We constructed a binary vector to express hairpin RNA of a 472 bp MiStarget sequence of MeloidogyneincognitaPolA1 gene (Chukwurah et al. submitted, Figure 1). Briefly, MiS sequence was PCR-amplified from pUC57 vector using MiS5P and MiS3P primers and inserted into the entry vector, pCR8, by TA cloning. MiS target on pCR8 was inserted into pANDA35HK RNAi binary vector using Gateway LR Clonase II enzyme mix (Thermo Fisher Scientific). We checked both pCR8: MiS and pANDA35HK: MiS plasmids from transformed E.

coli by PCR and restriction analyses to confirmsequence correctness as well MiS integration in both sense and anti-sense orientations, respectively (Figure 2). The PolA1 silencing construct, pANDA35HK: MiS, was transformed into Agrobacterium tumefaciens strain EHA 105 using the freeze-thaw method.

We transformed tobacco (Nicotianatabacum “Petit Havana”) using the Agrobacterium-mediated leaf disc method [14] . Transgenic tobacco calli were selected on MS basal medium supplemented with 3% sucrose, 0.1 mg·l⁻¹ NAA, 1 mg·l⁻¹ BA, 40 mg·l⁻¹ hygromycin, 20 mg·l⁻¹ meropenem and solidified with 0.8% agar. Regenerated shoots were transferred to 1/2 MS medium supplemented with 40 mg·l⁻¹ hygromycin, 20 mg·l⁻¹ meropenem and solidified with 0.8% agar but without growth regulators for root development.

We extracted genomic DNA from 100 mg young leaves of putative transgenic and wild type plants using Sodium Dodecyl Sulfate (SDS) method [15] . Using different sets of primers (Table 1), we targeted regions of the binary vector corresponding to sense and anti-sense orientations of MiS sequence in the plants’ genome (Figure 1). We also amplified a region corresponding to the hygromycin marker gene. Amplification of these regions was also carried out in the binary vector as a positive control.

We selected PCR positive transgenic lines and extracted genomic DNA using acetyl trimethyl ammonium bromide (CTAB) protocol [16] . We digested extracted genomic DNA (15 µg) of both transgenic and WT plants with SacI enzyme (TAKARA) at 37˚C. Digestion was done overnight following which DNA fragments were separated on 0.7% agarose gel and transferred to a nylon membrane. A 472 bp MiS probe was labelled using PCR DIG Probe Synthesis Kit (Roche). Probe hybridization, stringency washes and chemiluminescence detection with CDP-Star were carried out following manufacturer’s instructions.

We used reverse transcription PCR (RT-PCR) to confirm expression of MiS dsRNA in the transgenic plants. Total RNA was extracted from 100 mg young leaves of transgenic and WT plants using the RNeasy Plant Mini Kit (Qiagen). One microgram of the purified RNA (DNAse-treated) was used as template for the synthesis of first strand cDNA using Superscript III First-Strand cDNA Synthesis Kit (Invitrogen). First strand cDNA (2 µl) from both positive and negative control reactions (without enzyme) were used as template in a 50 µl total volume for PCR amplification of a region of the GUS linker using GUS5P and GUS3P primers (Figure 1(B)). We also amplified elongation factor 1α (EF1α) using primers EF1a5P and EF1a3P (Table 1) as an internal control.

We extracted small RNAs (<200 bp) from the selected transgenic and WT plants using ISOGEN II reagent (Nippon Gene). Thirty micrograms of small RNA from each sample was resolved on 17% denaturing polyacrylamide gel (acrylamide: bis 19:1) containing 7M urea. Transfer of separated small RNAs to a nylon membrane was done using a semi-dry cell (Nippon Eido) for 1 hour at 10 V/400mA. Northern hybridization was done using DIG-labelled MiS RNA probe (472 bp) obtained via in vitro transcription of the MiS target sequence using T7 RNA polymerase according to DIG Northern Starter Kit Version 10 (Roche) protocol. Pre-hybridization (30 min) and hybridization (overnight) were performed at 50˚C. Post-hybridization stringency washes and chemiluminescent detection of siRNA using CDP-Star were performed following protocols outlined in the DIG Northern Starter Kit (Roche) manual.

A pure culture of root knot nematode (Melodoigyneincognita) race 2 was maintained on a highly susceptible tomato cultivar “Kyoryoku-beiju” in a glass house for 2 months to enable gall formation. We prepared nematode egg suspension used asinfection inoculum by extraction from freshly uprooted galled roots. Infected roots were washed, chopped and macerated in an electric blender for 5 mins at full speed. Nematode eggs were recovered by filtration on a 25 µm sieve. Recovered eggs were reconstituted into suspension and one ml egg suspension was used to determine egg concentration by counting under a microscope.

One-internode stem cuttings of 3 selected transgenic (T1, T4 and T7) and WT tobacco lines were rooted in 1/2 MS medium containing 1% sucrose and 0.8% agar for two weeks. Rooted plants were acclimatized on sterile vermiculite [17] , transferred to pots containing 3000 ml of sterile commercial garden soil and allowed further acclimatization in the glasshouse for 12 days. They were then individually infected with approximately 10,000 Meloidogyne incognita eggs of same batch via 3 holes made around each plant root system. All tested plants were replicated 10 times and confined in the glass house for a total period of 7 weeks. Infection experiment was repeated.

After 7 weeks of nematode infection, transgenic and wild type plants were harvested, and analyzed for nematode parasitic success and key agronomic characters. Galling index (%) was scored according to [18] . Number of eggs per egg mass was evaluated by counting from ten randomly selected egg masses after roots were stained with 15 mg/L Phloxine B and egg masses treated with 1% sodium hypochlorite [19] . We used SYBR Green-based real time PCR to quantify the amount of root knot nematode juveniles in the soil by measuring nematode DNA amount per gram soil. We homogenized and bulked soil samples in which transgenic and wild type plants were grown, separately. Genomic DNA was extracted from 3 replicate soil samples from each lot using ISOIL for Beads Beating Soil DNA extraction kit (Nippon Gene). We adjusted genomic DNA concentration in all samples to 2.8 ng/µl, and used 2 µl as template in 20 µl qPCR cocktail containing 10 µl KOD SYBRqPCR Mix, 4 pmol forward primer (RKN-2F), 4 pmol reverse primer (RKN-2R), 0.4 µl 50x ROX reference dye and sterile water. Real time PCR reaction was performed in a Step One Plus Real-Time PCR system (AB Applied Biosystem). A dilution series of known concentration of M. incognita genomic DNA was prepared and a standard curve of the log of each known concentration in the dilution series (x-axis) was plotted against the C_t (threshold) value for that concentration (y-axis). Absolute quantification of M. incognita genomic DNA concentration was done by comparison with the standard curve.

We acclimatized T₀ plants (T1and T7) and transferred them to the green house for T₀ seed production. We germinated T₀ seeds of lines T1 and T7 in 0.5x MS medium supplemented with 1% sucrose and 100 mg/L kanamycin to generate their T₁ progeny plants. We confirmed via PCR the presence of MiS sense and anti-sense regions as well as hygromycin gene in the genomes of the T₁ plants.

T₁ plants showing presence of all three amplicons were transferred to 300 ml autoclaved garden soil and acclimatized under high humid conditions in growth room for 10 days. The plants were further acclimatized in the green house for 11 days after which they were individually infected with approximately 500 freshly extracted eggs of M. incognita. Nematode-infected plants were grown in confinement for 49 days and analyzed for nematode parasitic success. Nematode parasitic parameters evaluated include number of nematode galls on roots, number of nematode egg masses, number of nematodes eggs per mass and nematode multiplication. We counted nematode galls by root inspection with the aid of magnifying lens. Nematode multiplication was evaluated by multiplying the number of egg masses per gram root by number of eggs per egg mass and dividing by initial amount of egg inoculum used for infection. Infection experiment was repeated twice.

We extracted adult female nematodes feeding on roots of WT and T₁ transgenic tobacco plants under a stereo microscope (Olympus SZX9) and stored them in −80˚C after flash freezing in liquid Nitrogen. Total RNA was extracted from the samples using ISOGEN (Nippon Gene), and 300 ng from each was converted to cDNA using PrimeScript^TM RT reagent Kit with gDNA Eraser (TaKaRa). Quantitative real time PCR (qRT-PCR)was then performed to amplify a 152 bp target region of the PolA1 gene (Figure 10(A)) using SYBR Green technology in StepOnePlus^TM thermal cycler (Applied Biosystems).

We prepared PCR cocktail for each reaction by mixing 10 µl KOD SYBR qPCR Mix (TOYOBO), 0.2 µM each of forward and reverse primers (PolA RT-F and PolA RT-R), 0.4 µl 50x ROX reference dye, 1 µl cDNA (10x dilution) and distilled water to a total of 20 µl. Amplification reaction was carried out at a hot start of 98˚C for 2 min, followed by 40 cycles of 98˚C for 10 s, 55˚C for 10 s and 68˚C for 30 s in a 96-well µltra Amp PCR plate (Sorenson Bioscience). We assessed specificity of the amplification by melt curve analysis at 60˚C - 95˚C after 40 cycles.

Three biological and three technical replicates were used with each sample. We used mean C_t values (normalized against internal reference gene) to calculate the fold change in PolA1 expression in the nematodes using the 2^−∆∆CT method. Root knot nematode elongation factor was used as the internal reference gene. We expressed PolA1 transcript abundance in nematodes extracted from transgenic roots as a percentage relative to the transcript level in nematodes extracted from wild type plants.

All experimental units were laid in a completely randomized design (CRD) in the green house, and data generated after nematode infection were analyzed by a one-way analysis of variance (ANOVA) using SigmaPlot 14.0 software (SYSTAT). Significantly different means were separated using the Duncan’s Multiple Range Test (DMRT).

Following agrobacterium transformation, selection and regeneration of tobacco plantlets, we generated and genotyped 13 primary transgenic lines. We used 2 sets of primers GUSLK5P/MiS3P and MiS3P/GUSLK3P (Figure 1(B)) to amplify 1083 bp and 796 bpgenomic regions corresponding to the sense and anti-sense orientations of MiS target. We also amplified a 687 bp genomic region corresponding to hygromycin marker gene (hpt) using primers HPT5P and HPT3P (Figure 1(B)). Sense, anti-sense and hpt amplicons for 13 transgenic lines are shown in Figure 3(A). Plasmid DNA was amplified as a positive control while untransformed plants (WT) showed no amplification.

We subjected nine PCR-positive transgenic lines to Southern hybridization to analyze their MiST-DNA integration patterns. Using probes specific to MiS target, single, double and triple copy T-DNA insertions were observed with the different lines (Figure 3(B)). Wild type plants showed no hybridization signal with the probe.

We derived complementary DNA (cDNA) from selected T₀ plants (T1, T4 and T7) and conducted reverse-transcription PCR (RT-PCR) analysis to confirm expression of MiS dsRNA in the transgenic lines. Using primers GUS5P and GUS3P (Figure 1(B)), we amplified a 636 bp fragment corresponding to the GUS linker region between sense and anti-sense orientations of the MiS target sequence in the selected lines (Figure 3(C)). Wild type plants showed no amplification for the GUS linker region. Elongation factor (EF1a) used as internal

control reference gene was amplified using primers EF1a5P and EF1a3P (Table 1). Transgenic and wild type plants both showed a 481 bp amplicon of EF1a. We also amplified 2 genomic DNA controls containing a 77 bp intron with the same primers and obtained a higher 558 bp amplicon (Figure 3(D)).

DIG-labelled MiSRNA probe hybridized to sequence-specific siRNAsin the transgenic plants that expressed MiSdsRNA (Figure 4). We did not detect any hybridization signal with WT plants. Ethidium-bromide staining of the polyacrylamide gel after electrophoresis showed equivalent loading of small RNAs across all samples.

An inspection of the test plants’ roots showed wild type plants with reduced and necrotic root system having larger multiple fused galls compared to the transgenic lines (Figure 5(A)). Nematode galling index was equally highest on roots of wild type tobacco plants (70.0%). The transgenic lines showed reduced root galling indices particularly T1 and T7 (54.3% and 58.6% respectively. Galling index on T4 was 62.7% (Figure 5(B)). Nematode eggs per mass was highest on roots of WT plants (613) but significantly reduced (p < 0.05) by 26.4% in transgenic line T1. Nematodes eggs on lines T7 and T4 were reduced by 17.3% and 2.5% respectively but not significantly (Figure 5(C)). Quantification of nematode DNA by real time PCR showed that soil pooled from wild type plants contained significantly more (p < 0.05) nematode DNA per gram (617.6 ng/g soil) than those of transgenic plants T1 (235.0 ng/g), T7 (149.8 ng/g) and T4 (303.4 ng/g) (Figure 5(D)). This represented a 61.9%, 75.7% and 50.9% reduction respectively.

The transgenic plants were generally more vigorous post-infection compared to WT plants (Figure 6(A)). Number of leaves determined for all treatment groups before nematode infection was comparable (p > 0.05). All plants had an average of 13 healthy leaves (Figure 6(B)). After 7 weeks of nematode infection,

however, mean percentage of green, standing leaves reduced significantly (p < 0.001) in WT plants by 32.5% and 20.7% respectively compared to T1 and T7 (Figure 6(C)). Fresh root biomass was also significantly reduced (p < 0.05) in the WT plants by 23.1% and 21.4% compared to lines T1 and T7 respectively, but comparable to T4 (Figure 6(D)).

Disease condition was more outstanding on WT leaves compared to transgenic plants (Figure 7(A)). We measured the distance from base of each plant up the stem to the last diseased leaf. This parameter, termed disease progression, was significantly reduced (p < 0.001) in transgenic lines T1 and T7 by about 80.0% compared to WT plants. Compared to T4, disease progression was significantly reduced by 12.0% (Figure 7(B)). Fresh leaf biomass was significantly reduced (p < 0.001) in WT plants compared to those of T1 and T7 by 22.0% and 14.2% respectively. Compared to T4, WT plants showed 6.9% reduction in fresh leaf biomass (Figure 7(C)). In the same vein, dry leaf biomass in WT plants was reduced significantly by 19.9% and 14.2% compared to T1 and T7 respectively, but comparable to T4 (Figure 7(D)).

We extracted genomic DNA from 100 mg young leaves of T₁ tobacco progeny plants generated from transgenic lines T1 and T7. We excluded T4 from further

analyses owing to its comparatively weaker T₀ phenotype. PCR amplification was carried out with same primer sets used with T₀ plants. We obtained correct amplicons for hygromycin gene (687 bp) as well as MiS target in both sense (1083 bp) and anti-sense orientations (796 bp) with the transgenic lines. Wild type plants showed no amplification signal (Figure 8(A)).

Nematode galls per gram root were significantly reduced (p < 0.05) in T₁ plants of transgenic lines T7 and T1 by 25.5% and 20.6% respectively compared to WT plants. T7 and T1 were comparable (p > 0.05) in mean number of galls on their roots (Figure 8(B)). Nematode egg masses were also significantly reduced (p < 0.05) by 13.8% and 11.4% on transgenic lines T1 and T7 respectively, compared to WT plants (Figure 8(C) and Figure 8(A)). The transgenic plants had significantly reduced (p < 0.05) number of nematode eggs permass. Compared to WT plants, nematode eggs per mass on T1 and T7 were reduced by 20.9% and 17.7% respectively (Figure 8(D)). Nematode multiplication on transgenic lines T1 and T7 was also significantly reduced (p < 0.05) by 30.7% and 26.9% respectively compared to WT plants (Figure 8(E)).

Agronomic traits evaluated in the test plants included shoot weights, fresh root weights and root lengths. All T₁ test plants were comparable (p > 0.05) in these characters (Figures 9(B)-(D)).

We used qRT-PCR analyses to compare PolA1 transcript expression in adult female nematodes feeding on roots of transgenic and wild type plants. Relative to expression in nematodes feeding on roots of WT plants, PolA1expression was significantly reduced (p < 0.05) by 34.3% and 31.5% respectively in nematodes feeding on transgenic lines T1 and T7 (Figure 10(B)). Analysis of melt curves for reference and PolA1 after qRT-PCR showed single peaks that indicate specific target amplification.