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Expression of an insecticidal fern protein in cotton protects against whitefly

Nature Biotechnology volume 34, pages 1046–1051 (2016)Cite this article

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Abstract

Whitefly (Bemisia tabaci) damages field crops by sucking sap and transmitting viral diseases. None of the insecticidal proteins used in genetically modified (GM) crop plants to date are effective against whitefly. We report the identification of a protein (Tma12) from an edible fern, Tectaria macrodonta (Fee) C. Chr., that is insecticidal to whitefly (median lethal concentration = 1.49 μg/ml in in vitro feeding assays) and interferes with its life cycle at sublethal doses. Transgenic cotton lines that express Tma12 at ∼0.01% of total soluble leaf protein were resistant to whitefly infestation in contained field trials, with no detectable yield penalty. The transgenic cotton lines were also protected from whitefly-borne cotton leaf curl viral disease. Rats fed Tma12 showed no detectable histological or biochemical changes, and this, together with the predicted absence of allergenic domains in Tma12, indicates that Tma12 might be well suited for deployment in GM crops to control whitefly and the viruses it carries.

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Figure 1: Purification and characterization of Tma12 from T. macrodonta.
Figure 2: Transgenic cotton plants expressing Tma12 resist whitefly.
Figure 3: Tma12 expression in cotton interferes with the whitefly life cycle.
Figure 4: Toxicological study of Tma12 in rats.

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Acknowledgements

The authors acknowledge H.K. Yadav, R. Kamal and S. Sharma for help in statistical analysis; M. Lal, Rajesh K. Srivastava, S.H.M. Abidi, A. Bano (deceased) and Rakesh K. Srivastava for technical assistance; and A.C. Little for photography. The authors thank the Council of Scientific and Industrial Research, India, for funding under the Supra Institutional Project (SIP05), the Network Projects (NWP03 and PlaGen BSC0107) and the Council of Scientific and Industrial Research–National Botanical Research Institute for providing research facilities. A.K.S., S.K.U., R.S., M.M., S. Saurabh, H.S., N.T., P.P., A.L.H. and V.R. are supported by CSIR. P.R. and S.K.Y. are supported by the Indian Council of Medical Research. S. Srivastava is supported by the University Grant Commission. R.T. is supported by a Department of Science and Technology (DST) JC Bose National Fellowship.

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Author notes

  1. Santosh Kumar Upadhyay, K Chandrashekar & Rakesh Tuli

    Present address: Present addresses: Department of Botany, Panjab University, Chandigarh, India (S.K.U.), Regional Station, Indian Agricultural Research Institute, Pune, India (K.C.), University Institute of Engineering and Technology, Panjab University, Chandigarh, India (R.T.).,

  2. Anoop Kumar Shukla, Santosh Kumar Upadhyay, Manisha Mishra, Sharad Saurabh, Rahul Singh, Harpal Singh and Nidhi Thakur: These authors contributed equally to this work.

Authors and Affiliations

  1. Genetics and Plant Molecular Biology, Council of Scientific and Industrial Research (CSIR)–National Botanical Research Institute, Lucknow, India

    Anoop Kumar Shukla, Santosh Kumar Upadhyay, Manisha Mishra, Sharad Saurabh, Rahul Singh, Harpal Singh, Nidhi Thakur, Preeti Rai, Paras Pandey, Aradhana L Hans, Subhi Srivastava, Vikram Rajapure, Sunil Kumar Yadav, Mithlesh Kumar Singh, K Chandrashekar, Praveen C Verma, Shirish A Ranade, Rakesh Tuli & Pradhyumna Kumar Singh

  2. Academy of Scientific and Innovative Research, New Delhi, India

    Manisha Mishra, Nidhi Thakur, Paras Pandey, Aradhana L Hans, Sunil Kumar Yadav, Mithlesh Kumar Singh, Praveen C Verma, Ajit Pratap Singh, K N Nair, Shirish A Ranade & Pradhyumna Kumar Singh

  3. National Agri-Food Biotechnology Institute, Mohali, Punjab, India.,

    Jitendra Kumar

  4. Systematics and Herbarium Division, Plant Diversity, CSIR–National Botanical Research Institute, Lucknow, India

    Ajit Pratap Singh & K N Nair

  5. Toxicology Division, CSIR–Central Drug Research Institute, Lucknow, India

    Smrati Bhadauria, Sarika Singh & Sharad Sharma

  6. Pharmacokinetics and Metabolism Division, CSIR–Central Drug Research Institute, Lucknow, India

    Muhammad Wahajuddin

  7. Department of Zoology, Ladybird Beetle Research Laboratory, University of Lucknow, Lucknow, India

    Omkar

  8. Department of Botany, Banaras Hindu University, Varanasi, India

    Ram Sanmukh Upadhyay

Authors
  1. Anoop Kumar Shukla
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Contributions

A.K.S.: development of cotton transformation protocols, transgenic cotton lines and acquisition of data. S.K.U.: purification of Tma12 and characterization, cloning of tma12. M.M.: MS analysis of Tma12, study on whitefly life cycle on transgenic cotton in clip cage and manuscript preparation. S. Saurabh, R.S., H.S., P.R.: bioprospection of ferns for insecticidal activity, establishment of screening protocol, identification of the potential ferns, execution of experiments and manuscript preparation. N.T.: clip cage assay, analysis of transgenic cotton lines and manuscript preparation. S. Srivastava: analysis of transgenic cotton lines and manuscript preparation. A.L.H., P.P.: study of whitefly life cycle on transgenic cotton in the field. V.R., M.K.S.: evaluation of transgenic cotton in field trials. S.K.Y.: chitin-binding assay, analysis of impact of Tma12 on whitefly at sublethal doses. J.K.: virus studies. K.C.: designing and execution of whitefly bioassays and statistical analysis. P.C.V.: designing and supervision of whitefly life cycle study on transgenic cotton and transgenic cotton development. A.P.S.: collection, identification, mass multiplication and in-house maintenance of ferns. K.N.N.: review of experimental design and writing of manuscript. S.B., M.W., S. Singh, S. Sharma: biosafety experiments. O.: designing and execution of experiments on beneficial insect ladybird beetle. R.S.U.: supervised A.K.S. in cotton tissue culture and field evaluation of transgenic cotton. S.A.R.: critical review of manuscript. R.T.: critical review of results, intellectual input and manuscript editing. P.K.S.: study concept and experimental design, data analysis, study supervision and manuscript writing and editing.

Corresponding author

Correspondence to Pradhyumna Kumar Singh.

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Competing interests

A patent application (WO 2013098858A2; P.K.S., S.K.U., K.C., S. Saurabh, R.S., P.R., H.S., M.M., A.P.S., P.C.V., K.N.N. and R.T. are inventors) based on the Tma12-GM crop strategy for field control of whiteflies is under examination. A.K.S., S.K.U., M.M., S. Saurabh, R.S., H.S., N.T., P.R., P.P., A.L.H., S. Srivastava, V.R., S.K.Y., M.K.S., K.C., P.C.V., A.P.S., K.N.N., R.T., P.K.S., CSIR–National Botanical Research Institute and Council of Scientific and Industrial Research (the funding agency), Ministry of Science and Technology, Government of India, hope to translate the technology jointly with industry to develop whitefly-resistant GM crops.

Integrated supplementary information

Supplementary Figure 1 Overview of the study.

Schematic representation of key steps in the strategy for prospecting ferns for insecticidal proteins and development of transgenic cotton.

Supplementary Figure 2 N-terminal sequencing of Tma12.

Detection of 1st to 12th amino acid sequence ‘HGSMEDPISRVY’ in Tma12 is shown in b to m. Arrows show the probable peak of the identified amino acid residue. The amino acid standards are shown in a. The sequencing was done by Proteomics International, Australia.

Supplementary Figure 3 De novo sequencing of peptides of Tma12.

Schematic representation of fragmentation chemistry of peptides before and after derivatization on MALDI TOF/TOF. Panel (a) represents simultaneous fragmentation of the peptide at both the ends generating b and y ions that made the spectra condensed. The data was analyzed in-silico (DeNovo Explorertm Software) and the top 10 sequences were predicted, as listed above. Panel (b) represents the strategy followed to improve de novo sequencing. The peptide was modified at N-terminus (SPITC modification) and then fragmented. The modification prevented all the b ions (generated from N-terminus fragmentation) from entering into drift tube. This allowed y-ions to produce a clear spectrum with high signal to noise ratio. The series of ions was analyzed manually and amino acid sequence of the peptide was deduced.

Supplementary Figure 4 Architecture of insecticidal protein-encoding gene.

(a) Structure of tma12 gene. (b) Complete ORF of Tma12 showing signal peptide of 24 amino acids (red fonts) and mature protein of 192 amino acid residues. Protein sequences shown in blue fonts are the three peptides sequenced through N-terminal and de novo sequencing. (c) Conserved domain search of Tma12 showing the best match with bacterial chitin binding domain 3 protein of Herpetosiphon aurantiacus (GI:159899166) with similarity score 55.

Supplementary Figure 5 Characterization of Tma12.

(a) Chitin binding assay and SDS-PAGE analysis showing binding of Tma12 on chitin magnetic beads and its elution. (b) Thermal stability of Tma12; enzymatic activity after incubation at different temperatures for 30 min. Both exo and endo-chitinase activities started declining sharply at 50oC and were lost at 60oC. Chitinolytic activity of Tma12, Michaelis-Menten and Lineweaver-Burk plot are shown for (c) colloidal chitin (d) 4-methylumbelliferyl β-D-N, N’-diacetylchitobioside hydrate and (e) 4-methylumbelliferyl β-D-N, N’, N’’-triacetylchitotriose.

Supplementary Figure 6 Development of putative transgenic cotton lines, insect resistance and generation advancement.

A total of 16 transgenic plants were developed, 9 of them exhibited high resistance against whitefly. However, eight plants shown in top panels did not flower. Four other plants (left lower panel) developed flowers but did not advance into next generation. Four plants (right lower panel) produced bolls and advanced to next generation. The transgenic line number is given on the pots. Scale bars,10 cm.

Supplementary Figure 7 Expression analysis of transgene in event 14.

(a) qRT-PCR of different parts of transgenic plant, the expression of tma12 in leaf tissue is considered as unity and relative expression of tma12 in all other plant parts is plotted in the graph. The relative expression of tma12 was highest in root followed by stem, fruit wall, leaf, petal, anthers and square. (b) Immuno-blot analysis of the different parts of Event14. Tma12 was purified from stem, leaf, root and seed of the transgenic plant and quantified using purified Tma12 as standard. The expression of Tma12 was reasonably well in leaves, moderate in stem and barely detectable in seeds. There was no detectable expression in root despite of high transcript level.

Supplementary Figure 8 Whitefly resistance in transgenic cotton prevents infection by CLCuV.

Detection of the cotton leaf curl virus (type CLCuKoV-Bu) by PCR. Presence of the virus is seen in non transgenic cotton plants, with the PCR product of ∼2.8 kb DNA representing the viral genome. (b) Amplification of ∼1.3 kb alpha satellite DNA and (c) the ∼1.3 kb beta satellite DNA, associated with CLCuV from non-transgenic cotton plants. (d-f) Transgenic cotton plants showed no amplification of the genome of CLCuV, the alpha satellite or the beta satellite. M is λ DNA/HindIII-EcoRI digested molecular weight markers.

Supplementary Figure 9 Effect of Tma12-transgenic cotton on Helicoverpa armigera.

Leaves of transgenic and control cotton plants were challenged with neonatal larvae of H. armigera. Insect larvae feed on both the leaves. This indicated that Tma12-cotton is not efficacious to H. armigera. Scale bar, 2.5 cm.

Supplementary Figure 10 Effect of Tma12-transgenic cotton on nontarget sucking insect pests.

(a) 4th instar aphids were inoculated on transgenic or control plants (20 per plant) and the population was recorded on 9 plants at days 7 and 22. Data shown as mean ± SD per plant. No significant difference in aphid population was observed between control and transgenic cotton plants (P value >0.05). (b) Representative photograph of transgenic and control leaves infested with aphids. Scale bars, 2.5 cm. (c) Three leaves of transgenic or control cotton plants were challenged with six mature female mealybugs (2 per leaf). The insects were counted on days 6 and 24. Data shown as mean ± SD per plant. (d) Representative photograph of transgenic and control leaves infested with mealybugs. Insect population increased rapidly till day 6, while decreased and stabilized on day 24. Scale bars, 2.5 cm.

Supplementary Figure 11 Field evaluation of Tma12 transgenic cotton in T1 and T2.

(a) Transgenic and (b) Nontransgenic cotton plants in T1 generation. (c) Transgenic and (d) Nontransgenic cotton plants in T2 generation. Scale bars, 1 m.

Supplementary Figure 12 Evaluation of Tma12 transgenic cotton in contained field trials.

(a) Transgenic (b) Nontransgenic and (c) Pesticide-protected nontransgenic cotton plants in T3 generation. (d) Nontransgenic and (e) transgenic cotton plants in T4 generation. Scale bars for a, b & c; 0.5 m and d & e, 1 m.

Supplementary Figure 13 Pepsin sensitivity assay.

SDS-PAGE showing time course digestion of Tma12; the insecticidal protein was digested completely, within 30 sec of incubation with pepsin.

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Supplementary Figures 1–13, Supplementary Tables 1–7 and Supplementary Note 1 (PDF 3754 kb)

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Shukla, A., Upadhyay, S., Mishra, M. et al. Expression of an insecticidal fern protein in cotton protects against whitefly. Nat Biotechnol 34, 1046–1051 (2016). https://doi.org/10.1038/nbt.3665

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