Transgenic rice seeds cv IR64 used for this study were developed by the present investigating group [17]. BPH were obtained from Regional Rice Research Station at Chinsurah, West Bengal for all the experiments.

Rice seeds (T₃) were surface sterilized for 30 minutes in 1.5% (v/v) sodium hypochlorite solution, rinsed thoroughly (3 - 4 times) in sterile water and sown on half strength MS [18] germinating medium with 2% sucrose and 0.7% w/v agar [17]. 30 days old plants were used for molecular analysis.

Multiplex PCR was carried out with seven T₃ plants (randomly chosen) of T₂ parental line No. L03C04 (6) using ASAL gene specific primer pairs. Genomic DNA was isolated from young green leaves of T₃ progeny plants and control rice plants followed by CTAB extraction method [19]. Multiplex PCR was subjected using Qiagen Multiplex PCR Kit (GmbH, Hilden, Germany). Each 50 µl PCR reaction mixture contained 1x PCR master mix including Hot Star Taq DNA polymerase. Primers (Table 1) were used in 0.2 µM concentration. The reaction started with incubation for 15 minutes at 94˚C followed by 35 cycles of [30 s at 94˚C, 90 s at 58˚C, 90 s at 72˚C] and final extension with 10 minutes at 72˚C in My Cycler (Bio Rad, Hercules, CA, USA). DNA of pBKhg ASAL and pBK16.2 plasmids was taken as positive control for ASAL and cre gene respectively. DNA of control rice plant was taken as negative control. The segregation patterns of ASAL and cre genes in the T₃ hybrid lines were observed by analysing the presence or absence of respective gene-specific amplicons resolved in 1.2% Agarose gel.

Protein from leaves of 1 month old control and T₃ progeny plants were extracted in Tris-cl buffer pH-7. After quantification using Bradford reagent [20], western blotting was carried out with total protein extracted from each of the T₃ progeny plants probed with anti-ASAL antibody according to the protocol described by Dutta et al. 2005 [11]. About 50 µg of total plant protein re

Table 1. List of PCR primers.

solved in 15% SDS-PAGE was electrophoretically transferred to Hybond-C membrane (GE Healthcare, UK) using Hoeffer (Hoefer Inc., Holliston, MA, USA) electroblot apparatus. The membrane was transiently stained with Ponceau S (Sigma-Aldrich, USA) for confirming the transfer of proteins onto it. Blocking of membrane was done with 5% non-fat milk (Merck, Germany) dissolved in 1x PBST at 37˚C for 3 hours. After washing with 1x PBST, the membrane was then incubated with anti-ASAL polyclonal antiboby (1:4000) for 2 hours at 37˚C. Further probing of the primary antibody was done for 2 hours with anti-rabbit IgG-HRP conjugate (1:20,000) (Sigma-Aldrich, USA) and developed using Hybond ECL kit on Hybond ECL film (GE Healthcare, Germany). Purified native ASAL protein (0.5 µg) was used as positive control and total protein (50 µg) from untransformed plants was used as negative control.

100 µg of crude protein extracted from T₃ transgenic leaves of each plant were coated in the wells of microtitre plate with coating buffer (15 mM Sodium carbonate, 35 mM sodium bicarbonate, 3 mM sodium azide). After charging the protein extract with primary and secondary antibody, substrate [O-phenylen ediamine diamine hydrochloride (OPD, BRL)] dissolved in citrate buffer (pH –9.6) was added and coloured reaction was developed. Optical density was measured at 415 nm in a Bio-Rad microtitre plate reader model no. 680 [11].

The heamagglutination assay was done with the total protein extracts (100 µg/well) of five T₃ ASAL expressing lines. Blood sample was collected from the rabbit by a syringe and immediately transferred to a glass tube pre filled with 0.9% saline as described by Banerjee et al. [21]. The erythrocytes were incubated with plant extracts in microtiter well and after 1 hour of incubation at 37˚C, the hemagglutination activity was checked.

T₃ plants expressing ASAL and control plants were subjected to insect bioassay with BPH as described by Sengupta et al. [17]. Five marker gene free plants (line no. SD2, SD4, SD5, SD7 and SD8) were tested with BPH nymph and compared the mortality of the insects incubated on control plants. Each set was infested with 20 BPH nymphs of first instar into an individual insectproof cage and survivability of insects was monitored at every 2 days from the release of insects up to 16 days. Effect of ASAL on fecundity of BPH was assessed by measuring total nymph production by adult insects on the transgenic rice plants (line no. SD2, SD4, SD5, SD7 and SD8). Each experimental set was repeated five times and statistical analysis was done in order to compare the significance of differential mortality among all transgenic and controls plants.

Total brush border membrane vesicle (BBMV) from Nilaparvata lugens was isolated according to Bandopadhyay et al. [22] using MgCl₂ sequential precipitation method suited for luminal membranes. About 20 µg of total BBMV resolved in 12% SDS-PAGE was electrophoretically transferred to Hybond-C membrane (GE Healthcare, UK) using Hoeffer (Hoefer Inc., Holliston, MA, USA) electroblot apparatus. The membrane was temporarily stained with Ponceau S (Sigma-Aldrich, USA) to ensure the protein transfer from gel to Hybond-C membrane. Blocking of membrane was done with 5% non-fat milk (Merck, Germany) dissolved in 1x PBST at 37˚C for 2 hours. The membrane was further subjected to incubation with 100 µg of total protein extracted from transgenic ASAL positive SD7 line (ASAL expression level ~5 ng∙µg⁻¹) for 2 hours following extensive washing with 1x PBST. The membrane was then incubated with anti-ASAL antibody (1:4000) for 2 hours at 37˚C. Further probing of the secondary antibody was done with anti-rabbit IgG-HRP conjugate (1:20,000) (Sigma-Aldrich, USA) and film was developed using Hybond ECL kit (GE Healthcare, Germany) as described by Mondal et al. [23]. Ligand blot assay with total protein (100 µg) from non-transgenic rice plant served as negative control.

Receptor protein band detected via ligand blot assay when challenged with total protein from ASAL positive transgenic rice line were excised, trypsinised and processed for LC MS/MS analysis as described in details by Sarkar et al. [24]. The extracted peptides were analyzed by LC MS/MS on Orbitrap Velos. Individual MS/MS spectra were searched through Mascot search using UNIPROT_2011_08 database with preset parameters such as standard global modifications, minimum of 5 peptide match and 99% protein thresholds. The results were observed using Scaffold (version Scaffold_3.6.4, Proteome software Inc., Portland, OR).

Seven T₃ plants were taken from T₂ parental line no. L03C04 (6) (randomly chosen) for multiplex PCR analysis. Five plants were found to be positive for ASAL and two plants were found to be negative suggesting about the segregation of ASAL in T₃ generation (Figure 1). Absence of cre gene was also observed in the ASAL positive lines.

Western blot of total protein extract from 1 month old leaves of the five T₃ lines were analysed which showed ASAL positive protein band at 12.5 kDa region (Figure 2(a)). ELISA result of five plants showed ASAL expression level in the range of 0.4% to 0.5% of the total soluble protein fraction (Figure 2(b)). Among these five lines, SD7 rice line showed highest expression level of ASAL (0.5%).

In the heamagglutination assay, ASAL positive T₃ lines showed agglutination of rabbit erythrocytes by forming a carpet of erythrocytes cells in the wells (Figure 3: Panel A, well 1 - 5) whereas the control line showed a tight button of rabbit erythrocytes cells at the centre of the well (Figure 3: Panel B, well 1 - 5) indicating the negative agglutination reaction. This result confirmed the retention of lectin activity of the expressed ASAL from transgenic rice lines.

In planta insect bioassay was carried out on 1-month-old five ASAL-expressing T₃ progeny rice plants. All five transgenic plants inoculated with BPH, showed significant reduction in survivability and fecundity of the pest compared to that of the insects incubated on untrans-

formed control plants. The number of living insect declined to 5.3 ± 0.33 (27%) to 6 ± 1.0 (30%) whereas in control plant the number of living insect was 13.66 ± 0.3 (68.3%) (Figure 4(a)). The mean number of survival of BPH on transgenic plants [t_(0.01) 18.37] was significantly (p < 0.01) different than the control plants (unpaired t test). After 16 days of the experiment SD7 line represents maximum mortality of BPH. Fecundity of BPH on ASAL expressing plants was also examined by counting the total nymphs produced by the adult insects on transformed and untransformed control rice plants. After 30 days of assay period, the mean number of BPH nymphs on ASAL positive T₃ plants was found to be 50 ± 2.8 (42.4%) to 40.67 ± 1.2 (35%) where as in control plants the number increased up to 118 ± 4.1 (100%) (Figure 4(b)) suggesting the lower reproductive rate of BPH fed on transgenic rice plants compared to the control rice plants.

Approximately 56 kDa receptor protein from the brush border membrane vesicle (BBMV) of N. lugens was lightened up after ligand blot assay carried out with total protein from transgenic ASAL positive SD7 rice lines (Figure 5). Identified receptor protein band was subjected to LC MS/MS analysis. Sequence information of peptide fragments obtained through MS/MS were individually searched against Uniport database and receptor protein identified as NADH-quinone oxidoreductase from N. lugens [Accession no: Q5W1K6_NILLU] with a sequence coverage of 45% (Table 2, Supply Figure 1).

Analysing T₃ marker free transgenic rice lines, it was found that 71% of the transgenic rice plants showed presence of ASAL gene and absence of cre gene in this generation suggesting about segregation of ASAL among T₃ progenies which is close to attain homozygosity. Significant level of ASAL expression was observed in T₃ rice lines revealed by ELISA and western blot assay.

In planta insect bioassay experiments demonstrated significant insecticidal efficacy of different transgenic ASAL positive rice lines against BPH. Furthermore, fecundity rate was also observed to be reduced among the insects (BPH) fed on transgenic rice plants which seem to be comparable with the earlier reports from the present investigating group [13,17].

The insecticidal efficacy of ASAL in SD2, SD4, SD5, SD7 and SD8 T₃ rice lines had already been documented by in planta insect bioassay experiment. To understand the basis of this toxic effect, ligand blot assay had been done and ~56 kDa putative receptor protein band from N. lugens BBMV fraction was detected. LC MS/MS analysis has shown the receptor protein band (56 kDa) was of NADH quinone oxidoreductase enzyme of N. lugens [27].

NADH quinone oxidoreductase (NQO) is the first enzyme of electron transport respiratory chain. It contributes to ATP synthesis. Studies revealed that some secondary metabolites like rotenone and piericidin A from microbial and plant sources are important components that act on the respiratory chain by inhibiting NQO [28]. It was also documented by earlier investigators that NQO gene expression was enhanced in BPH, feeding on B5 (BPH resistance) rice lines, indicating that there might be some compounds having a similar structure and function as rotenone or piericidin A generated in B5 plants, which might inhibit the respiratory chain enzymes in BPH to

certain degree. This result emphasises that NQO may play important role in the defence response of BPH against resistant rice lines [27]. So it is quite obvious that binding of ASAL to NQO may upset insect defence mechanism and costs its survival.

It has also been reported in Drosophila melanogaster that the mitochondrial acyl carrier protein (mtACP), a subunit of NADH ubiquinone oxidoreductase is involved in de novo fatty acid synthesis in the mitochondrion. APelement-induced loss-of-function mutation in the mtacp1 gene resulted in lethality, indicating that the gene is essential for viability of the insect [29]. Additional studies showed that mtacp1 is expressed at higher levels during late embryogenesis, in the pupa and adult mosquitoes, indicating its requirement for both male and female gametogenesis and possibly for tracheal development in Drosophila. Correlating the earlier observations with our present findings, it is assumed that ASAL binding to NQO of BPH can affect normal development and gametogenesis, which results into loss of fecundity of insects feeding on presently, mentioned ASAL rice lines.