Inoculation of Maize with Sugarcane Mosaic Virus Constructs and Application for RNA Interference in Fall Armyworms

Materials and reagents

1. Plants, insects, vectors, and cell lines

   1. Zea mays inbred line P39 seeds (Maize COOP, http://maizecoop.cropsci.uiuc.edu)

   2. Sweet corn variety Golden Bantam (Burpee Seeds, Warminster Township, PA, or www.amazon.com)

   3. Fall armyworm (Spodoptera frugiperda) eggs (Benzon Research, Carlisle, PA, USA, www.benzonresearch.com)

   4. pSCMV-CS3 (plasmid with a SCMV cloning site #3). This plasmid can be obtained with a material transfer agreement from Iowa State University; contact Dr. Steve Whitham (swhitham@iastate.edu)

   5. Escherichia coli DH5α competent cells (Thermo Fisher, catalog number: 18265017)

   6. pSCMV-GFP and pSCMV-GOI (Gene of Interest)

      
      

2. Oligonucleotides (Integrated DNA Technologies, USA)

   1. Flanking multiple cloning site of pSCMV-CS3

      SP7788: 5′-GCACAAATGGTTTCCAACG-3′

      SP7789: 5′-ATGTTGCATGTCTTGCATG-3′

   2. Reference maize gene primers for reverse-transcriptase PCR (RT-PCR)

      Actin-F, 5′-GGTTTCGCTGGTGATGATGC-3′

      Actin-R, 5′-CAATGCCATGCTCAATCGGG-3′

      EF-1α-F, 5′-TGGGCCTACTGGTCTTACTACTGA-3′

      EF-1α-R, 5′-ACATACCCACGCTTCAGATCCT-3′

   3. Reference fall armyworm gene primers for quantitative reverse transcriptase-PCR (qPCR)

      qrSF-RPL13-F, 5′-GCCTTAACCCTGCTTTTGCTAG-3′

      qrSF-RPL13-R, 5′-GCTTCGCCCTTCAATACCTTC-3′

      qrSF-EF1α-F, 5′-TGGGCGTCAACAAAATGGA-3′

      qrSF-EF1α-R, 5′-TCTCCGTGCCAGCCAGAAAT-3′

   4. Primers for VIGS target gene cloning and quantitative PCR (will vary based on the gene of interest)

      
      

3. Materials

   1. Wizard^® SV Gel and PCR clean-up system (Promega, catalog number: A9282)

   2. Wizard^® Plus SV Minipreps DNA purification system (Promega, catalog number: A1460)

   3. Wizard^® SV total RNA isolation system (Promega, catalog number: Z3100)

   4. High-Capacity cDNA Reverse Transcription kits (Applied Biosystems, catalog number: 4374966)

   5. GoTaq^® Green Master Mix (Promega, catalog number: M7123)

   6. PowerUp^TM SYBR^TM Green Master Mix (Applied Biosystems, catalog number: A25742)

   7. Restriction enzymes: PspOMI, SbfI (NEB Biolabs, catalog number: R0653S and R3642)

   8. T4 DNA ligase (Promega, catalog number: M1804)

   9. 100 bp DNA ladder (Promega, catalog number: G210A)

   10. Nuclease-free water

   11. Absolute ethanol (Sigma-Aldrich, catalog number: E7023)

   12. Liquid nitrogen

   13. Carborundum powder (Fisher, catalog number: C192-500)

   14. Mortar and pestle

   15. Pellet pestle (Fisher Scientific, catalog number: 12-141-363)

   16. Biolistic Optimization kit [1.0 μm Gold Microcarriers, Macrocarrier disks, stopping screens, and 7,600 kPa (1,100 psi) rupture disks (Bio-Rad Laboratories, catalog number: 165-2279)]

   17. 0.1 M spermidine (Sigma-Aldrich, catalog number: 05292-1ML-F)

   18. Pipettes and pipette tips (various)

   19. Agarose low-EEO/multi-purpose/molecular biology grade (Fisher BioReagents^TM, catalog number BP160-100)

   20. Microperforated plastic vented bread bags (30 cm × 60 cm) (www.amazon.com)

   21. Plant trays without holes (28 cm wide × 54 cm long × 6 cm deep), plastic pots (9 cm square, 8 cm deep) (www.amazon.com)

   22. qPCR plates (MicroAmp^TM optical 384-well reaction plate with barcode (Applied Biosystems, catalog number: 43-098-49)

   23. Cornell maize soil mix: [0.16 m³ Metro-Mix 360 (Scotts, Marysville, OH, USA), 0.45 kg finely ground lime, 0.45 kg Peters Unimix (Griffin Greenhouse Supplies, Auburn, NY, USA), 68 kg Turface MVP (Banfield-Baker Corp., Horseheads, NY, USA), 23 kg coarse quartz sand, and 0.018 m³ pasteurized field soil]

   24. Fall armyworm artificial diet (Southland Products Inc, Lake Village, AR, USA, www.southlandproducts.net)

   25. 50 mg/mL kanamycin stock solution (Sigma-Aldrich, catalog number: K0254-20ML)

   26. CaCl₂·6H₂O (Sigma-Aldrich, catalog number: 21108-500G)

   27. Tryptone (Sigma-Aldrich, catalog number: T7293-1KG)

   28. NaCl (Sigma-Aldrich, catalog number: S9888-1G)

   29. Yeast extract (Sigma-Aldrich, catalog number: 70161-500G)

   30. Agar (Sigma-Aldrich, catalog number: A1296-500G)

   31. 15 cm Petri dishes (Fisher Scientific, catalog number FB0875714)

   32. 0.22 μm syringe filters (Corning^®, catalog number: CLS431219-50EA)

   33. Monobasic potassium phosphate (KH₂PO₄) (Sigma-Aldrich, catalog number: P0662-500G)

   34. Dibasic potassium phosphate, (K₂HPO₄) (Sigma-Aldrich, catalog number: P3786-500G)

   35. Tris base (Sigma-Aldrich, catalog number: T1503-500G)

   36. Acetic acid (Sigma-Aldrich, catalog number: A6283-1L)

   37. EDTA (Sigma-Aldrich, catalog number: E4884-500G)

   38. Glycerol (Sigma-Aldrich, catalog number: G7893-IL)

       
       

4. Buffers and solutions

   1. 2.5 M CaCl₂ (see Recipes)

   2. LB medium (see Recipes)

   3. Inoculation buffer (see Recipes)

   4. 1× TAE buffer (see Recipes)

   5. 50% (v/v) glycerol (see Recipes)

   6. 70% ethanol (see Recipes)

   7. LB agar plates (see Recipes)

Recipes

1. 2.5 M CaCl₂

   Dissolve 11.0 g of CaCl₂·6H₂O in deionized water and make the volume up to 20 mL, filter sterilize with a 0.22 μm filter, and store at 4 °C.

   
   

2. LB medium

   1% (w/v) tryptone

   1% (w/v) NaCl

   0.5% (w/v) yeast extract

   pH 7.5

   
   

3. Inoculation buffer (50 mM potassium phosphate)

   43.4 mL of 1 M KH₂PO₄

   6.6 mL of 1 M K₂HPO₄

   Bring volume to 1 L with deionized water

   pH 6.0

   
   

4. 1× Tris-Acetate-EDTA (TAE) buffer

   242 g of Tris base

   57.1 mL of glacial acetic acid

   18.6 g of EDTA

   Bring final volume to 1 L with deionized water

   This is a 50 × TAE stock solution, dilute 1:50 to make 1× TAE buffer

   
   

5. 50% (v/v) glycerol

   50 mL of 100% glycerol in 50 mL of deionized water

   
   

6. 70% ethanol

   70 mL of absolute ethanol in 30 mL of deionized water

   
   

7. LB agar plates

   LB medium (see Recipe 2)

   1.5% w/v agar

   Boil to dissolve the agar

   Pour approximately 25 mL of heated LB agar into each Petri dish

Equipment

1. NanoDrop ND-1000 spectrophotometer (Thermo Fisher, USA)

2. PDS-1000/He biolistic particle delivery system with metal mesh (Bio-Rad laboratories, USA)

3. 1600 MiniG^® automated tissue homogenizer and cell lyser (SPEX^® Sample Prep, USA)

4. QuantStudio 6 Flex real-time PCR system (Applied Biosystems, USA)

5. Ultrasonic water bath with 40 kHz frequency and 284 W heater power (FS140H, Fisher Scientific, USA)

6. Plate centrifuge (Benchmark Scientific, USA)

7. Vortex (American Scientific Products, USA)

8. Benchtop centrifuge (Thermo Scientific, USA)

9. Incubator with temperature set at 37 °C

10. Incubator with temperature set at 28 °C

11. Shaker with temperature set at 37 °C

12. Plant growth chambers

Procedure

1. Selection of target sequence and primer design

   1. Select a sequence of size between 200 and 400 bp from the fall armyworm gene of interest, which should be a multiple of three base pairs, avoiding stop codons (in the antisense direction), and avoiding restriction sites of selected restriction enzymes. Check for off targets in the selected sequence against the fall armyworm and maize genomes using blastn at the National Center for Biotechnology Information (NCBI; https://blast.ncbi.nlm.nih.gov). Select a sequence with no off targets.

      Design PCR primers to specifically amplify the selected sequence and clone the sequence into the pSCMV-CS3 vector (Figure 1 and Supplementary information) in the antisense direction using the SbfI and PspOMI restriction enzymes.

      
      

      
      Figure 1. Plasmid map of pSCMV-CS3, showing the restriction sites used for cloning gene fragments into sugarcane mosaic virus (SCMV)

      
      

   2. Following the same parameters, clone a 240 bp fragment of the jellyfish green fluorescent protein (Gfp) gene into the pSCMV-CS3 vector to use as negative control for VIGS experiments.

      
      

2. Synthesis of SCMV-VIGS constructs

   1. Hatch fall armyworm eggs on fall armyworm diet at 28 °C in an incubator. Use 7-day-old fall armyworm caterpillars for RNA isolation. Freeze caterpillars using liquid nitrogen. Grind the frozen larval tissue in a microfuge tube using a pellet pestle and isolate the total RNA using Wizard^® SV Total RNA isolation system following the manufacturer’s instructions (Note 1).

   2. Quantify the RNA and use 1 μg for reverse transcription using High-Capacity cDNA Reverse Transcription kits following the manufacturer’s instructions.

   3. Using the cDNA as a template, amplify the fragment of target gene using 400 nM of each target-specific primer with GoTaq^® Green Master Mix.

   4. Analyze the specific amplification by 1.5% agarose electrophoresis using 1× TAE buffer, checking the correct size of the fragments by running a 100 bp DNA ladder in a parallel lane on the gel.

   5. Purify the amplicon using Wizard^® SV Gel and PCR clean-up system following the manufacturer’s instructions.

   6. Double-digest the purified amplicons and pSCMV-CS3 vector with the selected restriction enzymes and, after analyzing the digestion by 1.5% agarose gel electrophoresis, purify fragments from gel using Wizard^® SV Gel and PCR clean-up system following the manufacturer’s instructions.

   7. Quantify the restricted purified products and ligate the target fragments in linearized pSCMV-CS3 using a 1:3 equimolar end ratio and T4 DNA ligase according to the manufacturer’s instructions.

   8. Transform the ligation mixture into E. coli Top 10 competent cells (1:10 dilution) using a heat shock method and select the transformants after overnight growth at 37 °C on LB agar plates containing 50 μg/mL kanamycin.

   9. Verify insertion of the fragment of interest in pSCMV-CS3 by screening a few colonies by PCR using the SP7788/SP7789 primers (flanking the multiple cloning site of the pSCMV-CS3 vector).

   10. Verify the sequence of the inserted fragments by Sanger sequencing with the SP7788 primer, using plasmid DNA isolated using the Wizard^® Plus SV Minipreps DNA purification system from positive colonies after overnight growth at 37 °C with shaking at 180 rpm in LB broth containing 50 μg/mL kanamycin.

       
       

3. Biolistic transformation of SCMV-VIGS constructs into maize and in planta detection of RNA expression

   1. Prepare seedlings at the two-leaf stage by sowing five seeds of maize variety Golden Bantam in a pot containing Cornell maize mix. Allow seeds to germinate and grow for a week in a growth chamber with a 16:8 light/dark cycle at 23 °C with 60% humidity (Figure 2) (Notes 2 and 3). Prepare Gold microcarriers (1.0 μm) at the concentration of 100 mg/mL in 50% glycerol. For this, first vortex 50 mg of gold particles in 1 mL of 70% ethanol for 5 min at 2,000 rpm. Then, keep at room temperature for 15 min and collect the gold particles by centrifugation at 900× g for 20 s. Wash the gold particles three times with 1 mL of sterile water by following this sequence: vortex for 1 min at 2,000 rpm, incubation at room temperature for 1 min, and centrifugation for 10 s. Finally, resuspend particles in 500 μL of 50% glycerol (Note 4).

      
      

      
      Figure 2. Maize variety Golden Bantam seedlings at the two-leaf stage for biolistic transformation. Five seedlings are planted in each pot.

      
      

   2. Place 30 μL of prepared gold microcarriers in a 1.5 mL microfuge tube and vortex for 30 s at 2,000 rpm, followed by sonication for 10 s in an ultrasonic water bath at room temperature with 40 kHz frequency.

   3. Add 5 μg of the pSCMV-VIGS construct during sonication. After 10 s of further sonication, add 25 μL of 2.5 M CaCl₂ and 10 μL of 0.1 M spermidine and vortex for 3 min at 2,000 rpm, followed by centrifugation at 2,700× g for 30 s to collect the pellet of gold microcarriers.

   4. Discard supernatant and wash the gold particles with 150 μL of absolute ethanol followed by vortexing at 2,000 rpm for 10 s and centrifugation at 2,700× g for 10 s to collect the gold microcarriers.

   5. Remove the supernatant and resuspend the DNA-coated gold particles in 50 μL of absolute ethanol (see Figure 3 for a flowchart of these steps).

      
      

      
      Figure 3. Steps for coating of DNA on gold microcarriers

      
      

   6. Place the microfuge tubes having DNA-coated gold microcarriers in an ultrasonic water bath, remove μL of microcarrier suspension, evenly spread it on macrocarrier, and air dry. Prepare four more macrocarriers with the remaining suspension of DNA-coated gold microcarriers.

   7. Arrange the PDS-1000/He biolistic particle delivery system (gene gun) with a microcarrier disk, stopping screen, and 7,600 kPa rupture disk following the manufacturer’s instructions (https://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1652249.pdf). Place one pot of maize seedlings in the instrument chamber and keep the leaves flat against the solid support with a metal mesh that is supplied along with the biolistic particle delivery system (Figure 4). After closing the door, turn on the vacuum and shoot the gold particles when the vacuum reaches 7,600 kPa.

      
      

      
      Figure 4. Maize variety Golden Bantam seedlings in the chamber of the PDS-1000/He biolistic particle delivery system

      
      

   8. When the helium pressure reaches approximately 7,600 kPa, a clear pop is heard. Release vacuum at this point, remove the pot, mist with water, and transfer the plants to a growth chamber.

   9. Transfer the inoculated plants to individual pots the next day and grow them in a growth chamber under a 16:8 h photoperiod at 23 °C for three weeks, until the appearance of viral symptoms.

   10. After three weeks, collect the tissue from the seventh leaf of each plant and immediately place it in liquid nitrogen.

   11. Grind the leaf tissue using 1600 MiniG^® automated tissue homogenizer and cell lyser and extract the total RNA using Wizard^® SV Total RNA isolation system following the manufacturer’s instructions.

   12. Quantify the RNA and use 1 μg of RNA for reverse transcription using High-Capacity cDNA Reverse Transcription kit following the manufacturer’s instructions.

   13. Use SP7788 & SP7789 primers for PCR amplification and analyze the expression of target sequence in maize. Maize actin and EF-1α gene are amplified as reference genes using Actin-F, Actin-R & EF-1α-F, and EF-1α-R primer pairs. Analyze the amplicons by 1.5% agarose gel electrophoresis (Note 5).

       
       

4. Preparation of sap and rub inoculation of maize with SCMV-VIGS constructs

   1. Separate the whole seventh leaf from the infected plant in which the expression of dsRNA has been confirmed, grind the tissue finely in a mortar and pestle in the presence of liquid nitrogen, wait for a few minutes until sap comes out of tissue, add inoculation buffer in 1:10 ratio (w/v), and grind again to prepare sap for inoculation (Figure 5A) (Note 6).

   2. Grow maize inbred line P39 plants in individual pots in a growth chamber with a 16:8 h photoperiod at 23 °C for one week until the two-leaf stage (Note 7).

   3. Spray the seedling leaves with deionized water and dust (light sprinkling) the leaves with 600 mesh carborundum.

   4. Take a cotton swab, wet it with prepared maize sap, and use it to rub the top surface of both leaves (Figure 5B) (Note 8).

      
      

      
      Figure 5. Sap preparation (A) and rub inoculation (B). Seven-day-old maize seedlings of inbred line P-39 at the two-leaf stage are rub inoculated with sap prepared from plants infected with pSCMV-VIGS constructs.

      
      

   5. Transfer the plants to the growth chamber and look for the appearance of viral infection symptoms after three weeks under a 16:8 h photoperiod at 23 °C.

   6. The expression of viral RNA can be confirmed by PCR as described in section C, step 15, as plants without viral symptoms may also have active infections and expression of the gene of interest (Figure 6).

      
      

      
      Figure 6. Maize leaves collected from sugarcane mosaic virus (SCMV)-induced gene silencing (VIGS)-infected plants and confirmation of viral infection by PCR. Leaves without (A) and with (B) mosaic symptoms can be positive for SCMV infection. (C) 1.5% agarose gel electrophoresis analysis of PCR for dsRNA from leaves with (lane 1) and without (lane 2) viral symptoms; negative control using water (lane C). (D) 1.5% agarose gel electrophoresis analysis of PCR for maize reference genes actin (lane 1) and EF-1α (lane 2) in the same virus-infected maize leaves.

      
      

5. Caterpillar bioassay for SCMV VIGS in maize and measurement of gene silencing in caterpillars by qRT-PCR

   1. Three weeks after infecting the maize plants with pSCMV-GOI and pSCMV-GFP control constructs by rub inoculation, put five pre-weighed two-day-old caterpillars onto each plant. One caterpillar will be used for measurement of gene silencing after three days of feeding. Growth of the other caterpillars will be monitored after seven days of feeding. Cover the plants with the perforated plastic bags and tie the bags around the stems of the plants with wire twist ties to prevent caterpillar escape (Figure 7).

      
      

      
      Figure 7. Bioassays with Spodoptera frugiperda caterpillars. (A) Experimental setup for caterpillar bioassay and gene expression analysis, starting three weeks post rub inoculation. Five two-day-old caterpillars are confined on each plant. The plants are covered with perforated bread bags, which are tied around the plant stem with wire twist ties. (B) Spodoptera exigua (fall armyworm) on a maize leaf.

      
      

   2. Transfer the plants back to the growth chamber under the same conditions (16:8 h photoperiod at 23 °C) and monitor the growth of caterpillars after seven days by collecting and weighing the surviving caterpillars.

   3. For measurement of gene silencing, collect one caterpillar from each plant after three days of feeding on pSCMV-VIGS- and pSCMV-GFP-infected plants, place it in a 1.5 mL microfuge tube, and immediately place the tube in liquid nitrogen.

   4. Grind collected caterpillars with a pellet pestle tissue homogenizer and extract the total RNA using Wizard^® SV Total RNA isolation system following the manufacturer’s instructions.

   5. Quantify the RNA using a Nanodrop and synthesize the cDNA with 1 μg of RNA using High-Capacity cDNA Reverse Transcription kit following the manufacturer’s instructions.

   6. Perform quantitative PCR using a QuantStudio 6 Flex real-time PCR system, with a five-fold dilution of cDNA samples using qPCR primers and PowerUp^TM SYBR^TM Green Master Mix. The expression levels are normalized using the fall armyworm RPL13 and EF1α reference genes (with primers qrSF-RPL13-F, qrSF-RPL13-R, qrSF-EF1α-F, and qrSF-EF1α-R) as internal controls.

   7. The gene expression levels are calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001) (Figure 8). Gene expression levels of caterpillars fed on pSCMV-GOI infected plants is compared to those on control plants (pSCMV-GFP infected plants).

      
      

      
      Figure 8. Sample quantitative PCR amplification plots from a QuantStudio 6 Flex real-time PCR system. (A) Amplification of a control housekeeping gene, showing a threshold cycle (CT) value of 19.5. (B) Amplification of a gene of interest from control and experimental (RNA interference) samples, showing CT values of 23.5 and 26, respectively. (C) Formula used to calculate the relative gene expression in the RNAi sample compared to that of the untreated control.

   Notes

   1. A similar approach can be used for isolating RNA from other insect species if their gene expression will be targeted by VIGS.

   2. Use pots (9 cm square × 8 cm deep) to grow seedlings. Prepare five pots of seedling per construct transformation. In our hands, Golden Bantam works well for biolistic transformation of maize, but inbred line P39 can also be used.

   3. The efficiency of transformation decreases after the two-leaf stage of seedling. Keep the seedlings in the dark for 24 h before biolistic transformation.

   4. The prepared gold microcarriers can be stored at -20 °C and used for up to three months. Three milligrams of prepared microcarrier is sufficient for five bombardments per construct.

   5. The amplified band is expected at 475 bp + the size of the target gene fragment. You can also confirm the sequence of the target fragment by Sanger sequencing.

   6. Maize leaf sap can be stored indefinitely at -80 °C. Avoid frequent freeze thaw of the sap, as it will reduce the infection efficiency. You can also store the infected leaf tissue (0.25 g of leaf tissue per tube) at -80 °C after snap freezing in liquid nitrogen. This frozen tissue can be used to prepare fresh sap for inoculation of plants. Infection efficiency in both cases will be same.

   7. Use of seedlings after the two-leaf stage results in low infection efficiency.

   8. Change gloves and the cotton swab for sap from different pSCMV-VIGS constructs to avoid cross-contamination.

   Acknowledgments

   This work was supported by an International Postdoc Scholarship from the Punjab Higher Education Commission to I.G., NSF award 2019516 to G.J., and USDA award 2021-67014- 342237 to G.J.

   Competing interests

   The authors declare that they have no competing interests.

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