed aggravated disease incidence as compared to the pathogen-only-treated (90% FC) plants (Figure 6A and B). Control and drought-treated plants did not show any symptoms (Figure 6A and B). Roots under combined stress had more necrotic spots and rot as compared to pathogen only-plants (Figure 6B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61702/61702fig01.jpg" /> 
Figure 1: Characteristic morphological features of Rhizoctonia bataticola.&#160;The causal agent of dry root rot (Rhizoctonia bataticola, ITCC 8635) was isolated from the field (National Institute of Plant Genome Research, New Delhi, 28.6139&#176;N, 77.2090&#176;E). From the cultures, the fungus was isolated using the hyphal tip method8. Images show the 4-days-old R. bataticola culture in potato dextrose agar in a Petri plate (A), 5-days-old liquid culture (B), and 10-days-old slant culture (C). Fungal mycelia (D) and microsclerotia (black arrows) (E) was teased on a microscope slide and stained with WGA-FITC and aniline blue, respectively. Images (E, F, scale bar, 20, and 50 &#181;m) were captured under the 20x and 40x objective lens of an epifluorescent microscope. White arrows show the cross walls in the mycelia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61702/61702fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61702/61702fig02v2.jpg" /> 
Figure 2: Steps involved in R. bataticola inoculation and DRR assays in the blotting paper technique.&#160;Surface sterilize the seeds by passing them through running tap water and washing with 2% sodium hypochlorite, followed by washing with sterile RO water 3&#8211;4 times (step 1). Then, sow 30 seeds in 15-cm-high pots containing Soilrite (step 2) and allow the seeds to grow for eight days in a growth room with 28 &#176;C &#177; 2 &#176;C temperature, 16 h photoperiod with a light intensity of 150 &#956;mol m&#8722;2 s&#8722;1, and relative humidity of 70% (step 3). Uproot the plants and wash them with sterilized water (step 4). Prepare the fungal inoculum by inoculating 500 mL PDB media with fungus (step 5). For the infection, use 5-days-old fungal broth culture. Then, inoculate the plants by dipping the roots into the fungal inoculum in a beaker for 30 s and remove excess inoculum by touching the inner sidewall of the beaker (step 6). After infection, place fungal-inoculated and mock-inoculated plants in different blotting papers in separate clean trays (step 7). Moisten the blotting paper with adequate sterile water daily and observe the symptoms, viz., shedding off of lateral roots, yellowing and wilting of plant leaves, rotten seeds, and root rot at eight days post-infection (step 8) (A). Images represent essential steps (steps 3&#8211;7) (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61702/61702fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61702/61702fig03.jpg" /> 
Figure 3: DRR disease symptoms in chickpea-based on the blotting paper technique.&#160;Following the protocol depicted in Figure 2, DRR-susceptible plants (genotype, JG 62) were subjected to infection using the blotting paper technique, and symptoms were captured eight days after infection. Images show the representative control plants (mock-inoculated) with healthy shoots (red arrow) and roots with more lateral roots (yellow arrow) (A). Images show the representative infected plants with typical symptoms, such as wilting, yellowing, and drying of leaves (blue arrows) and dried/necrotic roots with fewer lateral roots (white arrows) (B). The scale bar is 1 cm. Experiments were repeated at least five times. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61702/61702fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61702/61702fig04.jpg" /> 
Figure 4: Overview of sick pot preparation for R. bataticola inoculation and DRR disease assays.&#160;Prepare the fungal inoculum by inoculating a PDA plate with a 5 mm fungal disc of actively growing fungal culture and incubate at 28 &#176;C for ten days. Then, for preparing the substrate, wash the chickpea seeds with tap water, soak the seeds in water overnight, and autoclave at 121 lbs for 15 min. Then, inoculate 100 g of the substrate with three agar plugs from 10-day-old culture and mix well. Then, incubate the inoculated substrate at 30 &#176;C for 15 days in an incubator. Crush the sick culture (fungal grown substrate), dry and powder it, and store at 4 &#176;C. Then, mix 50 g of sick culture with 100 g of dry Soilrite thoroughly (sick pot) (A). Then, sow the surface-sterilized susceptible chickpea seeds and observe the symptoms, viz., tap root rot, lateral root necrosis, and leaf yellowing. Assimilate the plants showing symptoms in the same pot. For further experiments, use the pots showing 90% infection as the susceptible genotype. Images represent the inoculum (i), the substrate (ii), and control and inoculated sick culture (iii) (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61702/61702fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61702/61702fig05v3.jpg" /> 
Figure 5: DRR disease symptoms in chickpea-based on the sick pot technique.&#160;For making a sick pot, the protocol depicted in Figure 4 was used. Then, surface-sterilized DRR-susceptible chickpea genotype (JG 62) seeds were sown in the control (A) and sick pot (B). Each plant in the pot represents one replicate, and dead or stunted plant growth was observed in the sick pot. Plants on the right side of the panel (C) indicate the presence and absence of lateral roots (yellow arrow) in the control and treatment, respectively. The graph shows the number of dead plants in the sick pot treatment as compared to the control (D). The sick pot was prepared on sterilized field soil collected from the NIPGR field, and sick culture was mixed. Surface-sterilized seeds were sown, and disease symptoms were captured 48 days after sowing. The image shows the control plants (E), and the typical DRR foliar symptoms, namely, the drying of plants, are indicated (white arrows). Statistical significance was determined using Student&#8217;s t-test. The bar represents the SEM of nine biological replicates, and the asterisk denotes a statistically significant value at P &#60; 0.0001. The yellow arrow indicates necrotic/rotten, dried primary root without any lateral roots. Experiments were repeated at least ten times, with similar results. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61702/61702fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61702/61702fig06v3.jpg" /> 
Figure 6: The sick pot method is useful to study the influence of drought stress on plant-pathogen interaction.&#160;A pot experiment was conducted to study the effect of drought on R. bataticola infection. The experiment comprised control, drought-only, pathogen-only (R. bataticola, pathogen), and combined drought and R. bataticola stress (combined stress). Plants were grown in a growth room with 28 &#176;C &#177; 2 &#176;C temperature, 16 h photoperiod with a light intensity of 150 &#956;mol m&#8722;2 s&#8722;1, and 70% relative humidity. The sick pot was prepared following the protocol depicted in Figure 4. Then, surface-sterilized DDR-susceptible chickpea genotype (JG 62) seeds were sown in control and sick pots. Control and pathogen treatments were irrigated throughout the experiment. Drought stress was imposed on plants under drought and combined stress treatment. Water was withheld 18 days after sowing, and the desired drought level was reached 24 days after sowing. Plants were observed for symptoms 29 days after sowing. The image shows the foliar and root changes under treatments (A). Images show unstained plant roots observed under the 0.5X objective lens of an SMZ25/SMZ18 research stereomicroscope (B). The red arrow indicates the uninfected lateral roots, and the black arrows indicate the infected lateral roots. Experiments were repeated at least ten times, with similar results. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61702/61702fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1: Inoculum size and reduction in lateral root number in chickpea. Following the protocol depicted in figure 2, DRR susceptible plants (genotype, JG 62) were subjected to infection with varied size of inoculum using blotting paper technique, and symptoms were captured eight days after infection. Plants were inoculated with varied inoculum size (0.1%, 1%, 10% and 20% w/v in water)Images show the representative infected plants with typical symptoms such as wilting, yellowing, and drying of leaves and dried/necrotic roots with less lateral roots (B). Graph shows the number of lateral roots in plants under infection with inoculum variation (B). The scale bar is 1 cm. n=10. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61702/61702supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure S2: Foliar symptoms of DRR in chickpea in soilrite. Following the protocol depicted in figure 4, surface sterilized seeds were sown, and disease symptoms were captured on 14 days after sowing. The representative image shows the control plants (A), and the picture shows the typical DRR foliar symptoms, namely drying of plants (white arrows) (B). Disease score was developed based on necrotic spots on the roots. The scoring was developed across the days (C). The photographs (A&#38;B) are from the same experiment. Scale bar= 3 cm. n= 10. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61702/61702supfig02.jpg" target="_blank">Please click here to download this figure.</a>

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Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

dry-root-rot-disease-assays-in-chickpea-a-detailed-methodology

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7.1K Views.  National Institute of Plant Genome Research. This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea–Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

Video: Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

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

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

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

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

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

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

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

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

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

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

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Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

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Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

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