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Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato

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Abstract

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a devastating agricultural insect pest with broad spectrum of host range, causing million dollars crop loss annually. Limitations in the present conventional and transgenic approaches have made it crucial to develop sustainable and environmental friendly methods for crop improvement. In the present study, host-induced RNA interference (HI-RNAi) approach was used to develop H. armigera resistant tobacco and tomato plants. Chitinase (HaCHI) gene, critically required for insect molting and metamorphosis was selected as a potential target. Hair-pin RNAi construct was prepared from the conserved off-target free partial HaCHI gene sequence and was used to generate several HaCHI-RNAi tobacco and tomato plants. Northern hybridization confirmed the production of HaCHI gene-specific siRNAs in HaCHI-RNAi tobacco and tomato lines. Continuous feeding on leaves of RNAi lines drastically reduced the target gene transcripts and consequently, affected the overall growth and survival of H. armigera. Various developmental deformities were also manifested in H. armigera larvae after feeding on the leaves of RNAi lines. These results demonstrated the role of chitinase in insect development and potential of HI-RNAi for effective management of H. armigera.

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Acknowledgments

We are grateful to the Department of Biotechnology (Grant No. BT/AGR/TF/2006), New Delhi and Sri Biotech Laboratory India Ltd. Hyderabad for financial assistance (to MVR). Mamta acknowledges the University Grants Commission, New Delhi for the senior research fellowship under the special assistance programme (SAP) of University Grants Commission (UGC). We also thank the UGC for SAP (DRS-III), Department of Science and Technology (DST), New Delhi for FIST (Level 2) programme and DU-DST PURSE (Phase II) Grant.

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  1. Department of Genetics, University of Delhi South Campus, Benito Juarez Marg, New Delhi, 110021, India

    Mamta & M. V. Rajam

  2. Sri Biotech Laboratory India Ltd., Street No. 2, Sagar Society, Road No. 2, Banjara Hills, Hyderabad, 500034, India

    K. R. K. Reddy

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  1. Mamta
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Correspondence to M. V. Rajam.

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Supplementary Fig. 1

Similarity of selected HaCHI sequences [approximately 144 bp] with Spodoptera spp. and other insects (JPEG 97 kb)

Supplementary Fig. 2

Generation and molecular analyses of HaCHI-RNAi tobacco and tomato plants. (a) T-DNA map of HaCHI-RNAi construct, harbouring partial HaCHI cDNA in sense (S-CHI) and antisense (AS-CHI) orientation. 35S P- CaMV 35S promoter, NPT-II- Neomycin phosphotransferase, Intron- Chalcone synthase intron, LB- Left border, RB- Right border; Confirmation of transgene presence in primary HaCHI-RNAi tobacco (b) and tomato (c) transformants by PCR analysis with HaCHI and NPT-II gene-specific primers. M- 100 bp and 1 kb DNA ladder, B- Blank control (PCR without DNA), P- positive control (PCR with plasmid of HaCHI-RNAi vector), UT- Untransformed control, RNAi 13 to 80 are independent HaCHI-RNAi tobacco lines, RNAi 1 to 12 are independent HaCHI-RNAi tomato lines. Determination of transgene copy number by Southern blot hybridization in EcoRI digested genomic DNA from untransformed control and HaCHI-RNAi tobacco (d) and tomato (e) lines probed with radiolabeled probe prepared from partial coding sequence of NPT-II gene. UT- Untransformed control, RNAi 13 to 80 are independent HaCHI-RNAi tobacco lines, RNAi 2 to 22 are independent HaCHI-RNAi tomato lines (JPEG 132 kb)

Supplementary Fig. 3

Initial screening of different HaCHI-RNAi tobacco and tomato lines for insect resistance, based on detached leaf feeding bioassay. Mortality based initial screening of different HaCHI-RNAi tobacco (a) and tomato (b) lines for resistance against H. armigera, Data represent mean percentage mortality ± SE from three independent experiments with n=10 larvae per experiment, UT- Untransformed plant, RNAi 4 to 88 are independent HaCHI-RNAi tobacco lines, RNAi 1 to 38 are independent HaCHI-RNAi tomato lines. *Represent significant differences between HaCHI-RNAi lines and UT (P< 0.05) (JPEG 133 kb)

Supplementary material 4 (DOCX 14 kb)

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Mamta, Reddy, K.R.K. & Rajam, M.V. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato. Plant Mol Biol 90, 281–292 (2016). https://doi.org/10.1007/s11103-015-0414-y

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