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Host-mediated RNAi for simultaneous silencing of different functional groups of genes in Meloidogyne incognita using fusion cassettes in Nicotiana tabacum

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Abstract

Key message

This study establishes possibility of combinatorial silencing of more than one functional gene for their efficacy against root-knot nematode, M. incognita .

Abstract

Root-knot nematodes (RKN) of the genus Meloidogyne are the key important plant parasitic nematodes (PPNs) in agricultural and horticultural crops worldwide. Among RKNs, M. incognita is the most notorious that demand exploration of novel strategies for their management. Due to its sustainable and target-specific nature, RNA interference (RNAi) has gained unprecedented importance to combat RKNs. However, based on the available genomic information and interaction studies, it can be presumed that RKNs are dynamic and not dependent on single genes for accomplishing a particular function. Therefore, it becomes extremely important to consider silencing of more than one gene to establish any synergistic or additive effect on nematode parasitism. In this direction, we have combined three effectors specific to subventral gland cells of M. incognita, Mi-msp1, Mi-msp16, Mi-msp20 as fusion cassettes-1 and two FMRFamide-like peptides, Mi-flp14, Mi-flp18, and Mi-msp20 as fusion cassettes-2 to establish their possible utility for M. incognita management. In vitro RNAi assay in tomato and adzuki bean using these two fusion gene negatively altered nematode behavior in terms of reduced attraction, invasion, development, and reproduction. Subsequently, Nicotiana tabacum plants were transformed with these two fusion gene hairpin RNA-expressing vectors (hpRNA), and characterized via PCR, qRT-PCR, and Southern blot hybridization. Production of siRNAs specific to Mi-flp18 and Mi-msp1 was also confirmed by Northern hybridization. Further, transgenic events expressing single copy insertions of hpRNA constructs of fusion 1 and fusion-2 conferred up to 85% reduction in M. incognita multiplication. Besides, expression quantification revealed a significant reduction in mRNA abundance of target genes (up to 1.8-fold) in M. incognita females extracted from transgenic plants, and provided additional evidence for successful gene silencing.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

Authors are acknowledged the Department of Biotechnology (DBT), Government of India for financial support.

Funding

Funding from the Department of Biotechnology (DBT), Government of India to UR through grant number BT/PR5908/AGR/36/727/2012 is acknowledged. Authors also acknowledge the financial support received from DBT-COE project BT/PR-18924/COE/34/48/2017

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Authors and Affiliations

  1. Division of Nematology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India

    Alkesh Hada, Divya Singh, Pradeep K. Papolu, Prakash Banakar, Ankita Raj & Uma Rao

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  1. Alkesh Hada
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  2. Divya Singh
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  3. Pradeep K. Papolu
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  4. Prakash Banakar
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  5. Ankita Raj
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  6. Uma Rao
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Contributions

UR: conceptualization, supervision, and fund acquisition. AH and PKP: plant transformation and molecular characterization. DS and AR: assisted with in vitro studies. PB: design the constructs. AH: nematode bioassays, data analysis, and wrote the original draft of the manuscript. All authors read and approved the manuscript.

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Correspondence to Uma Rao.

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Hada, A., Singh, D., Papolu, P.K. et al. Host-mediated RNAi for simultaneous silencing of different functional groups of genes in Meloidogyne incognita using fusion cassettes in Nicotiana tabacum. Plant Cell Rep 40, 2287–2302 (2021). https://doi.org/10.1007/s00299-021-02767-5

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