Insect sampling and rearing on potato plants
M. persicae was collected into 1.5 mL sterilized centrifuge tubes from potato leaves grown in different rural regions in the cities of north-east Punjab (Sialkot & Gujrat). Solanum tuberosum pure line Desiree (red variety) were sown in plastic pots (LxW: 3x5″) with potting mixture containing mixing sand, clay, and farm yard manure in the ratio of 1:1:1. The mixture was then sterilized with formalin and adult apterous M. persicae (collected from fields) were maintained on 3-W-Old in-vivo potato plants grown under white fluorescent light (300 µmol m-2 s-1, 16 h light/8 h dark at 22oC and 70% relative humidity) and were covered with insect-proof mesh to avoid contamination. Adults obtained from a single colonial lineage were transferred to fresh 3-W-Old potato plants and allowed to reproduce within 24h, after which the new nymphs were transferred to another potato plant; until reach the third instar. 50-100 mg of these aphids were used for RNA extraction.
Total RNA Extraction and Complementary DNA (cDNA) Synthesis
RNA from M. persicae was extracted by PureLink™ RNA Mini Kit (Cat # 12183018A; Thermo ScientificTM). RNA was converted to full-length cDNA via RevertAid First Strand cDNA Synthesis Kit (Cat # K1621; Thermo ScientificTM).
Polymerase Chain Reaction (PCR)
FASTA sequences of partial mRNA of OBP8 were retrieved from NCBI, and primers were designed using Primer 3. The primers were checked for their Tm values, GC content, 3' and 5' modifications and potential hairpin formation on OligoCalc (Oligonucleotide Properties Calculator online) (Table 1). To amplify OBP8 reverse transcriptase (RT) PCR reaction was carried out using Taq DNA Polymerase, recombinant (5U/µL) kit (Thermo ScientificTM). For the amplification of OBP8 cDNA, the initial denaturation step was carried out at 95ºC for 3 min, followed by 35 cycles of annealing involving heating at 94ºC for 40s, 55ºC for 45s and 72ºC for 40s. The final extension step was carried out 72ºC for 10 min. 5 µL of product with 1 µL of 6X loading dye was loaded on 1% agarose gel along with 1kb DNA ladder.
Cloning of OBP8
OBP8 was purified from gel by (GEL DNA Extraction Kit; Molecular Diagnostics) and cloned in pTZ57R/T by Fermentas InsTA clone PCR Cloning Kit (Cat #K1214). DH5α competent cells were prepared by heat shock method. The primary culture was prepared by placing individual colony in lysogeny broth liquid medium followed by incubation at 37°C. Heat-Shock was used to transform pTZ57R/T ligated with target genes in competent cells. After colony PCR plasmid preparation was done (GeneJET Plasmid Miniprep kit; Cat # K0502).
Sequencing and Multiple Sequence Alignment
Purified plasmid DNA (40µL); with gene of interest was sent to Macrogen Korea for sequencing. After sequencing, bioinformatics tools were used for phylogenetic analysis, structural and RNAi target prediction was done. Phylogenetic tree was constructed to find out evolutionary relationship with other aphid species using Maximum Likelihood method via Phylogeny.fr. Multiple Sequence Alignment (MSA) was performed to find conservancy among proteins of closely resembled aphid species.
siRNA Target Prediction
Potential RNAi targets of OBP8 was find out by ERNAi tool using in-silico approach (Boutros lab, E-RNAi-Version 3.2) (Horn and Boutros 2010). Tool helped to find out suspected siRNA targets produced by dsRNA; their position, off targets and graphical view of designed targets.
Artificial feed stabilization and dsRNA Assay
Percentage survival of control sample was calculated for 8D on basis of time and sucrose concentration consumed by aphids. For dsRNA preparation: cDNA was used as a template for OBP8 gene amplification with primers attached with T7 promoter region (5′TAATACGACTCACTATAG 3′) (Table 1). RTPCR was run for candidate genes amplification using T7 promoter containing primers with optimum Tm (57°C) for OBP8 (Table 1). These PCR products were then purified from 1% agarose gel as above and processed according to instructions (MEGAscript® kit (Ambion) and stored at -80°C for subsequent dietary experiments. DNA and single-stranded RNA were removed from the transcription reaction by DNase I and RNaseA treatment. For dsRNA feeding assays; dsRNA-OBP8 were added to the artificial diet (15ng/µL+ 20% Sucrose) without dsRNA as -ve control. Ten aphids replicated were used per dsRNA treatment along with control. The mortality rate was checked for aphids and nymphs between 2-8D (Tariq et al. 2019).
OBP8 expression analysis by Quantitative Real-Time PCR (RT-qPCR)
Quantitative reverse transcription PCRs (RT-qPCR) was performed to analyze the expression level of OBP8 before and after feeding dsRNAOBP8, sucrose and dsRNAGFP. Primers for the OBP8, GFP and Actin gene as internal control were designed online using Primer 3 (Table 1). RT-qPCR reactions were performed using SYBR Green supermix in an Applied Biosystems RT qPCR (Thermo Fisher Scientific), following the manufacturer’s instructions. PCR conditions were 95°C for 10min, 40 cycles with (95°C (15s), 65°C (30s), and 72°C (30s)). The expression analysis was done in triplicate for statistical analysis and confirmation.
RNAi vector construct for OBP8 silencing
Solanum tuberosum seeds were planted under standard conditions in the potting mixture of sand, soil and manure in equal amounts in a greenhouse (16 h light/8 h darkness at 25±1oC and 70% relative humidity). Plant mediated gene silencing can be used as a protective tool in agriculture for the control of insects (Poreddy et al. 2017). To test a target for silencing agroinfiltration can give a good picture whether to produce stable transformants or not (Hoffmann et al. 2006). The 4-week-old plants were used for agroinfiltration. Post transcriptional gene silencing by using a long intron containing constructs are found be most effective in plants (Zhao et al. 2020). In the present study construct, pRNAiBAB7ihp made in the Department of Biochemistry and Biotechnology, was used. For the insertion of gene in vector, primers were designed where in addition to the sequences of OBP8, restriction site of enzyme Eco31I and an adapter sequence where the enzyme will cut were also added to the 5 prime end (Table 1). As with the Golden Gate (GG) technology, restriction and ligation can be done in a single tube. 25 µl restriction ligation reaction was prepared which contained 75ng of Plasmid, 75ng of PCR product, 0.1 X Ligase buffer, 1000 U of T4 DNA Ligase and 30U of Eco31I. The tube was incubated for two hours at 37˚C followed by two hours at 23oC. Enzyme inactivation was done at 60oC for 5 min. The mixture was transformed into electro competent cells of Escherichia coli followed by plating for blue white screening. The white colonies were the recombinants where LacZ gene was replaced. The insertion and correct orientation was confirmed by the Polymerase Chain Reaction (Table 1; Fig 5). Primer 1 and primer 4 were used for the confirmation of recombinants. Primer 1 and Primer 2 were used to check the sense strand while it’s antisense by Primer 2 and Primer 4. Primer 3 was used to check the orientation of pdk intron (Fig 4; 5). OBP8 gene presence as an inverted repeat flanking the pdk intron was confirmed by sequencing with M13 primer set. pRNAiBAB7ihp was transformed into Agrobacterium tumefaciens (EHA105) and was used for the agro infiltration.
Infiltration Procedures and To Generation
The Agrobacterium strain containing pRNAiBAB7ihp was cultured in lysogeny broth medium containing kanamycin (100 mgL-1) and hygromycin (50 mgL-1) was cultured at 28oC at 200 rpm until OD600 reached 0.6. The Agrobacterium cells were centrifuged at 3000g followed by suspension in buffer containing 200 µM acetosyringone and OD600 of 0.2; used for syringe agroinfiltration. The Agrobacterium transformed cells were injected in tobacco leaves using a syringe. Agroinfiltred S. tuberosum was taken as To generation; leaves were harvested for RT-qPCR assay.
OBP8 expression post S. tuberosum feeding
Expression of OBP8 was analyzed after 2, 4, 6 and 8 days feeding of transgenic potato by RT-qPCR. Primers for the OBP8, actin and GFP genes were used as before in dsRNA feeding assays (Table 1). RT-qPCR reactions were performed using Applied Biosystems RT qPCR using SYBR Green PCR Supermix (Thermo Fisher Scientific) according to the protocol. The experiment was done in triplicate using n= 50 M. persicae per replicate post feeding. The reaction mixture consist of cDNA (25 ng), forward and reverse primers (0.5 µM), SYBR Green PCR master mix (10 μL), and cDNA template (2 µL) with total volume of 20 µL. PCR conditions were 95°C for 10 min along with 40 cycles (95°C for 15s, 60°C and 72°C for the 30s).
Mortality using artificial diet feeding assay statistically significant difference was find out by using MS Excel by calculation of the means and standard deviation (STDEV) among the 3 replicate experiment using 20 aphids per replicate. The mRNA expression after ds RNA feeding of the artificial diet and the transgenic potato was done in triplicate. Again 40 aphids per replicate were used to collect mRNA followed by RT qPCR. STDEV was used for the error bars in MS Excel. One-way analysis of variance (ANOVA) was used to find out the significant difference between the +ve control and artificial diet dsRNA assay as well transgenic potato feeding assay. Different alphabets are representing the significant difference of the control with feeding assays.