This research was funded by the National Natural Science Foundation of China (31870637, 31200487) and jointly funded by the Zhejiang Key Research Plan (2019C02024, LGN22C160004).

The study was approved by the council for animal experimentation of Zhejiang Agricultural &#38; Forestry University. The B. xylophilus isolate NXY61 was originally extracted from a diseased Pinus massoniana in the Ningbo area of Zhejiang province, China11.
1. Gene cloning
NOTE: See the Table of Materials for details about the primers used in this protocol.
<ol>
	<li>Collect nematodes.
	<ol>
		<li>Culture the B. xylophilus strain on the mycelia of Botrytis cinerea on Potato Dextrose Agar (PDA) plates at 25 &#176;C for 3-5 days.</li>
		<li>Collect the nematodes using the Bellman funnel method14.
		<ol>
			<li>Place a clamped rubber tube below a funnel and place two layers of filter paper in the mouth of the funnel. Transfer the fungal cultures to the funnel and add water to immerse the fungal mat. Wait for 2 h, then collect the nematodes.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Extract the total RNA from the nematodes using a total RNA extraction reagent (see the Table of Materials)11 according to the following steps.
	<ol>
		<li>Add 500 &#181;L of extraction reagent and 100 &#181;L of magnetic beads to a 2 mL centrifuge tube. Aspirate 20 &#181;L of the nematodes and transfer the sample to a grinder for grinding at 9,000 &#215; g for 30 s. Incubate for 5 min and then centrifuge for 10 min at 12,000 &#215; g and 4 &#176;C.</li>
		<li>Transfer the supernatant to a new centrifuge tube. Add 100 &#181;L of chloroform, cap the tube, and mix by inverting the tube several times. Incubate for 3 min and then centrifuge for 10 min at 12,000 &#215; g at 4 &#176;C.</li>
		<li>Transfer the supernatant to a new centrifuge tube. Add 250 &#181;L of isopropyl alcohol and vortex vigorously. Centrifuge at 12,000 &#215; g for 10 min.&#160;</li>
		<li>Discard the supernatant. Add 500 &#181;L of 75% ethanol to wash the RNA and then vortex the sample. Centrifuge it for 5 min at 12,000 &#215; g and 4 &#176;C.</li>
		<li>Air-dry the RNA pellet for 5 min.</li>
		<li>Resuspend the pellet in 30 &#181;L of RNase-free water.</li>
		<li>Calculate the RNA concentration using the formula: A260 &#215; dilution &#215; 40 = &#181;g RNA/mL. Calculate the A260/A280 ratio. 
		&#8203;NOTE: A ratio of ~2 is considered pure.</li>
	</ol>
	</li>
	<li>Perform reverse transcription of good quality RNA to obtain the cDNA template.
	<ol>
		<li>Design and use a pair of specific primers, ppm-1-F/R (see the Table of Materials), to amplify the partial coding sequence of the Bx-ppm-1 gene in B. xylophilus (GenBank accession number QTZ96795).</li>
		<li>Clone the ppm-1 gene sequences into the pGEM-Teasy vector containing the T7 promoter following a standard cloning protocol11.
		<ol>
			<li>Set up the PCR reactions as follows: 2 &#181;L of cDNA, 25 &#181;L of 2x Ex Taq Polymerase Premix, 2 &#181;L of each primer (10 pmol/l), and sterile distilled water to a final volume of 50 &#181;L.</li>
			<li>Perform the amplification procedure as follows: 5 min at 94 &#176;C; followed by 35 cycles of 30 s at 94 &#176;C, 30 s at 55 &#176;C, and 1 min at 72 &#176;C; and a final extension step at 72 &#176;C for 5 min.</li>
			<li>Clone the amplified products into a pGEM-T Easy vector for sequencing.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Synthesis of dsRNA
<ol>
	<li>Prepare the DNA template for dsRNA synthesis using PCR with primers designed to add T7 promoter sites at both ends. Add the T7 promoter sequence to the 5&#39; end of the primers.</li>
	<li>Use the plasmid containing the ppm-1 gene fragment (894 bp) as the template for PCR and recover the fragment containing the T7 promoter11. Use the PCR procedure and system described above.</li>
	<li>Use an in vitro transcription kit to synthesize dsRNA11.
	<ol>
		<li>Thaw the frozen reagents on ice.</li>
		<li>Add 2 &#181;L of 10x reaction buffer, 2 &#181;L of enzyme mix, and 1 &#181;g of DNA to a centrifuge tube. Add nuclease-free water to produce a standard 4 &#181;L reaction. Then, mix equal volumes of the four ribonucleotide solutions (ATP, CTP, GTP, and UTP) together and add 8 &#181;L of the mixture to the tube. Mix thoroughly and incubate at 37 &#176;C for 4 h.</li>
		<li>Add 1 &#181;L of DNase, mix well, and incubate for 15 min at 37 &#176;C.</li>
		<li>Stop the reaction and add 30 &#181;L of nuclease-free water and 30 &#181;L of LiCl precipitation solution to precipitate the RNA. Mix thoroughly. Incubate at -20 &#176;C overnight.</li>
		<li>Centrifuge for 15 min at 12,000 &#215; g and 4 &#176;C. Discard the supernatant.</li>
		<li>Add 1 mL of 75% ethanol to wash the RNA. Vortex the sample and centrifuge it for 10 min at 12,000 &#215; g and 4 &#176;C.</li>
		<li>Air-dry the RNA pellet for 3 min.</li>
		<li>Resuspend the pellet in 30 &#181;L of RNase-free water.</li>
		<li>Analyze the quality of the dsRNA using a spectrophotometer. Pipette 1 &#181;L of the dsRNA sample onto the measurement pedestal and set the wavelength to 340 nm. Visualize the products on a 1.0% agarose gel.</li>
	</ol>
	</li>
</ol>
3. RNAi by soaking
<ol>
	<li>Mix 4 &#181;L of 5x soaking buffer (0.05% gelatin, 5.5 mM KH2PO4, 2.1 mM NaCl, 4.7 mM NH4Cl, 3 mM spermidine) with the dsRNA and ddH2O to produce a total volume of 20 &#181;L and a final RNA concentration of 0.8 &#181;g/mL.</li>
	<li>Acquire J2 larvae.
	<ol>
		<li>Collect the nematodes from the fungal cultures and transfer them to a glass Petri dish 6 cm in diameter. Add 10 mL of water to the dish to ensure that the nematodes can swim freely. Keep the nematodes in the dish for 30 min and wait for the eggs to adhere to the bottom.</li>
		<li>Remove the water and nematodes carefully, making sure not to disturb the eggs. Repeat the steps until all the larvae and adults are removed, leaving only the eggs in the dish.</li>
		<li>Hatch the collected eggs for 24 h in the dark at 25 &#176;C to obtain J2 larvae. Collect the J2 larvae, place them in a tube, and wash them three times with ddH2O for the RNAi experiment15.</li>
		<li>Transfer the J2 larvae to the 2 mL tube containing the dsRNA solution and add resorcinol solution (wrapped in tinfoil and dissolved in water) to produce a final concentration of 1.0%. Incubate the larvae with centrifugation at 15&#160;&#215;&#160;g on a shaking table for 24 h at 25 &#176;C to ensure that the larvae effectively absorb the dsRNA.</li>
		<li>Soak the same quantity of nematodes in the soaking buffer without the dsRNA probe or with a GFP dsRNA probe as a control. Use the GFP gene (gfp, M62653.1) as a nonendogenous control and synthesize the dsRNA of gfp using gene-specific primers T7-GFP-F/R.</li>
	</ol>
	</li>
</ol>
4. qPCR detection
<ol>
	<li>Clean the J2 larvae with ddH2O, including those with target gene interference, GFP gene interference, and the undisturbed control group. Extract the total RNAs from each group using the method described above.</li>
	<li>Start the qPCR.
	<ol>
		<li>Design the q-ppm-1-F/R primers using the desired software (see the Table of Materials).</li>
		<li>Set up the qPCR reaction in 12 &#181;L containing 1 &#181;L of cDNA, 6 &#181;L of fluorescent premix, 0.4 &#181;L of each primer (10 pmol/l), and sterile distilled water.</li>
		<li>Perform qPCR as follows: 2 min at 95 &#176;C; followed by 40 cycles of 10 s at 95 &#176;C, 30 s at 55 &#176;C, and 1 min at 72 &#176;C.</li>
		<li>Use the actb gene (GenBank accession number EU100952) and tbb-2 gene (GenBank accession number MT769316),&#160;or other genes, as internal reference genes to evaluate the changes in gene expression level after RNAi15.</li>
	</ol>
	</li>
	<li>According to the cycle threshold (Ct) value and the dissolution curve, use the 2-&#916;&#916;Ct method to estimate the relative expression level of the target gene and verify the interference efficiency.
	<ol>
		<li>Subtract the Ct value of the internal reference gene of each sample from the Ct value of the target gene to obtain the &#916;Ct value. Then, subtract the &#916;Ct value of the interference group from the &#916;Ct value of the control group to obtain the &#916;&#916;Ct value. 
		&#8203;NOTE: A &#916;&#916;Ct value greater than 0 indicates that the interference is effective.</li>
	</ol>
	</li>
</ol>
5. Evaluate the body length of nematode adults following RNAi
<ol>
	<li>After RNAi, culture the J2 larvae until adulthood on B. cinerea lawns in PDA plates for 60 h at 25 &#176;C15.</li>
	<li>Collect the adults using the Bellman funnel method (see step 1.1.2.1)14.</li>
	<li>Acquire images of the adult nematodes under a microscope and use ImageJ software (or other measurement software) to measure the body lengths.
	<ol>
		<li>Measure the length using ImageJ by selecting Analyze | Set Scale. If the distance is known, enter the length value of the drawn straight line. Enter the unit of length.</li>
		<li>Check the Global check box (use this standard for all pictures), and click OK to select the length to be measured with a straight line on the image to be measured.</li>
		<li>Use the command Ctrl+M (measurement) to display the results and record them in the results window.</li>
	</ol>
	</li>
	<li>Take measurements of 50 male and 50 female nematodes for statistical analysis12.</li>
	<li>Analyze the data by calculating the mean and standard deviation for each sample. Compare the means of the samples from the different groups using the Student&#39;s t-test.</li>
</ol>

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No conflicts of interest were declared.

Although the life history and parasitic environment of B. xylophilus are different from those of other nematodes, there has been limited research on the molecular pathogenesis of this plant pathogen. Despite great progress made in the application of CRISPR/Cas9 genome editing technology in C. elegans and other nematodes, only RNAi technology applied to B. xylophilus has been published to date17. RNAi is one of the most powerful tools available to study the gene function of nematodes and has been widely used in research elucidating B. xylophilus gene function, signal transduction pathways, and gene therapy18,19,20. In contrast to the gene knockout approaches used in invertebrate models, RNAi-mediated knockdown of target genes in nematodes enables the rapid evaluation of gene function.
There are three ways to conduct RNAi in C. elegans: injection6, soaking21, and feeding6. Because J2 larvae only feed after infection, the soaking method is typically used for the RNAi of plant-parasitic nematodes. The key step in the soaking method is to add a nerve agent to stimulate the nematodes to feed. Urwin first used octopamine to stimulate the J2 of two oral cyst nematode species, Globodera pallida, and Heterodera glycines, resulting in the uptake of dsRNA from the soaking solution22. The same method has been successfully used to induce J2 of the root-knot nematode Meloidogyne incognita to absorb dsRNA 23. Resorcinol can also induce the uptake of dsRNA by J2 of&#160;M. incognita and may be more effective than octopamine for this nematode24. Furthermore, the addition of spermidine to the soaking buffer with a prolonged incubation time improved the efficiency of RNAi in nematodes25. After 24 h of soaking, dsRNA effectively enters B. xylophilus, thereby silencing the ppm-1 gene.
This protocol, therefore, provides a reference for the future study of the RNAi of other plant-parasitic nematodes with phagocytosis behavior. In addition, the suspension effect of the shaking table plays an important role in maximizing the advantage of the soaking method. This method can allow for the simultaneous treatment of a large number of nematodes. RNAi is easier to operate when focused on the target genes for which mutants cannot easily be obtained and for evaluating the effects at different points in development because nematodes can be soaked at any life stage. Thus, RNAi can be used to study the function of a specific gene at a specific developmental stage. Wang et al. analyzed the influence of MAPK on the fecundity of B. xylophilus and its important role in the growth and development of nematodes using dsRNA soaking26. Qiu et al. found that the migration speed and fecundity of B. xylophilus in pine trees decreased after downregulation of the Bxpel1 gene, which indicates that this gene is an important pathogenic factor of B. xylophilus27.
However, not all RNAi in nematodes works through dsRNA. Dulovic and Streit applied small interfering RNAs (siRNAs) rather than the longer dsRNAs to successfully interfere with Strongyloides ratti28. The limitation of using dsRNA for RNAi&#160;in Strongyloides may be related to the absence of genes such as rsd-6, sid-1, or sid-2 that are known to be involved in dsRNA uptake. RNAi is also associated with the disadvantages of a positional effect and temporary and incomplete knockout and has limited effectiveness for some genes and cell types (such as neurons)29. However, until a breakthrough in the transgenic technology of B. xylophilus is achieved, RNAi represents a relatively effective research strategy.
RNAi has shown great potential for crop protection. Using RNAi technology, transgenic potatoes capable of producing dsRNA that targets root-knot nematode genes have been bred to produce complete resistance to root-knot nematodes30. Expression of dsRNAs that target insect genes can provide crop protection in the absence of chemical insecticides and offers the additional advantage of not producing foreign proteins, with an almost unlimited number of target genes31. Therefore, accelerated research in the field of applied RNAi for pest control will provide better biosecurity for plant nematode control than transgenic methods or other chemical control methods.
In conclusion, this protocol describes the preparation of dsRNA of the ppm-1 gene to achieve RNAi by directly soaking the larvae of B. xylophilus in dsRNA solution. The interference effect was confirmed based on a significant reduction in the body size of the larvae after they developed into adults compared with that of the control, demonstrating that&#160;the ppm-1 gene exerts effects on the growth and development of B. xylophilus. This study provides a theoretical basis for further revealing the function of ppm-1 with additional practical value in guiding the biological control of this plant parasite. It is believed that with the further disclosure and improvement of the RNAi technology mechanism, its application in the field of B. xylophilus control will have broad prospects.

Plant-parasitic nematodes (PPNs) are a continuing threat to food security and forest ecosystems. They cause an estimated 100 billion USD in economic losses each year1, the most problematic of which are primarily root-knot nematodes, cyst nematodes, and pinewood nematodes. The pinewood nematode, Bursaphelenchus xylophilus, is a migratory, endoparasitic nematode, which is the causal pathogen of pine wilt disease2. It has caused great harm to pine forests worldwide3. Using the terminology of Van Megen et al.4, B. xylophilus is a member of the Parasitaphelenchidae and belongs to clade 10, whereas most other major plant parasites belong to clade 12.
As an independent and recently evolved plant parasite, B. xylophilus is an attractive model for comparative studies. To date, there has been substantial research on root-knot nematodes and cyst nematodes belonging to clade 12, which are obligate, sedentary endoparasites and are some of the most intensely studied nematodes. However, conducting further research in this important area comes with a major challenge: the function of parasitism genes is a research bottleneck. Functional studies generally include ectopic expression and knockdown/out experiments but rely on effective genetic transformation protocols for the nematode. As a result, reverse genetics in PPNs almost exclusively relies on gene silencing by RNAi.
RNAi, a mechanism widely present in eukaryotic cells, silences gene expression by introducing double-stranded RNA (dsRNA)5. To date, the posttranscriptional gene-silencing mechanism induced by dsRNA has been found in all studied eukaryotes, and RNAi technology, as a tool of functional genomics research and other applications, has developed rapidly in many organisms. Since the discovery of the RNAi machinery in Caenorhabditis elegans in 19986, RNAi techniques have become effective methods for identifying the gene function of nematodes and are proposed as a new way to effectively control pathogenic nematodes7.
RNAi is technically facile-soaking the juveniles in dsRNA can suffice; however, the efficacy and reproducibility of this approach vary widely with the nematode species and the target gene8. The silencing of 20 genes involved in the RNAi pathways of the root-knot nematode, Meloidogyne incognita, was investigated using long dsRNAs as triggers, resulting in diverse responses, including an increase and no change in the expression of some genes9. These results show that target genes may respond to RNAi knockdown differently, necessitating an exhaustive assessment of their suitability as targets for nematode control via RNAi. However, there is currently a paucity of research on the developmental and reproductive biology of B. xylophilus.
As a continuation of previous work10,11,12,13, we describe here a protocol for applying RNAi to study the function of&#160;the ppm-1 gene of B. xylophilus, including the synthesis of dsRNA, synthetic neurostimulant soaking, and quantitative polymerase chain reaction (qPCR) detection. The knowledge gained from this experimental approach will likely contribute markedly to understanding basic biological systems and preventing pine wilt disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Baermann funnel</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>to isolate nematodes</td>
		</tr>
		<tr>
			<td>Beacon Designer 7.9</td>
			<td>Shanghai kangyusheng information technology co.</td>
			<td>n/a</td>
			<td>to design qPCR primers</td>
		</tr>
		<tr>
			<td>Botrytis cinerea</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>as food for nematodes</td>
		</tr>
		<tr>
			<td>Bursaphelenchus xylophilus</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>its number was NXY61 and was it was originally extracted from diseased 
			Pinus massoniana in Ningbo, Zhejiang province, China.</td>
		</tr>
		<tr>
			<td>constant temperature incubator</td>
			<td>Shanghai Jing Hong Laboratory Instrument Co.</td>
			<td>H1703544</td>
			<td>to cultur nematodes</td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Bio-Rad Laboratories</td>
			<td>1704466</td>
			<td>to achieve electrophoretic analysis</td>
		</tr>
		<tr>
			<td>Ethanol, 75%</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>80176961</td>
			<td>to extract RNA</td>
		</tr>
		<tr>
			<td>Ex Taq Polymerase Premix</td>
			<td>Takara Bio Inc.</td>
			<td>RR030A</td>
			<td>for PCR</td>
		</tr>
		<tr>
			<td>Ex Taq Polymerase Premix</td>
			<td>Takara Bio Inc.</td>
			<td>RR390A</td>
			<td>for PCR</td>
		</tr>
		<tr>
			<td>Gel imager</td>
			<td>LongGene Scientific Instruments Co.</td>
			<td>LG2020</td>
			<td>to make nucleic acid bands visible</td>
		</tr>
		<tr>
			<td>GraphPad Prism 8</td>
			<td>GraphPad Prism</td>
			<td>n/a</td>
			<td>to analyze the data and make figurs</td>
		</tr>
		<tr>
			<td>High Speed Centrifuge</td>
			<td>Hangzhou Allsheng Instruments Co.</td>
			<td>AS0813000</td>
			<td>centrifug</td>
		</tr>
		<tr>
			<td>High-flux tissue grinder</td>
			<td>Bertin</td>
			<td></td>
			<td>to extract RNA</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td>n/a</td>
			<td>to measure the body lengths</td>
		</tr>
		<tr>
			<td>isopropyl alcohol</td>
			<td>Shanghai Aladdin Biochemical Technology Co.</td>
			<td>L1909022</td>
			<td>to extract RNA</td>
		</tr>
		<tr>
			<td>Leica DM4B microscope</td>
			<td>Leica Microsystems Inc.</td>
			<td></td>
			<td>to observe nematodes</td>
		</tr>
		<tr>
			<td>magnetic beads</td>
			<td>Aoran science technology co.</td>
			<td>150010C</td>
			<td>to extract RNA</td>
		</tr>
		<tr>
			<td>MEGAscript T7 High Yield Transcription Kit</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>AM1333</td>
			<td>to synthesize dsRNA in vitro</td>
		</tr>
		<tr>
			<td>NanoDrop ND-2000 spectrophotometer</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>NanoDrop 2000/2000C</td>
			<td>to analyze the quality of the dsRNA</td>
		</tr>
		<tr>
			<td>PCR Amplifier</td>
			<td>Bio-Rad Life Medical Products Co.</td>
			<td>1851148</td>
			<td>to amplify nucleic acid sequence</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>to cultur nematodes</td>
		</tr>
		<tr>
			<td>pGEM-T Easy vector</td>
			<td>Promega Corporation</td>
			<td>A1360</td>
			<td>for cloning</td>
		</tr>
		<tr>
			<td>Potato Dextrose Agar (Medium)</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>to cultur Botrytis cinerea</td>
		</tr>
		<tr>
			<td>Prime Script RT reagent Kit with gDNA Eraser</td>
			<td>Takara Bio Inc.</td>
			<td>RR047B</td>
			<td>to synthetic cDNA</td>
		</tr>
		<tr>
			<td>Primer Premier 5.0</td>
			<td>PREMIER Biosoft</td>
			<td>n/a</td>
			<td>to design PCR primers</td>
		</tr>
		<tr>
			<td>primers:ppm-1-F/R</td>
			<td>Tsingke Biotechnology Co.</td>
			<td>n/a</td>
			<td>F: 5&#39;-GATGCGAAGTTGCCAATCATTCT -3&#39;; R: 5&#39;- CCAGATCCAGTCCACCATACACC -3</td>
		</tr>
		<tr>
			<td>q-ppm-1-F/R</td>
			<td>Tsingke Biotechnology Co.</td>
			<td>n/a</td>
			<td>F: 5&#39;-CATCCGAATGGCAATACAG-3&#39;; R: 5&#39;-ACTATCCTCAGCGTTAGC-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time thermal cycler&#160;qTOWER 2.2</td>
			<td>Analytique Jena Instruments (Beijing) Co.</td>
			<td></td>
			<td>for qPCR</td>
		</tr>
		<tr>
			<td>shaking table</td>
			<td>Shanghai Zhicheng analytical instrument manufacturing co.</td>
			<td></td>
			<td>to soak nematodes</td>
		</tr>
		<tr>
			<td>stereoscopic microscope</td>
			<td>Chongqing Optec Instrument Co.</td>
			<td>1814120</td>
			<td>to observe nematodes</td>
		</tr>
		<tr>
			<td>T7-GFP-F/R</td>
			<td>Tsingke Biotechnology Co.</td>
			<td>n/a</td>
			<td>F: 5&#39;-TAATACGACTCACTATAGGGAAA 
			GGAGAAGAACTTTTCAC-3&#39;; R: 5&#39;-TAATACGACTCACTATAGGGCTG 
			TTACAAACTCAAGAAGG-3&#39;</td>
		</tr>
		<tr>
			<td>&#160;T7 promoter</td>
			<td>Tsingke Biotechnology Co.</td>
			<td>n/a</td>
			<td>TAATACGACTCACTATAGGG</td>
		</tr>
		<tr>
			<td>Takara MiniBEST Agarose Gel DNA Extraction Kit</td>
			<td>Takara Bio Inc.</td>
			<td>9762</td>
			<td>to recover DNA</td>
		</tr>
		<tr>
			<td>TaKaRa TB Green Premix Ex Taq (Tli RNaseH Plus)</td>
			<td>Takara Bio Inc.</td>
			<td>RR820A</td>
			<td>for qPCR</td>
		</tr>
		<tr>
			<td>trichloroethane</td>
			<td>Shanghai LingFeng Chemical Reagent Co.</td>
			<td></td>
			<td>to extract RNA</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>15596026</td>
			<td>total RNA extraction reagent,to extract RNA</td>
		</tr>
	</tbody>
</table>

application of rna interference in the pinewood nematode, bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Analysis of ppm-1 expression of B. xylophilus after RNAi 
The relative expression level of the ppm-1 gene of B. xylophilus soaked with GFP dsRNA and that soaked with target gene dsRNA was 0.92 and 0.52, respectively (the ppm-1 gene expression level of the ddH2O-treated control group was set to 1) (Figure 1). Thus, exogenous dsRNA has no effect on the ppm-1 expression of B. xylophilus; however, ppm-1 dsRNA can effectively inhibit the expression of the target gene.
Effect of ppm-1 expression on growth and development of B. xylophilus 
After RNAi, the size of the adults markedly decreased (Figure 2), resulting in the SMA (small body size) mutant phenotype. Although RNAi-treated individuals developed to sexual maturity, their body length was substantially smaller than normal adults. Specifically, after RNAi in J2-stage nematodes, the mean body length of females and males was 544.61 &#181;m and 526.24 &#181;m, respectively. In contrast, the mean body length of females and males in the control group was 971.86 &#181;m and 912.31 &#181;m, respectively (Figure 3), representing significant differences (P = 0.0322).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63645/63645fig01.jpg" /> 
Figure 1: Expression of the ppm-1 gene following RNAi of Bursaphelenchus xylophilus. **P &#60; 0.001. Abbreviations: RNAi = RNA interference; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/63645/63645fig01large.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/63645/63645fig02.jpg" /> 
Figure 2: Body length reduction of adults after the interference of ppm-1 in Bursaphelenchus xylophilus. Images of an adult male (A) and female (C) of the RNAi group. Images of an adult male (B) and female (D) of the control group. Scale bars = 50 &#181;m. Abbreviation: RNAi = RNA interference. <a href="https://www.jove.com/files/ftp_upload/63645/63645fig02large.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/63645/63645fig03.jpg" /> 
Figure 3: Quantification and statistical analysis of the body length of adults of Bursaphelenchus xylophilus after RNAi of the ppm-1 gene. (*P = 0.0322). Abbreviation: RNAi = RNA interference. <a href="https://www.jove.com/files/ftp_upload/63645/63645fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus at JoVE.com

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

application-rna-interference-pinewood-nematode-bursaphelenchus

Nanyang Technological University

Research

JoVE Journal

Biology

2.3K Views.  Zhejiang Agricultural & Forestry University. Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions. 

Video: Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.