Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Podsumowanie

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

Streszczenie

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

Wprowadzenie

Hemipteran insects comprise some of the most economically significant pests of agriculturebecause of their ability to attain elevated population growth and spread diseases in plants. The BMSB, H. halys Stål, is an invasive pest that was accidentally introduced in the Western hemisphere in Allentown, Pennsylvania from Asia (China, Taiwan, Korea, and Japan) with the first sighting reported in 1996¹. Since its introduction, BMSB has been detected in 43 states, with the highest populations in the Mid-Atlantic (DE, MD, PA, NJ, VA, and WV), as well as in Canada and Europe, and represents a potential threat to agriculture². As a polyphagous pest, BMSB can instigate damage to approximately 300 identified plant hosts including high-value crops such as apples, grapes, ornamental plants, seed crops, soybeans, and corn. Damage is caused primarily due the mode of feeding known as lacerate and flush where the animal pierces the host crop with its needle-like stylet to gain access to the nutrients from the vascular tissues^2,3. BMSB is also an indoor pest as they may find residence in living areas such as schools and houses during autumn through winter². Chemicals and aeroallergens released by BMSB were reported to illicit allergic reaction in fruit crop workers. BMSB may also contribute to allergic disease leading to contact dermatitis, conjunctivitis, and rhinitis in sensitive individuals^4,5. Another hemipteran insect, the ACP, D. citri Kuwayama (Hemiptera: Liviidae), is a serious pest of citrus fruits, and transmits the phloem-limited bacteria (Candidatus Liberibacter asiaticus) causing Huanglongbing (HLB), better known as citrus greening disease^6,7. HLB was first reported from Southern China and has spread to 40 different Asian, African, Oceanian, South and North American countries⁷. Citrus greening is a worldwide problem with threatening economic and financial losses due to citrus fruit loss; hence, management of ACP is considered of utmost importance to prevent and control HLB.

Measures for effective control of these insect pests usually requires the application of chemical pesticides that are relatively short lived. Chemical insecticide control strategies often lack safe environmental management strategies or have decreased susceptibility due to pesticide resistance in pest populations^8,9. Hence, the biological control of pests with molecular biopesticides is a potential alternative, but its use globally remains modest, and various species of parasitoids (e.g., Trisolcus japonicus) may also be effective as natural biological controls. RNAi is a potential emerging technology for managing invasive insect pests with molecular biopesticides¹⁰. RNAi is a well described gene regulatory mechanism that facilitates the effective posttranscriptional gene silencing of endogenous as well as invading dsRNAs in a sequence-specific manner, that eventually leads to the regulation of gene expression at the mRNA level^11,12. Briefly, when exogenous dsRNA is internalized into a cell it is processed into siRNAs by a member of the bidentate nuclease RNase III superfamily, called Dicer, which is evolutionarily conserved in worms, flies, plants, fungi, and mammals^13,14,15. These 21-25 nucleotide siRNA duplexes are then unwound and integrated in the RNA-induced silencing complex (RISC) as guide RNAs. This RISC-RNA complex allows Watson-Crick base pairing to the complementary target mRNA; this eventually leads to cleavage by the Argonaute protein, a multi domain protein containing an RNase H-like domain, which degrades the corresponding mRNA and reduces protein translation, thereby leading to posttranscriptional gene silencing^16,17,18.

RNAi for pest management requires the introduction of dsRNA in vivo to silence the gene of interest, thereby activating the siRNA pathway. Various methods that have been used for dsRNA delivery to insects and insect cells to induce systemic RNAi include feeding^10,19, soaking^20,21, microinjection²², carriers such as liposomes²³, and other techniques²⁴. RNAi was first demonstrated in Caenorhabditis elegans to silence unc-22 gene expression by Fire and Mello²⁵, followed by knockdown in expression of the frizzled genes in Drosophila melanogaster²⁶. Initial functional studies utilized microinjection to deliver dsRNA in insects, such as Apis mellifera^22,27, Acyrthosiphon pisum²⁸, Blattella germanica²⁹, H. halys³⁰, and lepidopteran insects (reviewed by Terenius et al.³¹). Microinjection is advantageous to deliver an accurate and precise dose to the site of interest in the insect. Albeit such septic punctures may elicit expression of immune related genes due to trauma³², hence, ruling out its practicality in agricultural biopesticides development.

Another method of delivering dsRNA in vivo is by soaking, which involves ingestion or absorption of dsRNA by suspension of animals or cells generally in extracellular medium containing dsRNA. Soaking has been used to efficiently induce RNAi in Drosophila S2 tissue culture cells to inhibit Downstream-of-Raf1 (DSOR1) mitogen-activated protein kinase kinase (MAPKK)²⁰, as well as in C. elegans to silence the pos-1 gene³³. However, dsRNA delivered using soaking is less efficient to induce RNAi compared to microinjection²⁰. RNAi mediated silencing in a chewing insect was first shown in the Western corn rootworm (WCR) (Diabrotica virgifera virgifera) by infusing the dsRNA into an artificial agar diet¹⁰. Earlier reports have summarized methods to deliver dsRNA infused in natural diets specific to arthropods³⁴. These delivery methods were further determined to be comparably effective to artificial means of delivery; such as the case of the tsetse fly (Glossina morsitans morsitans), where equal knockdown of an immune-related gene was observed when dsRNA was delivered either through blood meal or microinjected³⁵. Similarly, delivery of dsRNA through droplets in light brown apple moth (Epiphyas postvittana)³⁶, diamondback moth (Plutella xylostella) larvae³⁷, as well as honey bees^38,39 induced efficient RNAi. Most effective RNAi experiments in hemipteran have utilized injection of dsRNA⁴⁰ because oral delivery of dsRNA in hemipteran insects is arduous since it must be delivered through the host plant's vascular tissues. Effective RNAi was also observed in ACP and glassy-winged sharpshooter leafhopper (GWSS), Homalodisca vitripennis: dsRNA was delivered through citrus and grapevines that had absorbed dsRNA into the vascular tissues through root drench, foliar sprays, trunk injections, or absorption by cuttings^{41,42,43,44,45,46}. This also resulted in the first patent for dsRNA against the ACP (2016, US 20170211082 A1). Delivery of siRNA and dsRNA using carriers such as nanoparticles and liposomes imparts stability, and increases in delivered dsRNA efficacy are rapidly emerging^{23,47,48,49,50}. A new class of nanoparticle-based delivery vehicles for nucleic acids for in vitro and in vivo that was summarized specifically for therapeutic applications may impart immense potential as suitable delivery vectors⁵¹. Nanoparticles as a delivery vehicle for dsRNA may have disadvantages including solubility, hydrophobicity, or limited bioaccumulation⁵², but a suitable polymer aiding delivery may compensate these disadvantages. Development and use of self-delivering nucleotides are also emerging called 'antisense oligonucleotides', which are single stranded RNA/DNA duplexes⁴⁶.

Vitellogenesis in arthropods is a key process controlling reproduction and regulated by juvenile hormone (JH) or ecdysone, which are the key inducers of Vg synthesis by the body fat; the Vg is eventually taken up by the developing oocyte via Vg receptor mediated endocytosis⁵³. Vg is a group of polypeptides synthesized extraovarially, which is essential for development of the major egg yolk protein, vitellin^54,55, and therefore, it is important in reproduction and aging⁵⁶. Vg has been successfully silenced in nematodes⁵⁷ as well as in honey bee (Apis mellifera) where RNAi mediated depletion of Vg was observed in adults and eggs²². RNAi mediated posttranscriptional gene silencing of Vg was tested because it was thought its depletion would lead to an observable phenotypic effect such as reduced fertility and fecundity, to potentially aid in BMSB control. The JHAMT gene that encodes the S-adenosyl-L-methionine (SAM)-dependent JH acid O-methyltransferase, catalyzes the final step of the JH biosynthesis pathway⁵⁸. In this pathway farnesyl pyrophosphate (FPP) is sequentially transformed from farnesol, to farnesoic acid followed by conversion of methyl farnesoate to JH by JHAMT. This pathway is conserved in insects and arthropods specifically for metamorphosis, a process that is developmentally regulated by hormones^59,60,61. In B. mori, JHAMT gene expression and the JH biosynthetic activity in the Corpora allata suggest that the transcriptional suppression of the JHAMT gene is crucial for the termination of JH biosynthesis⁵⁸. Therefore, the JHAMT and Vg genes were selected for targeted depletion using RNAi. RNAi was also tested in citrus trees for control of ACP and GWSS. Citrus trees were treated with dsRNA through root drench, stem tap (trunk injections), as well as foliar sprays with dsRNAs against insect specific arginine kinase (AK) transcripts^42,44. The topical application of dsRNA was detected all over the canopy of citrus trees, indicating efficient delivery through the plants vascular tissues, and resulted in increased mortality in ACP and GWSS^41,42,45.

In the current study, we have identified a natural diet delivery method for treatments such as dsRNA. This newly developed technique was subsequently used for silencing the JHAMT and Vg mRNA using gene specific dsRNAs in BMSB nymphs as demonstrated earlier⁶². These new delivery protocols demonstrated here supersede conventional RNA delivery systems that use topical sprays or microinjections. Vegetables and fruits, stem tap, soil drenching, and clay absorbents in may be used for delivery of dsRNA, which is critical to the continued development of biopesticide pest and pathogen management.

Protokół

1. BMSB Rearing

1. Rear BMSB insects as per standard lab practice and previously described⁶³.
2. Raise ACP (D. citri) insects on Citrus macrophylla in a glasshouse (22 °C) and natural light. Use adult ACP, at approximately 5-7 days post eclosion.

2. Selection of Gene Regions and In Vitro Synthesis of dsRNA

1. Select genes specific to BMSB from previously published transcriptome profiles³².
2. Ensure the regions of interest selected vary between 200 to 500 base pairs.
3. Perform polymerase chain reaction (PCR) using the conditions described below to generate fragments associated with the selected gene of interest from genomic DNA. See Table 1 for the gene-specific oligonucleotides.
   1. PCR reaction: In a 0.25 mL PCR tube, combine 5 µL of 10X PCR Buffer, 4 µL of dNTP Mixture (2.5 mM each), 2 µL of DNA template (50 ng/µL), 2.5 µL each of Primers 1 and 2 (10 µM), 0.25 µL of DNA polymerase (5 U/µL), and DNase/RNase free water up to 50 µL.
   2. PCR condition: Cycle the PCR reaction to amplify the region of interest at 95 °C for 3 min followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, 72°C for 1 min. Incubate the reaction at 72 °C for an additional 10 min. Purify the PCR reaction using a purification kit.
4. Amplify the obtained PCR fragments further with gene specific primers flanked with the T7 RNA polymerase promoter sequence (5'-GAA TTA ATA CGA CTC ACT ATA GGG AGA-3') as mentioned earlier⁶².
5. Use LacZ gene as a negative control (mock) for RNAi.
   NOTE: LacZ is a gene that encodes β-galactosidase amplified from Escherichiacoli genomic DNA (the primers used are listed in Table 1).
6. Perform in vitro transcription to yield dsRNA as described earlier⁶².
7. Dissolve and resuspend the resulting dsRNA in 150 µL DNase/RNase free water, measure the concentration, and store at -80 °C for future use.

3. Delivery of dsRNA Using Green Beans

1. Select early 4^th instar BMSB nymphs hatched from the same egg mass and starve them for 24 h prior to dsRNA feeding.
2. Select slender certified organic green beans (Phaseolus vulgaris L.) and wash with 0.2% sodium hypochlorite solution for 5 min.
   NOTE: Slender green beans were selected so that the beans can easily be accommodated in the 2 mL microcentrifuge tubes.
3. Wash 3 times with ddH₂O and allow to air dry.
4. Trim the green beans from the calyx end to a total length of 7.5 cm using a clean razor blade.
5. Immerse the washed and trimmed green beans in a cap-less 2 mL microcentrifuge tube containing 300 µL of control solution (a 1:10 dilution of green food coloring (ingredients: Water, Propylene Glycol, Fd&C Yellow 5, Fd&C Blue 1, and Propylparaben as preservative)).
6. Make dilutions of the in vitro synthesized LacZ, JHAMT, or Vg dsRNAs by diluting 5 µg or 20 µg in 300 µL of RNase/DNase free water to yield final concentrations of 0.017 µg/µL or 0.067 µg/µL, respectively.
7. Immerse the washed and trimmed green beans in a cap-less 2 mL microcentrifuge tube containing 300 µL of dsRNA solution (from step 3.6).
8. Wrap and seal the edges of the microcentrifuge tubes enclosing the immersed beans to avoid evaporation of the dsRNA solution and to prevent the animals from entering the microcentrifuge tube.
9. Position these tubes in an upright manner at room temperature for 3 h to allow the dsRNA solution to be loaded throughout the green bean by capillary action.
10. Place these tubes in clean culture vessels (polypropylene). Place three starved 4^th instar BMSB nymphs in the culture vessels.
11. Treat three animals per culture vessel each containing three green beans with green food coloring or dsRNA solution. Maintain the insects at 25 °C and 72% relative humidity, under a 16L:8D photoperiod in an incubator.
12. Allow the insects to feed on the green beans (immersed and absorbed with dsRNA) for 5 days but replenish with fresh diets of dsRNA treatment green beads after 3 days.

4. Real-time Quantitative (qPCR) Analysis of Gene Expression Following RNAi Mediated Silencing in BMSB

1. Measure the effect of RNAi on the levels of transcript expression by qPCR.
2. Isolate the total RNA from the dsRNA treated animals and synthesize the cDNA⁶².
3. Setup the qPCR reactions using a real-time PCR system and the primers listed in Table 1. Use the following qPCR cycling condition: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min, along with dissociation step including 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 15 s.
4. Determine the qPCR standards: use the serial dilution of cDNA prepared from total RNA isolated from a normal animal as a reference standard for the quantification.
5. Use BMSB 18s RNA as an internal standard to correct for differences in RNA recovery from tissues³².

5. Foliar Spray Application in Large Potted Citrus Trees and Seedlings

Note: Plants of the citrus cultivar 'Carrizo' citrange (Citrus sinensis XPoncirus trifoliata, Rutaceae), were maintained in a glasshouse under natural light and temperature, grown in 1.2 L containers. The plants were constantly pruned to promote growth of new foliar shoots, called 'flush'. ACP prefers to feed and oviposition on the new growth of citrus⁶⁴.

1. Select plants or seedlings and do not water them for 2-3 days prior to use to let the soil dry out to damp but not completely dry.
2. Using a hand pump spray bottle apply a 200 mL of dsRNA solution (0.5 mg/mL) to the lower canopy (Figure 4A).
   NOTE: Prepare the above mentioned dsRNA solutions in DNase/RNase free water.
3. Post-spray application, allow the applied dsRNA solution to be completely absorbed by the leaves.
   NOTE: Citrus trees absorbed the applied dsRNA, and then leaves from either new growth or from branches that were covered prior to application, were extracted; the dsRNA was detectable using qPCR and showed systemic movement into the tree top canopy leaves in 3-4 h^44,45,46.
4. Sample the new growth from previously topped trees after 25-40 days by collecting approximately 10 leaves from the tips of four branches. Extract the total RNA and analyze it by reverse transcription PCR (RT-PCR) and qPCR for the presence of the applied dsRNA trigger using the primers listed in Table 1 and previously described methods⁴⁶.
   NOTE: The sample collection, total RNA isolation, RT-PCR, and qPCR were performed as previously described⁴⁶.
5. Similarly, topically spray 10 mL dsRNA to the lower region of a seedling or small potted tree foliage.
   NOTE: Hemipteran insects (ACP and GWSS) were given feeding access normally at 24 h, post treatments on new growth (leaves) which had not been directly sprayed, or which grew weeks later, or whole plants. This produced insects which tested positive for the dsRNA at 3, 6, and 10 days, post feeding.

6. Soil/Root Drench Application in Large or Small Potted Citrus Trees and Seedlings

1. Select plants or seedlings and do not water them for 2-3 days prior to use to let the soil dry out to damp but not completely dry (this creates air space to hold the liquid solution to be applied).
2. Add 1 L of dsRNA solution (0.2 mg/mL) to the soil of large potted plants (approximately 2.5 m) and add 1 L of water (chaser) after 1 h.
3. Apply 100 mL of dsRNA solution (1.33 mg/mL) to the soil of 1 m tall potted trees in partially dry soils.
4. For small seedlings, apply 10 mL of dsRNA solution (1 mg/mL) to the soil in the cones or to the bare roots (Figure 4B, C).
5. Allow the plants that receive the dsRNA solution applied as a soil drench to soak for 30 min. Then apply plain water only treatment to aid absorption by roots (20 mL for plants in yellow containers, or 100 mL if larger plant pots are > 1 gallon).
   NOTE: Topically applied dsRNA to foliage resulted in detection at most distal tips of branches within 3-6 h post treatments, showing systemic movement through trees. New growth branches tested positive for dsRNA at 60-90 days post treatments. Cuttings are provided to insects (ACP and GWSS) in a dsRNA feeding bioassay⁴⁴.

7. Stem Tap (Tree Trunk Injection) Application in Large Potted Citrus Trees and Seedlings

1. Select citrus seedlings, new, or approximately 3.5 years old plants for injecting dsRNA using the stem tap (trunk injections) method.
2. Drill holes in the citrus plants using a drill and a 10 mm drill bit, taking care not to exceed 2 cm, or about half the diameter of the stem.
3. Wrap the copper tip of each injector 4-6 times with a 0.6 cm (¼ inch) wide strip of sealing film to prevent leakage near the tip.
4. Fill the tree trunk injectors with 6 mL (1.7 mg/mL) of dsRNA solution diluted in DNase/RNase free water (denoted here as colored solution).
5. Inject the solution into the trunk of the tree and leave the injector in the trunk for 6-10 h to allow absorption of the dsRNA solution. Allow the insects to feed on the cuttings from treated trees at 3, 10, and 30 days post treatment.
   NOTE: The dsRNA injected using the trunk injection method persisted in the trees for a period of 30-60 days^41,42,44. Validation for RNAi was performed by qPCR using the primers listed in Table 1.

8. dsRNA Treated Clay Granules for Delivery to Insects Through Soil

1. Pour out the clay absorbent into a 50-mL conical tube to the 35 mL mark (approximately 30 g of clay absorbent) on the tube.
2. Pour 20 mL of dsRNA solution diluted in DNase/RNase free water (100 µg/mL) into the tube to wet all the absorbent. Cap the conical tube, tip the tube to help remove air, and cap the tube.
3. Place the tube upright and let it stand for 1-2 min for the clay particles to absorb the solution.
4. Add enough dsRNA-soaked clay into the soil mix to fill a 1 gallon pot.
5. Mix and turn the soil by hand to thoroughly mix the dsRNA-soaked clay into the soil.
6. Use this soil to repot seedlings selected for the dsRNA treatment.
7. Water the soil with 200 mL of plain water without dsRNA. After 30 min to 1 h, follow with 100 mL of plain water. After 24 h, put the plant on a normal watering schedule.
8. Test 4-6 leaves of the treated potted plants with clay absorbents and dsRNA each month post treatment for dsRNA by collecting the most apical leaves of new plant growth.
   NOTE: Plants or cuttings from these treated plants are fed to insects any time after 24 h post treatment and have been able to delivery RNAi for up to a year to insects (unpublished data).

Wyniki

Vegetable mediated dsRNA delivery through feeding in BMSB 4^th instar nymphs was tested for the development of molecular biopesticides using RNAi for invasive insect pests. BMSBs feed using their needle-like stylets by a mechanism known as lacerate and flush, which causes considerable damage to crops. Slender organic green beans, P. vulgaris L., were used to test if nutrients or dsRNA could be delivered in vivo to BMSB through feeding³. ...

Dyskusje

RNAi has proven to be an important tool for exploring gene biological function and regulation, with great potential to be utilized for management of insect pests^{19,68,69,70,71}. The design and selection of an appropriate gene(s) for silencing in a given insect species and the method of delivery of the corresponding dsRNA(s) to the insect are both of utmost impo...

Materiały

Name	Company	Catalog Number	Comments
BMSB (H. halys) insects	USDA
ACP (D. citri) insects	USDA
organic green beans	N/A
Citrus plants	USDA
sodium hypochlorite solution	J.T. Baker
green food coloring	McCormick & Co., Inc
Thermo Forma chambers	Thermo Fisher Scientific
Magenta vessel (Culture)	Sigma
Primers	IDT DNA
SensiMix SYBR	Bioline
qPCR ABI 7500	Applied Biosystems
Spray bottle	N/A
Parafilm	American Can Company
TaKaRa Ex Taq	Clontech
QIAquick	Qiagen