In-planta expression of insecticidal proteins provides protection against lepidopteran insects

The development of advanced biotechnological control strategies opens a new era of environment friendly pest management. The current study is part of such an effort, in which we developed a control strategy based on gene pyramiding that confers broad-spectrum resistance against lepidopteran (Helicoverpa armigera and Spodoptera litura) and hemipteran (Myzus persicae, Phenacoccus solenopsis, and Bemisia tabaci) insect pests. Previously, we reported a double gene construct expressing Hvt and lectin in tobacco (Nicotiana tabacum) plants under phloem specific promoters which confers resistance against hemipteran insects. Here we extended our studies by evaluating the advanced generation of these tobacco plants expressing hvt-lectin against lepidopteran insects. Tobacco plants expressing both toxins were tested against H. armigera and S. litura. Insect bioassay results showed 100% mortality of H. armigera within 48–72 hours and 100% mortality of S. litura within 72–96 hours. Our results suggest that the use of both toxins as a gene pyramiding strategy to control both lepidopteran and hemipterans insects on commercial basis to reduce the use of chemical pesticides.

Numerous studies have been done to identify peptides and proteins with insecticidal properties. After the successful evaluation of the synergistic effects of phloem-specific expression of ωatracotoxin (Hvt) from the Australian funnel web spider (Hadronyche versuta) and plant lectin (onion leaf lectin) against hemipteran insects, the present study was initiated to evaluate the efficacy of the same constructs against two chewing insects, H. armigera and S. litura. Spider venom consists of a diverse mixture of chemical compounds, including peptides and proteins 17 . These compounds have insecticidal properties with a wide range of mechanisms, with significant potential to improve plant resistance against insect pests [18][19][20] . ω-ACTX-Hv1a (Hv1a), one of the peptides isolated from H. versuta venom, acts as a calcium channel blocker to target the central nervous system of insects, resulting in abrupt mortality 21 . Lectins, which have been identified in several plant species, are oligosaccharide-binding proteins present in plants that function in defense against pest attack 22 . Plant lectins are abundant in different plant parts, including roots, leaves, bulbs, tubers, and flowers. Different plant lectins have been found to have entomotoxic effect against different insect orders including both sucking and chewing species 19,[23][24][25][26] . The general mechanism of lectin in insects is to disrupt the epithelial lining of the midgut cells by binding glycoproteins present in the midgut, which leads to different functional and physiological abnormalities like swelling of epithelial cells, microvilli elongation and cell membrane permeability that allows harmful substances into hemolymph, and impaired nutrient absorption 27,28 . Observed entomotoxic activity of lectins includes reduced fecundity, delayed development, mortality, lack of feeding, and abrogated emergence 22 .
In previous study the Hvt-lectin genes were expressed in tobacco plants under phloem specific promoter confer resistance against sucking insects 19 . Here we have extended our previous study and used the same construct to evaluate the toxic effect of both Hvt-lectin, when expressed in combination under phloem specific promoter in tobacco plants against H. armigera and S. litura.

Plant selection and molecular analysis of transgenic tobacco plants.
For the present study, two transgenic lines of tobacco plants expressing the hvt-lectin construct, D6 and D15, were selected. Five biological replicates of D6 and D15 plants in the T 3 generation were subjected to PCR analysis using primers amplifying an internal segment of the Hvt and lectin genes. All of the selected transgenic plants showed amplification of Hvt and lectin genes with the expected 117 bp and 333 bp product sizes, respectively (Fig. 1A,B).

Insect bioassays.
Bioassays were conducted with transgenic and control plants to evaluate the toxicity of the Hvt and lectin against H. armigera and S. litura. Initially, H. armigera larvae fed voraciously on the leaves, but after 6 hours most of the larvae became sluggish and feeding gradually slowed. After 24 hours, 76% mortality were noted on D6 line and 53% mortality on the D15 line. By the third day, almost 100% of the larvae on D6 leaves were dead. In the case of D15, up to 98% mortality was observed on last two consecutive days (Fig. 2). The effects of Hvt and lectin were also assessed visually, with much less damage being apparent on the D6 and D15 leaves than on control leaves (Fig. 3).
The same tobacco plants were used for a detached leaf assay with S. litura. During the initial period after release, larvae started feeding actively. However, after few hours some larvae looked sluggish and their movement www.nature.com/scientificreports www.nature.com/scientificreports/ was restricted, whereas others showed some resistance and continued to feed at different intervals. After 24 hours (Day 1) feeding, 21% of the larvae dead on line D6, whereas line D15 showed only 13% mortality. After 96 hours of feeding, 100% mortality was achieved on the D6 line, whereas, on the D15 line there was 92% mortality. The remaining larvae on D15 leaves were allowed to feed for one additional day (120 h), but only 98% mortality was achieved at the end of the day 5 ( Fig. 4). As in the case of the H. armigera experiments, S. litura larvae consumed much less leaf tissue of the transgenic lines than of control leaves (Fig. 5).
Hvt acts as an antagonist of the insect-specific calcium channel and produce symptoms like lack of coordination, uncontrolled movement, decreased body mass, and browning of the body 19 . The previously described symptoms, including lack of feeding, stunted growth, and changing of color from green to brownish black (Fig. 6) were also observed with S. litura in the current study.

Discussion
Keeping in view the world population, the demand for food production is increasing day by day. With likely increases in pest attacks, there is a need for implementation of advanced strategies to counter these pests. Thus, a combination of toxin proteins with different modes of actions is one of the tools being used to improve resistance against chewing insects for the long term. The present study was designed to use the gene pyramiding technique and evaluate the efficacy of two toxin proteins (Hvt and lectin) expressed in tobacco plants under phloem-specific promoters against economically important chewing pests H. armigera and S. litura. Our results proved the toxicity of both proteins, and significant mortality was observed in both chewing insects (Figs 2 and 4). Both toxins, in combination, reduced the population of H. armigera and caused 100% mortality within 48-72 hours, whereas, in the case of S. litura mortality was confirmed within 72-96 hours. These results are consistent with those of Abbas et al. 29 , who reported the same symptoms on H. armigera and S. litura larvae within two hours after feeding, when   www.nature.com/scientificreports www.nature.com/scientificreports/ Lectins are another class of toxic proteins used for the management of different insect pests. Mostly, lectins have been used against sucking pests, and their role in controlling lepidopteran insects is still under investigation. Several studies confirmed the effectiveness of different lectins when used with other proteins, as well as the direct effect on physiology in lepidopteran insects 23,31-37 . Our results confirmed the quick toxic effects of both lectin and Hvt against H. armigera within 24-48 h. The mortality percentage was 76% and 92% after 24 and 48 h respectively, whereas, these results were contrasts with the results from Hvt alone, which caused larval mortality of only 31% or 6% after 24 h and 69% or 46% after 48 h 20,21,38 . As lectin has been reported to have different modes of action: (i) it may disturb the iron metabolism in insect guts specially in Lepidoptera by binding with ferritin 39 , (ii) it may act   www.nature.com/scientificreports www.nature.com/scientificreports/ as a carrier protein to facilitate other proteins reaching the hemolymph 38 , and (iii) it may act on the peritrophic membrane of the gut region, causing deformation in the membrane and disturbing the microvilli, thereby facilitating the movement of other molecules and toxin proteins into hemolymph 40 . As the peritrophic membrane is absent in hemipteran insects and present in lepidopteran insects 40 , it is possible that lectin may facilitate Hvt movement into hemolymph, thereby enhancing the effects on the insect nervous system.
In summary, our results confirmed the toxicity of Hvt and lectin genes against H. armigera and S. litura expressed under phloem specific promoter. Combine expression of Hvt-lectin resulted quick mortality of H. armigera within 24 h and S. litura in 48 h. Overall 100% mortality of both insects were achieved within 72-96 h. Therefore, expression of these proteins from phloem specific promoters can be used as an alternative approach for long-term management of chewing as well as sucking insects. This is an exciting possibility that needs further demonstration in crop plants under field conditions.  19 were used as an initial material for this experiment. T 1 seeds were germinated on MS0 medium supplemented with 100 mg/L kanamycin in petri dishes to detect at least one copy of the transgene. Transgenic seedlings growing on kanamycin-supplemented medium were maintained further in glass jars and ultimately shifted to the soil. Plants were raised to the T 2 generation and T 3 seeds were collected. T 3 plants of two transgenic events, D6 and D15, were grown in a glasshouse and subjected to PCR amplification for the detection of the transgenes, as well as insect bioassays. Genomic  Insect rearing. Two chewing insects H. armigera and S. litura were used to evaluate the transgenic tobacco plants expressing Hvt-lectin proteins. Different stages of both insects were collected from the field and reared on chickpea flour-based artificial diet 42 under controlled conditions of 28 ± 2 °C, 75 ± 5% R.H. and 14-10(L:D) h photoperiod. Adults were kept in jars and fed on 10% honey solution. After egg laying and the emergence of larvae, each larva was shifted to a plastic vial containing artificial diet until pupation. Pupae were shifted to adult rearing jars for adult emergence. Multiple generations were obtained to maintain the culture in laboratory conditions. Second-instar larvae of both insect species were used for insect bioassays.

Insect bioassays.
After kanamycin screening and transgene confirmation through PCR, both two transgenic lines D6 and D15 (T 3 stage) were shifted to a growth room for further growth at 25 ± 2 °C, 60 ± 5% R.H. and 16-8 (L:D) h photoperiod. Five plants (biological replicates) of each line at the 4-5 leaf stage were used for insect bioassays using the detached leaf method. Three fully expanded leaves from each plant/line were detached, washed with distilled water, and placed in a Petri dishes containing moist filter paper. Five second-instar larvae of H. armigera or S. litura were carefully shifted onto each leaf. Petri plates were covered with a lid and sealed with parafilm to prevent escape of the larvae. Mortality data and morphological observations were taken on every 24 hours till 120 hours (day 5). Equal numbers of non-transgenic control leaves were also used to compare the mortality data with transgenic plants. Mortality percentage of the larvae were calculated using the following formula: − × Number of larvae found dead on leaf (Total number of larvae allowed to feed on the leaf number of larvae that absconded) 100 statistical analysis. All data were analyzed by using the statistical software SPSS 21. To check the normality of data ShapiroWilk test was used. ANOVA and Kruskal-Wallis test were carried out to determine the significant differences between treatments at P-value < 0.05