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Host-delivered RNAi-mediated root-knot nematode resistance in Arabidopsis by targeting splicing factor and integrase genes

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Abstract

Root-knot nematodes (RKNs) are one of the most important biotic factors limiting crop productivity in many crop plants. The major RKN control strategies include development of resistant cultivars, application of nematicides and crop rotation, but each has its own limitations. In recent years, RNA interference (RNAi) has become a powerful approach for developing nematode resistance. The two housekeeping genes, splicing factor and integrase, of Meloidogyne incognita were targeted for engineering nematode resistance using a host-delivered RNAi (HD-RNAi) approach. Splicing factor and integrase genes are essential for nematode development as they are involved in RNA metabolism. Stable homozygous transgenic Arabidopsis lines expressing dsRNA for both genes were generated. In RNAi lines of splicing factor gene, the number of galls, females and egg masses was reduced by 71.4, 74.5 and 86.6%, respectively, as compared with the empty vector controls. Similarly, in RNAi lines of the integrase gene, the number of galls, females and egg masses was reduced up to 59.5, 66.8 and 63.4%, respectively, compared with the empty vector controls. Expression analysis revealed a reduction in mRNA abundance of both targeted genes in female nematodes feeding on transgenic plants expressing dsRNA constructs. The silencing of housekeeping genes in the nematodes through HD-RNAi significantly reduced root-knot nematode infectivity and suggests that they will be useful in developing RKN resistance in crop plants.

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Acknowledgements

We gratefully acknowledge the financial support from National Agricultural Innovative Project (NAIP) and NASF (National Agricultural Science Fund) previously called the National Fund for Basic Strategic and Frontier Application Research in Agriculture (NFBSFARA), Indian Council of Agricultural Research (ICAR), New Delhi, India. The authors thank Dr. K. V. Prabhu, Dr. Rajendra Singh and the staff of National Phytotron Facility (NPF), IARI, New Delhi, India for providing space in the greenhouse to maintain nematode culture on tomato plants and Arabidopsis thaliana in the growth chambers.

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  1. Atul Kakrana

    Present address: Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE, USA

Authors and Affiliations

  1. ICAR-NRC on Plant Biotechnology, Pusa Campus, New Delhi, 110012, India

    Anil Kumar, Atul Kakrana, R. Srinivasan & Pradeep K. Jain

  2. Division of Nematology, ICAR-Indian Agricultural Research Institute, Pusa Campus, New Delhi, 110012, India

    Anil Sirohi

  3. Department of Biotechnology, Indian Institute of Technology Madras, Chennai, 600036, India

    K. Subramaniam

  4. Department of Biotechnology, Faculty of Science, Centre for Transgenic Plant Development, Jamia Hamdard, New Delhi, 110062, India

    Anil Kumar & M. Z. Abdin

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Correspondence to Pradeep K. Jain.

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10327_2017_701_MOESM1_ESM.jpg

Supplementary Fig. 1 PCR-based confirmation of the presence of splicing factor and integrase genes in control and transgenic Arabidopsis plants expressing dsRNA of splicing factor and integrase genes. a) Amplification of splicing factor gene from DNA samples isolated from control and representative transgenic plants of three independent lines (SF-E1, SF-E2, SF-E3 and control samples were loaded in lanes 1, 2, 3 and 4, respectively). b) Amplification of integrase gene from DNA samples isolated from control and representative transgenic plants of three independent lines (integrase-E1, integrase-E2, integrase-E3 and control samples were loaded in lanes 1, 2, 3 and 4 respectively). Lane M: 100 bp ladder marker. (JPG 109 KB)

10327_2017_701_MOESM2_ESM.jpg

Supplementary Fig. 2 RT-PCR based expression analysis of splicing factor and integrase genes in control and transgenic Arabidopsis plants expressing dsRNA of splicing factor and integrase genes. a) Expression of splicing factor gene from the RNA samples isolated from control and representative transgenic plants of three independent lines (SF-E1, SF-E2, SF-E3 and control samples were loaded in lanes 1, 2, 3 and 4, respectively). b) Expression of integrase gene from RNA samples isolated from control and representative transgenic plants of three independent lines (integrase-E1, integrase-E2, integrase-E3 and control samples were loaded in lanes 1, 2, 3 and 4, respectively). Lane M: 100 bp ladder marker. (JPG 86 KB)

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Kumar, A., Kakrana, A., Sirohi, A. et al. Host-delivered RNAi-mediated root-knot nematode resistance in Arabidopsis by targeting splicing factor and integrase genes. J Gen Plant Pathol 83, 91–97 (2017). https://doi.org/10.1007/s10327-017-0701-3

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  • DOI: https://doi.org/10.1007/s10327-017-0701-3

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