Meloidogyne incognita Chitwood race I was maintained on young tomato plants (Solanum lycopersicum L. cv. Pusa Ruby) inoculated with 2 J2s/gram of soil. Infected plants were uprooted 30 days post inoculations and egg masses were handpicked after washing the roots with double distilled water. The egg masses were kept in a cavity block and surface sterilized with 0.1% HgCl₂ for 1 min. The surface sterilizing agent was then removed by washing the egg masses thrice with double distilled water. The egg masses were allowed to hatch at 26–28°C through a wire gauge covered with double layered tissue paper into a petri plate filled with double distilled water (Hooper, 1986). Freshly hatched J2s were used for further experiments. Adult females were also isolated from the roots of the infected tomato plants 30 days post inoculation under the microscope using a needle.

Cuticle collagen genes, Mi-col-1 and Lemmi-5 were selected for silencing through host delivered RNAi to demonstrate the effect of downregulation of these structural genes in the parasitic life cycle of M. incognita. The nucleotide sequences of both the genes and their corresponding amino acid sequences were retrieved from NCBI database. Domains were predicted from the amino acid sequences of these genes using SMART (Letunic et al., 2015). A phylogenetic tree was constructed using the amino acid sequences of the cuticle collagens of the plant parasitic and free living nematodes available in the database using MEGA6 (Tamura et al., 2013). TMHMM 2.0 server was used for prediction of transmembrane domains.

Total RNA was extracted from adult females of M. incognita using TRIzol reagent (Invitrogen) following manufacturer's protocol followed by DNase I (Thermo Fischer Scientific) treatment. The total RNA was quantified using Nanodrop spectrophotometer (Thermo Fischer Scientific) and first strand cDNA was synthesized with 1 μg of the total RNA using PrimeScript™ first strand cDNA synthesis kit (Takara). 416 and 799 bp long fragments of Lemmi-5 and Mi-col-1, respectively were amplified using gene specific primers (Table 1). The amplified products were run on 1% agarose gel and subsequently eluted by using GeneJET gel extraction kit (Thermo Fischer Scientific). The eluted products were cloned into pGEMT easy vector (Promega) following manufacturer's protocol. The ligated products were transformed into E. coli DH5α and positive colonies were screened through blue white selection and colony PCR. Plasmids were isolated from PCR positive colonies using GeneJET plasmid miniprep kit (Thermo Fischer Scientific) following manufacturer's protocol and confirmed for the presence of the inserts by restriction digestion by Eco RI. The inserts in the plasmid were custom sequenced (ABI SOLiD sequencing system) to reassert their identity.

Expression of Mi-col-1 and Lemmi-5 was quantified at different life stages of M. incognita through quantitative real time PCR (qRT-PCR) using cDNAs from J2s, egg masses and adult females. Total RNA was isolated and from these developmental stages, quantified and first stranded cDNA was synthesized as described above. A constitutively expressed gene, 18s rRNA was used as an internal control to normalize the gene expression levels. qRT-PCR was performed in a CFX96 real time system (Biorad) using 2X brilliant III SYBR Green q-PCR master mix (Agilent). 20 μL reaction mixture for each sample consisted of 10 μL of 2X SYBR green qPCR master mix, 500 nM of each gene specific primer (Table 1) and 100 ng of first strand cDNA. Two biological and three technical replicates were taken for each sample. The amplification reactions included initial denaturation of 95°C for 5 min, followed by 35 cycles of 95°C for 30 s and 60°C for 1 min. A melt curve analysis at 60–95°C was performed after 35 cycles for assessment of the amplification specificity. Fold change in gene expression was quantified by 2^−ΔΔCT method using mean Ct values for each amplification (Livak and Schmittgen, 2001).

Partial sequences of Mi-col-1 (799 bp) and Lemmi-5 (416 bp) were amplified from their respective pGEMT clones. Lemmi-5 fragment was further cloned into pDONR 221 entry vector using attB1 and attB2 sites at the upstream and downstream of the gene sequence, respectively. Primer details are given in Table 1. pHELLSGATE12 [Obtained from Dr. Tushar Kanti Dutta, Division of Nematology, Indian Agricultural Research Institute, New Delhi] was used as a destination binary vector for development of the Lemmi-5 RNAi construct through recombination based gateway cloning technology mediated by LR recombination reaction catalyzed by LR clonase enzyme (Invitrogen). Mi-col-1 RNAi construct was developed using conventional cloning method with the binary vector pBC-6 (Yadav et al., 2006). The sense strand for Mi-col-1 was cloned with BamHI and XhoI while the antisense strand was cloned with KpnI and SacI restriction enzymes. The recombinant clones for both the genes were transformed to E. coli DH5α cells. Presence of the inserts was confirmed by colony PCR and restriction digestion. Binary plasmids containing the RNAi constructs of Lemmi-5 and Mi-col-1 were independently transformed into Agrobacterium tumefaciens EHA 105 using freeze and thaw method (Jyothishwaran et al., 2007). Selection of positive clones was done through colony PCR and restriction digestion. The positive clones were maintained in yeast extract peptone medium supplemented with selection antibiotic rifampicin (25 μg mL⁻¹). In addition, spectinomycin (100 μg mL⁻¹) was used for the selection of pHELLSGATE12 clones while kanamycin (100 μg mL⁻¹) was used for the selection of pBC-6 clones.

Tomato (cv. Pusa Ruby) seeds were treated with tween 20 for 15 min followed by 3–4 washes with double distilled water. The seeds were treated 70% ethanol for 1 min and washed with autoclaved double distilled water for 3–4 times. These seeds were further sterilized with 4% NaOCl for 8–10 min followed by 4–5 washes with autoclaved double distilled water. Sterile seeds were germinated on half strength Murashige and Skoog (MS) agar medium (pH 5.8). Cotyledonary leaves taken from 12 to 15 days old tomato seedlings were pre-cultured in Petri dishes containing pre-cultivation media (MS + 0.5 mg L⁻¹ IAA + 1 mg L⁻¹ zeatin) with their abaxial surface in contact with the medium at 25°C for 2 days. Healthy explants after pre-culturing were infected with A. tumefaciens EHA105 harboring the RNAi constructs for 15 min with gentle shaking. These explants were blotted on sterile filter paper and co-cultured on the same pre-cultivation medium for 48 h at 28°C with 20–25 explants in each 9 cm Petri plate. After 48 h of co-cultivation under dark conditions, the co-cultivated explants were washed with 250 mg/L cefotaxime for 30 min and blotted on sterile filter paper and transferred to selection medium (MS + 0.5 mg L⁻¹ IAA + 1 mg L⁻¹ zeatin + 100 mg L⁻¹ kanamycin + 250 mg L⁻¹cefotaxime). The selection plates were incubated at 24°C with 16/8 h of light/dark photoperiod. Healthy calluses were cut and transferred to shooting medium (MS + 0.5 mg L⁻¹ IAA + 2 mg L⁻¹ zeatin + 100 mg L⁻¹ kanamycin + 250 mg L⁻¹ cefotaxime) for shoot induction and elongation. After every 15 days, the healthy explants were sub-cultured into fresh shooting medium for shoot induction and elongation. The regenerated shoots were excised from the callus and transferred on to the rooting medium (MS + 0.5 mg L⁻¹ IAA + 100 mg L⁻¹ kanamycin + 250 mg L⁻¹cefotaxime). After 20–25 days, tomato plantlets with well-developed shoots and roots were transferred to 10 cm diameter pots containing 50% soil rite mixed with autoclaved soil for hardening. After hardening, the plants were transferred to growth chambers and maintained under the controlled condition at 25 ± 2°C with a photoperiod of 16/8 h (light/dark) at National Phytotron Facility, ICAR-IARI, New Delhi.

Healthy leaves from T₀ events were used for genomic DNA isolation from cetyltrimethyl-ammonium bromide (CTAB) method (Murray and Thompson, 1980). Approximately one gram of fresh, green and healthy leaf tissues were ground to fine powder in pre-chilled mortar and pestle using liquid nitrogen. 15 mL of pre-warmed (65°C) CTAB-DNA extraction buffer (1 M Tris-HCl; 0.5 M EDTA pH 8.0; 4 M NaCl and 0.1 M β-mercaptoethanol) was added to each powdered leaf sample and each sample was transferred to autoclaved Oakridge centrifuge tubes. These tubes containing the samples were incubated at 65°C in the water bath for an hour with intermittent mixing. 15 mL of phenol: chloroform: isoamyl alcohol (25:24:1) was added to each sample and mixed gently by inverting the tubes followed by centrifugation at 20,000 × g for 15 min at room temperature. The upper aqueous layer from the tubes were carefully pipetted out and transferred into new Oakridge tubes. 12 mL of chloroform: isoamyl alcohol (24:1) mixture was added to each tube containing the aqueous layer obtained from previous step followed by centrifugation at 20,000 × g for 15 min at room temperature. The upper aqueous layer from the tubes were carefully pipetted out and transferred into new Oakridge tubes and 0.6 volume of chilled isopropanol was added to each tube and stored overnight at −20°C for precipitation. After overnight incubation, the tubes were centrifuged at 10,000 × g for 10 min at 4°C. The supernatant from each tube was discarded and pellets were washed with 2 mL of 70% ethanol by centrifugation at 10,000 × g for 10 min at 4°C. The supernatant from each tube was discarded completely and the pellets were allowed to air-dry. Each pellet sample was dissolved in 200 μl of nuclease free water. For purification of DNA, 1 μl of RNase A (10 mg/ml stock) was added to each tube and incubated at 37°C for 2 h in a dry bath. Equal volumes of phenol: chloroform: isoamyl alcohol in the ratio of 25:24:1 was added to each tube, mixed by inversion and centrifuged at 10,000 × g for 5 min at room temperature. The upper aqueous phase was collected in fresh microcentrifuge tubes, 2 volumes of chilled isopropanol was added to each tube, mixed properly by inverting and incubated overnight at −20°C. Next day, the tubes were subjected to centrifugation at 10,000 × g for 5 min at 4°C and each pellet was washed with 2 ml of 70% ethanol, air dried and dissolved in 100 μl of nuclease free water. Purity of the DNA was checked on agarose gel (0.8%) and quantification was done using nanodrop spectrophotometer (Thermo Fischer Scientific).

The presence of the transgenes in the putative T₀ plants was confirmed through PCR using gene specific primers (Table 1). Each PCR mixture (25 μl) contained 100 ng of first strand cDNA, 1 × Taq buffer, 10 mmol/L dNTP, 20 μmol/L of each primer, 3.5 mmol/L MgCl2 and 1.5 U Taq DNA polymerase (Fermentas). The PCR products were separated on 1.2% agarose gel through gel electrophoresis.

Clones of T₀ tomato plants were developed through hydroponics. Growing branches from T₀ plants were cut appropriately and put on conical flasks filled with water at the National Phytotron Facility. T₀ clones with well-developed roots were transferred to 10 cm pots after the development of healthy roots. Fruits were harvested from T₀ tomato plants, seeds were extracted, germinated and the developing seedlings were transferred to 10 cm pots.

Nucleotide sequences of Mi-col-1 (Accession no. U40766) and Lemmi-5 (Accession no. AF006727) were retrieved from NCBI Genbank. The nucleotide sequences of these two genes showed no significant similarities. Further, the conserved cystein patterns of the deduced amino acid sequences revealed that Mi-col-1 belongs to group 3 of nematode cuticle collagens, while Lemmi-5 belongs to group 1a according to the classification proposed by Johnstone (2000) (Figure S1). Primary structural properties of the deduced amino acid sequences are listed in Table S1. SMART domain analysis revealed the presence of nematode cuticle collagen N terminal domains in Mi-col-1 (between amino acids 12–64), as well as Lemmi-5 (between amino acids 5–58). In Mi-col-1, two pfam collagen domain between amino acids 160–219 and 220–219 were also predicted (Figure S2). Presence of a transmembrane domain was predicted for both the sequences (Figure S3), however, amino acid composition in the domain varied for Mi-col-1 and Lemmi-5. Phylogenetic tree based on predicted amino acid sequences showed two clusters. Lemmi-5 formed a separate sub-cluster within cluster I, while Mi-col-1 was placed in cluster II and showed close relationship with Mi-col-2 and Mj-col-3 (Figure 1).

Relative expression of Mi-col-1 and Lemmi-5 in egg masses, J2s and adult female stages of M. incognita was quantified through qRT-PCR. Expression of both the genes were found to be maximum in adult females followed by egg masses and J2s. Using expression levels in pre parasitic J2s as reference, 11.8 folds higher expression of Mi-col-1 was observed in adult females, while around 7 folds higher expression of Mi-col-1 was found in egg masses. Similarly, expression of Lemmi-5 was around 22.5 and 11.3 folds higher in adult females and egg masses, respectively compared to its expression in pre parasitic J2s (Figure 2).

dsRNA constructs of Mi-col-1 and Lemmi-5 were transformed into tomato plants through A. tumifaciens mediated transformation to generate T₀ population (Figure 3). Preliminary screening of T₀ events was carried out by PCR with target gene specific primers. Amplification of 799 bp gene specific DNA of Mi-col-1 was observed in all the nine T₀ events of Mi-col-1. Similarly, 416 bp long fragment was amplified from all the eight putative events of Lemmi-5 (Figure 4).

T₁ plants were generated from the seeds obtained from T₀ plants grown in the NPF, ICAR-IARI, New Delhi. A total of 7 T₁ events were generated for Mi-col-1, while 6 events were generated for Lemmi-5. Preliminary screening of T₁ plants was carried out by PCR with target gene specific primers. Genomic DNA was isolated from leaf tissues of putative T₁ plants of Mi-col-1 and Lemmi-5. Presence of transgene was detected in all the T₁ events for Mi-col-1 and Lemmi-5 RNAi transgenic lines. The PCR confirmed T₁ events for Mi-col-1 and Lemmi-5 were further subjected to southern blotting for T-DNA integration and copy number analysis. Out of the seven Mi-col-1 transgenic events, C1.4, had two insertions, while all other events had single insertions. All the six transgenic events for Lemmi-5 had single insertions. Hybridization bands were not detected in wild type untransformed and empty vector controls (Figure 5).

dsRNA transcript accumulation and expression was validated by qRT-PCR analysis of the selected transgenic events of Mi-col-1 and Lemmi-5. An increased transcript expression was observed in all the events for Mi-col-1 and Lemmi-5 as compared to the wild type. However, variation in the expression was observed among the respective events. Event C1.4 showed highest expression level of Mi-col-1 dsRNA among all the events in terms of average ΔCT values, while Lemmi-5 dsRNA expression was found to be highest in the event L5.16 (Figure 6). These results further insured the expression of the desired dsRNAs in the transgenic events of Mi-col-1 and Lemmi-5.

In order to study the effect of host delivered RNAi of Mi-col-1 and Lemmi-5 on M. incognita infection in tomato, T₁ plants confirmed for the presence and expression of Mi-col-1 and Lemmi-5 were inoculated with 1,000 freshly hatched J2s/pot. T₁ plants of seven independent events were evaluated in case of Mi-col-1, while six independent events were evaluated for Lemmi-5 transgenic plants 35 days post infection. Nematode infection was scored and assessed by means of number of galls, females, egg masses, eggs per egg masses and size of adult females in T₁ plants in comparison with wild type plants (Figures 7, 8). Reduction in the number of adult females was observed in T₁ plants as compared to the wild type and the percentage of reduction was 30.80–35.00% for Mi-col-1 transgenic lines and 34.15–38.54% for Lemmi-5 transgenic lines. Furthermore, the size of the adult females was heavily distorted and deformed in both Mi-col-1 and Lemmi-5 transgenic lines (Figure 9). Mean area of females isolated from wild type plants was calculated as 86,580.10 μm². Whereas, the mean area of females isolated from transgenic plants expressing Mi-col-1 and Lemmi-5 dsRNA were calculated as 50,854.10 and 37,681.40 μm², respectively. Similarly, mean diameter of females isolated from wild type plants was calculated as 360.10 μm; whereas, the mean diameter of females isolated from transgenic plants expressing Mi-col-1 and Lemmi-5 dsRNA were calculated as 231.00 and 199.70 μm, respectively. Statistical analysis revealed significant reduction in the area and diameter of the females isolated from transgenic plants expressing Mi-col-1 and Lemmi-5 as compared to those isolated from wild type plants (Table 2). The fecundity of the nematodes also got hampered as indicated by the reduction in number of egg masses/plant in the range of 50.06–65.73% and 57.30–66.44% in Mi-col-1 and Lemmi-5 RNAi transgenic lines, respectively. The number of eggs/egg masses also reduced significantly in the range of 76.07–82.59% and 67.13–79.56% in Mi-col-1 and Lemmi-5 RNAi lines, respectively. Nematode multiplication factor (MF) determines successful establishment of the concerned nematode in the host plants by representing parasitic and reproductive fitness of the nematode. The multiplication factor of the nematodes in the transgenic events expressing Mi-col-1 was calculated to be 1.51–2.17 among the events as compared to a multiplication factor of 20.39 in the wild type untransformed plants. Similarly, in transgenic events expressing Lemmi-5 dsRNA, the multiplication factor ranged from 1.48 to 2.49 as compared to a multiplication factor of 20.35 in the wild type untransformed plants. However, No significant reduction in number of galls was observed in the transgenic events as compared to the wild type.

qRT-PCR analysis of Mi-col-1 and Lemmi-5 gene expression in the females isolated from the roots of transgenic tomato events expressing the respective dsRNAs revealed significant down regulation of the target genes further indicating successful host delivered RNAi in transgenic plants. Expression levels of Mi-col-1 was reduced in the range of 67.69–91.30% in the females isolated from tomato lines expressing Mi-col-1 dsRNA. Similarly, the reduction in the expression levels of Lemmi-5 was found to be in the range of 79.55–96.12% in the females extracted from the tomato transgenic lines expressing Lemmi-5 dsRNA (Figure 10).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.