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 micr...

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 gr...

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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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7.0K 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 ...

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 ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...