This work was supported by the National Natural Science Foundation of China (31772291), the Research Project for Public Interest in Zhejiang Province (2017C32027), the Key Science Project of Plant Breeding in Zhejiang (2016C02051), and the National Program for the Support of Top-notch Young Professionals (to P.X.).

1. Seed Sterilization and Germination
<ol>
	<li>For surface sterilization, soak the bottle gourd seeds in a 500-mL beaker filled with water at 58 &#176;C with occasional stirring, until the water temperature drops to 40 &#176;C.</li>
	<li>Meanwhile, put 3 kg of peat soil into a nylon bag and, to sterilize it, autoclave it at 120 &#176;C/0.5 MPa for 20 min.</li>
	<li>Keep soaking the bottle gourd seeds for 4 - 5 h more with no stirring.
	<ol>
		<li>Once the water reaches room temperature, rinse the seeds 2x - 3x with distilled water.</li>
		<li>Drain the excess water and allow the seeds to sprout in a gauze bag at 28 &#176;C in a growth chamber in the dark.</li>
		<li>After germination, sow the seeds into plastic pots (6 cm in diameter) filled with sterilized peat soil.</li>
	</ol>
	</li>
	<li>When the bottle gourd seedlings have developed two flattened cotyledons, repeat steps 1.1 - 1.3 with watermelon seeds. 
	NOTE: This time management ensures that the sizes of the scion and rootstock match well for successful grafting.</li>
</ol>
2. Seedling Growth and Grafting
<ol>
	<li>Grow the seedlings in a growth chamber with a 16-h light /8-h dark cycle, keeping the temperature at 28 &#176;C during the day (light) and at 22 &#176;C during the night (dark). Irrigate the seedlings by adding water 1x a day in the late afternoon.</li>
	<li>Use the cut-grafting method22 to make heterografts when the seedlings of the bottle gourd (rootstock) are at the one true-leaf stage and the cotyledons of the watermelon (scion) have emerged (not yet flattened).
	<ol>
		<li>Cut the hypocotyls of the watermelon seedlings at 2 - 3 cm below the cotyledons, and the top of the bottle gourd seedlings at the site immediately above the true leaves.</li>
		<li>Use a toothpick to make a hole in the top of the trimmed bottle gourd seedlings. Insert the trimmed watermelon seedlings into the holes of the bottle gourd seedlings to make heterografts.</li>
	</ol>
	</li>
	<li>Use a similar method as presented in step 2.2 to make homografts. 
	NOTE: Homo- and heterografting combinations should always be made simultaneously (Figure 1), which, in this case, results in the following: watermelon/bottle gourd (WB, heterograft), watermelon/watermelon (WW, homograft) and bottle gourd /bottle gourd (BB, homograft).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58242/58242fig1.jpg" /> 
Figure 1: Illustration of the graft combinations and the grafted plant structures. WB = watermelon/bottle gourd heterografting; WW = watermelon/watermelon homografting; BB = bottle gourd/bottle gourd homo-grafting; WB-S = scion leaves of the watermelon/bottle gourd heterografts that were sampled; WB-R = rootstock leaves of the watermelon/bottle gourd heterografts that were sampled. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58242/58242fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Postgrafting Management, Cold Treatment, and Sampling
<ol>
	<li>Enwrap the grafted seedlings with transparent polyethylene bags to keep a relatively high humidity and maintain them for 7 d under environmental conditions of 16-h light/8-h dark cycles, keeping the temperature at 28 &#176;C during the day (light) and at 22 &#176;C during the night (dark).</li>
	<li>Uncover the transparent polyethylene bags on the 7th day. Let the plants grow for an additional 7 - 10 days under the same conditions.</li>
	<li>Divide the healthy uniform seedlings into two groups, one for cold treatment (stressed) and one for control (non-stressed). For the control group, leave the seedlings in the same growth chamber (at 28 &#176;C) for an additional 48 h, while for the cold-stressed group, transfer the seedlings to a growth chamber with a constant temperature at 6 &#176;C, with light/dark conditions as described in step 2.1.</li>
	<li>Sample the leaves of the scion and the rootstock from the grafts (Figure 1). Freeze the samples immediately in liquid nitrogen and store them at -70 &#176;C until use.</li>
</ol>
4. Library Preparation and High-throughput Sequencing
<ol>
	<li>Transfer the frozen samples to a 2-mL microcentrifuge tube in liquid nitrogen.</li>
	<li>Add a stainless-steel bead (5 mm in diameter) to each tube containing the tissues.</li>
	<li>Homogenize the tissues to a fine powder using a bead mill homogenizer for 30 s.</li>
	<li>For each grafting combination, take equal amounts (0.1 g) of ground sample from ten seedlings and mix them in a 10-mL centrifuge tube. Add an appropriate amount of guanidium hydrochloride reagent (Table of Materials) based on the manufacturer&#39;s suggestions corresponding to the tissue weight.
	<ol>
		<li>Remove genomic DNA contaminations by adding RNA-free DNase I to 150 U/mL at 37 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Determine the total RNA quantity on a microcapillary electrophoresis system to ensure the RNA integrity number &#62; 7.0. 
	NOTE: A RIN &#62; 7.0 ensures a high integrity of the RNA samples.</li>
	<li>Prepare small RNA libraries using a commercial kit (Table of Materials) according to the manufacturer&#39;s instructions. Use 1 &#181;g of total RNA per sample to initiate.
	<ol>
		<li>Thaw library normalization reagents and adapters according to the manufacturer&#39;s guidelines. Ligate small RNAs with the 5&#8242; and 3&#8242; adapters and elute and purify them. Then, reverse transcribe the 5&#8242; and 3&#39; ligated small RNAs following the manufacturer&#39;s guidelines.</li>
		<li>Perform PCR amplification according to the manufacturer&#39;s protocol. Assess the quality and quantity of the cDNA libraries using a microcapillary electrophoresis system.</li>
		<li>Load 1 &#181;L of an RNA library on a microcapillary electrophoresis system to ensure the RIN &#62; 7.0.</li>
	</ol>
	</li>
	<li>Sequence the small RNA libraries on a high-throughput sequencing instrument as described elsewhere23.</li>
</ol>
5. miRNA and Target Gene Prediction
<ol>
	<li>For each grafting combination, use the open source UEA sRNA workbench 2.4-plant version24 to remove poor-quality sequences and to trim adaptor sequences from the raw reads. Discard sequences that are smaller than 18 nt or larger than 32 nt.</li>
	<li>Compare the high-quality &#34;clean&#34; sequences to the open source Rfam 11.0 database to recognize and remove reads of rRNA, tRNA, snoRNA, and other snRNAs.</li>
	<li>Align the remaining reads to the reference genomes using a short-read sequence alignment tool25. No mismatch is allowed in this step. 
	NOTE: The watermelon &#34;97103&#34; genome assembly V126 was used for alignment with the reads from the scion, and the bottle gourd &#34;HZ&#34; genome assembly V127 was used for reads from the rootstock.</li>
	<li>Compare the remaining reads against known mature miRNAs in the open source miRBase 22.028. Reads that are homologous to known miRNAs are classified as conserved miRNAs.</li>
	<li>Compare the sequences that fail to match the known miRNA precursors with the genome sequence. Use the MIREAP29 algorithm to detect potential novel miRNAs under default settings.</li>
</ol>
6. Differential Expression and Gene Ontology Analysis
<ol>
	<li>Compare the expression levels of miRNAs based on their read counts. miRNAs with a P-value (Fisher&#39;s exact test) &#60; 0.05 and an absolute log2 value &#62; 2 are considered to be differentially expressed.</li>
	<li>Use an antisense oligonucleotide target site selection tool (TargetFinder)30 to predict potential complementary mRNAs (miRNA target genes) to the differentially expressed miRNAs under default parameters.</li>
	<li>Use the gene ontology (GO) enrichment analytical tool31 to reveal the miRNA target genes ontology (GO) patterns under a P-value threshold of 0.05 for statistical significance.</li>
</ol>

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<li>Savvas, D., et al. <a target="_blank" href="http://hortsci.ashspublications.org/content/44/7/1978.full">Interactive effects of grafting and manganese supply on growth, yield, and nutrient uptake by tomato.</a> HortScience. 44 (7), 1978-1982 (2009).</li>
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<li>Li, Y. S., Li, C. H., Bai, L. Q., He, C. X., Yu, X. C. <a target="_blank" href="https://link-springer-com.remotexs.ntu.edu.sg/article/10.1007/s11738-016-2070-5">MicroRNA and target gene responses to salt stress in grafted cucumber seedlings.</a> Acta Physiologiae Plantarum. 38 (2), 1-12 (2016).</li>
<li>Pagliarani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+accumulation+of+miRNAs+differentially+modulated+by+drought+stress+is+affected+by+grafting+in+grapevine%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">The accumulation of miRNAs differentially modulated by drought stress is affected by grafting in grapevine.</a> Plant Physiology. 173 (4), 2180-2195 (2017).</li>
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</ol>

The authors have nothing to disclose. The small RNA sequencing data is deposited in the GenBank under the accession number SRP136842.

In this protocol, we described in detail a highly efficient and reproducible method to make homo- and heterografts between watermelon and bottle gourd. This method, requiring no specific equipment, is very easy to operate and typically has a very high survival rate of grafting. The method can also be used to make grafts for other cucurbits, such as between watermelon, cucumber, and pumpkin.
It is worth noting that the relative size (age) of the rootstock and scion is critical to making a successful graft (step 2.2 of the Protocol). We observed that, if the rootstock used was too large compared to the scion, the graft union was more difficult to form because the stem of the scion was somewhat hollowed. Based on our previous proteomic data31, the inclusion of self-grafted scion and self-grafted rootstock as controls is strongly recommended (step 2.3 of the Protocol), because then, the impact of grafting injuries can be largely eliminated.
This protocol also provides a detailed experimental scheme and specific experimental procedures for investigating the abundances of miRNAs in the heterografting system. This method will also be useful for studies in other plant-grafting systems to reveal the mechanisms of local and long-distance miRNA regulation. In the Representative Results, we report the expression changes of only local miRNAs in the scion or rootstock in response to a low temperature. Accumulating reports have highlighted the involvement of long-distance small RNA transmission in grafting-related phenotypic changes. The protocol presented here, which combines the methods for grafting and high-throughput data analysis, can also be used for miRNA transmission analysis between the scion and the rootstock. The principle for differentiating transmitted miRNAs from local miRNAs is based on their sequence similarity to the reference genomes (i.e., a miRNA in the scion that is more like the rootstock genome is considered to be transferred from the rootstock, and vice versa).

Grafting has long been employed as an agricultural technique to improve crop production and tolerance to biotic and abiotic stresses1,2,3. In heterografting systems, elite rootstocks can enhance water and nutrients uptake of plants, strengthen resistance to soil pathogens, and limit the negative effects of metal toxicity4,5, which may confer the grafts an enhanced growth vigor and increased tolerance to environmental stresses. In many cases, heterografting can also impact fruit qualities in horticultural plants, leading to improved fruit flavor and increased content of health-related compounds6,7. It has been found that the long-distance transfer of phytohormones, RNAs, peptides, and proteins between the rootstock and the scion is a fundamental mechanism modulating the growth and development reprogramming of scion plants8,9,10. Grafting has been widely used in studies of long-distance signaling and transport in relation to environmental adaptation11. Grafting experiments are especially powerful for unambiguous detection of transmitted molecules in receiving tissue or vascular sap, and activation or suppression of molecular targets due to signal transmission12.
Non-coding RNAs, a big class of RNA that exert important regulatory functions in cells, have been reported to play a role in facilitating plant adaptation to abiotic stress13. miRNAs are endogenous small non-coding RNAs of about 20 - 24 nt. Studies have revealed the regulatory role of miRNAs in various aspects of plant activities, such as shoot growth, lateral root formation14,15,16, nutrient uptake, sulfate metabolism and homeostasis17, and responses to biotic and abiotic stress18. Recently, the expression of miRNAs and their target genes were related to salt stress tolerance in heterografted cucumber seedlings19. In the intervariety grafts of grape, the responses of miRNA expression to drought stress were found to be genotype-dependent20.
The rapid development and decreasing cost of high-throughput sequencing technology have provided a great opportunity for the study of miRNA regulations in agronomical plants. Watermelon (Citrullus lanatus [Thunb.] Mansf.), an important cucurbit crop grown throughout the world, is susceptible to low temperatures. Bottle gourd (Lagenaria siceraria [Molina] Standl.) is a more climate-resilient cucurbit commonly used by farmers to graft with watermelon. The primary goal of the current study is to establish a standard, efficient, and convenient method for making heterografts between watermelon (Citrullus lanatus [Thunb.] Mansf.) and bottle gourd (Lagenaria siceraria [Molina] Standl). This protocol also provides a detailed experimental scheme and analytical procedures for the study of the regulation of miRNA expressions following grafting, which is useful for revealing the mechanisms underlying heterografting advantages.
The plant materials used in this study include the watermelon cultivar and the bottle gourd landrace. Watermelon cultivar is a commercial cultivar with high yield but susceptible to low temperatures. Bottle gourd landrace is a popular rootstock for grafting with watermelon, cucumber, and bottle gourd, due to its excellent tolerance of low temperatures21.

<table><tbody><tr><td>TRIzol Reagent</td><td>Invitrogen</td><td>15596026</td><td></td></tr><tr><td>RNA-free DNase I</td><td>Takara</td><td>D2270A</td><td></td></tr><tr><td>Truseq Small RNA sample prep Kit</td><td>Illumina</td><td>RS-200-0012</td><td></td></tr><tr><td>2100 Bionalyser</td><td>Agilent</td><td>5067</td><td></td></tr><tr><td>DNA Polymerase</td><td>Thermo Fisher Scientific</td><td>F530S</td><td></td></tr><tr><td>UEA sRNA workbench 2.4-plant version&amp;nbsp;(software)</td><td>NA</td><td>NA</td><td>http://srna-workbench.cmp.uea.ac.uk/</td></tr><tr><td>Rfam 11.0 database&amp;nbsp;(website)</td><td>NA</td><td>NA</td><td>http://rfam.janelia.org</td></tr><tr><td>miRBase 22.0&amp;nbsp;(website)</td><td>NA</td><td>NA</td><td>http://www.mirbase.org/</td></tr><tr><td>MIREAP(software)</td><td>NA</td><td>NA</td><td>https://sourceforge.net/projects/mireap/</td></tr><tr><td>TargetFinder&amp;nbsp;(software)</td><td>NA</td><td>NA</td><td>http://targetfinder.org/</td></tr></tbody></table>

generating homo- and heterografts between watermelon and bottle gourd for the study of cold-responsive micrornas

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58242/58242fig2.jpg" /> 
Figure 2: Phenotypes of various grafts at room temperature and cold-stressed conditions. (a) This panel shows homo- and heterografted seedlings at room temperature as the control. (b) This panel shows homo- and heterografted seedlings after 48 h of cold treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58242/58242fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Using the described method, we obtained a high success (survival) rate of 98% for grafting. Phenotypes of various grafts at room temperature and cold-stressed conditions are shown in Figure 2. After 48 h of cold treatment, the homografted watermelon plants showed obvious growth retardation with wilted young leaves, while the homografted bottle gourd plants and the watermelon/bottle gourd heterografts exhibited much more vigorous growth. No symptoms of damage were observed in the leaves of the heterografts, which even outperformed the homografted bottle gourd plants, where the lowest true leaves were damaged. These results clearly demonstrate the advantage of heterografts in conferring cold tolerance.
Small RNA sequencing of the eight libraries yielded a total of 258 million raw reads. After the quality control (QC), a total of 146 million reads corresponding to approximately 30 million unique sequences were retained (Table 1). Based on this set of clean sRNA sequences, 323 miRNAs, including 10 known and 313 novel miRNAs, were predicted from the bottle gourd, and 20 known and 802 novel miRNAs were predicted from the watermelon.sRNAs of 24 nt made up the biggest class of sRNAs in all grafting combinations, regardless of room temperature or cold-stressed conditions (Figure 3).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Treatment</td>
			<td rowspan="2">Code</td>
			<td rowspan="2">No. reads</td>
			<td colspan="2">sRNA</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>Unique</td>
		</tr>
		<tr>
			<td></td>
			<td rowspan="3">WW-CK</td>
			<td>Raw</td>
			<td>30612962</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Clean</td>
			<td>19727501</td>
			<td>3858868</td>
		</tr>
		<tr>
			<td></td>
			<td>Mapped to genomic</td>
			<td>19059359</td>
			<td>3777952</td>
		</tr>
		<tr>
			<td></td>
			<td rowspan="3">BB-CK</td>
			<td>Raw</td>
			<td>30845546</td>
			<td></td>
		</tr>
		<tr>
			<td>CK</td>
			<td>Clean</td>
			<td>16832061</td>
			<td>3787866</td>
		</tr>
		<tr>
			<td></td>
			<td>Mapped to genomic</td>
			<td>16375142</td>
			<td>3694388</td>
		</tr>
		<tr>
			<td></td>
			<td rowspan="3">WB-CK-S</td>
			<td>Raw</td>
			<td>39492123</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Clean</td>
			<td>26783053</td>
			<td>6319473</td>
		</tr>
		<tr>
			<td></td>
			<td>Mapped to genomic</td>
			<td>25919944</td>
			<td>6132389</td>
		</tr>
		<tr>
			<td></td>
			<td rowspan="3">WB-CK-R</td>
			<td>Raw</td>
			<td>23763619</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Clean</td>
			<td>10187791</td>
			<td>1784447</td>
		</tr>
		<tr>
			<td></td>
			<td>Mapped to genomic</td>
			<td>8946929</td>
			<td>1537867</td>
		</tr>
		<tr>
			<td></td>
			<td rowspan="3">WW-CL</td>
			<td>Raw</td>
			<td>27557577</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Clean</td>
			<td>17879038</td>
			<td>3336242</td>
		</tr>
		<tr>
			<td></td>
			<td>Mapped to genomic</td>
			<td>17153763</td>
			<td>3259960</td>
		</tr>
		<tr>
			<td></td>
			<td rowspan="3">BB-CL</td>
			<td>Raw</td>
			<td>29780991</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Clean</td>
			<td>13342206</td>
			<td>3235570</td>
		</tr>
		<tr>
			<td>Cold</td>
			<td>Mapped to genomic</td>
			<td>12949972</td>
			<td>3164329</td>
		</tr>
		<tr>
			<td></td>
			<td rowspan="3">WB-CL-S</td>
			<td>Raw</td>
			<td>45708415</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Clean</td>
			<td>23071845</td>
			<td>4310276</td>
		</tr>
		<tr>
			<td></td>
			<td>Mapped to genomic</td>
			<td>22363113</td>
			<td>4224166</td>
		</tr>
		<tr>
			<td></td>
			<td rowspan="3">WB-CL-R</td>
			<td>Raw</td>
			<td>30585408</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Clean</td>
			<td>19029266</td>
			<td>3541729</td>
		</tr>
		<tr>
			<td></td>
			<td>Mapped to genomic</td>
			<td>17364239</td>
			<td>3196106</td>
		</tr>
	</tbody>
</table>
Table 1: Statistics of small RNAs in various grafts at room temperature or under cold treatment.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58242/58242fig3.jpg" /> 
Figure 3: Size distribution of the sRNA reads in various grafts. (a) This panel shows the size distribution of the sRNA reads in heterografts under control or cold conditions. (b) This panel shows the size distribution of the sRNA reads in homografts under control or cold conditions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58242/58242fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Upon a 48-h of cold treatment, 30 and 268 miRNAs were up- and downregulated, respectively, in the leaves of the scion in the heterografts. This was in sharp contrast to the results in the leaves of rootstock, where 31 and only 12 miRNAs were up- and downregulated, respectively (Figure 4). In the watermelon/watermelon homografts, 64 and 83 miRNAs were up- and downregulated, respectively. In the bottle gourd/bottle gourd homografts, these numbers were 30 and 28. Apparently, heterografting caused a profound reprogramming of the miRNA expressions. GO-enrichment analyses of the putative target genes of the differentially expressed miRNAs identified 78 enriched GO terms in the scion of the heterografts, with 40 classified into biological processes, 2 into cellular components, and 36 into molecular functions (Figure 5). We found that several known GO terms/pathways related to abiotic/biotic stress resistance and signal transduction, for instance, the chitin catabolic process (GO: 0006030, GO: 0006032), ethylene-activated signaling pathway (GO: 0009873), polyamine biosynthetic process (GO:0006596), and signal transduction by protein phosphorylation (GO: 0009755), were involved. Combined, our results suggest that the downregulation of miRNAs,by tuning the abundance of the transcripts of their target genes, may represent an important mechanism underlying enhanced cold tolerance. In the watermelon/bottle gourd grafts, the heterograft per se has a significant impact on the miRNA patterns that form the graft advantages.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58242/58242fig4.jpg" /> 
Figure 4: Comparison of the patterns of up- and downregulated miRNAs in response to cold stress in various grafts. WB-S = scion leaves of the watermelon/bottle gourd heterografts that were sampled; WB-R = rootstock leaves of the watermelon/bottle gourd heterografts that were sampled; WW = the watermelon/watermelon homografts; BB = the bottle gourd/bottle gourd homografts. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58242/58242fig4large.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/58242/58242fig5.jpg" /> 
Figure 5: GO enrichment analyses of the putative target genes of differentially expressed miRNAs in the scion leaves of heterografts upon cold stress. WB-CL-S = scion leaves of the watermelon/bottle gourd heterografts under cold treatment; WB-CK-S = scion leaves of the watermelon/bottle gourd heterografts at room temperature. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58242/58242fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs at JoVE.com

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...

generating-homo-heterografts-between-watermelon-bottle-gourd-for

Nanyang Technological University

Research

JoVE Journal

Environment

7.6K Views.  Zhejiang Academy of Agricultural Sciences. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in miRNA reactie op het vermogen om te stressen in plantentransplantaten, zoals hoe miRNA worden gereguleerd onder koude stress in watermeloen fles kalebasts. Het belangrijkste voordeel van deze techniek is dat het zeer efficiënte en reproduceerbare methode is om homo en heterografts te maken. Het vereist geen specifieke apparatuur, het is zeer eenvoudig uit te voeren, en meestal resulteert in een zeer hoge overlevingskans v...

Video: Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Biochemistry

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

Genetics

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Biology

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

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MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

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Immunology and Infection

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

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An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus&#160;and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the ...

mirMachine: A One-Stop Shop for Plant miRNA Annotation

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of gene expression, miRNAs play key roles in various cellular pathways, including both development and response to a/biotic stress, such as drought and diseases. Having high-quality reference genome sequences enabled identification and annotation of miRNAs in several plant species, where miRNA sequences are highly conserved. As computational miRNA identification and annotation processes are mostly error-prone processes, homology-based predictions increase prediction accuracy. We developed and have improved the miRNA annotation pipeline, SUmir, in the last decade, which has been used for several plant genomes since then. 
This study presents a fully automated, new miRNA pipeline, mirMachine (miRNA Machine), by (i) adding an additional filtering step on the secondary structure predictions, (ii) making it fully automated, and (iii) introducing new options to predict either known miRNA based on homology or novel miRNAs based on small RNA sequencing reads using the previous pipeline. The new miRNA pipeline, mirMachine, was tested using The Arabidopsis Information Resource, TAIR10, release of the Arabidopsis genome and the International Wheat Genome Sequencing Consortium (IWGSC) wheat reference genome v2.

Herein, we present a new and fully automated miRNA pipeline, mirMachine that 1) can identify known and novel miRNAs more accurately and 2) is fully automated and freely available. Users can now execute a short submission script to run the fully automated mirMachine pipeline.

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of ...

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

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