Abstract
Main conclusion
This study demonstrates the combinatorial management of multiple pests through a trans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy. Transgenic cotton events demonstrated improved efficacy against cotton leaf curl disease, cotton leaf hopper and root-knot nematode.
Abstract
Cotton (Gossypium hirsutum L.), an important commercial crop grown worldwide is confronted by several pests and pathogens, thus reiterating interventions for their management. In this study, we report, the utility of a novel Arabidopsis miRNA173-directed trans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy for the simultaneous management of cotton leaf curl disease (CLCuD), cotton leaf hopper (CLH; Amrasca biguttula biguttula) and root-knot nematode (RKN, Meloidogyne incognita). Cotton transgenics were developed with the MIGS construct targeting a total of 7 genes by an apical meristem-targeted in planta transformation strategy. Stable transgenics were selected using stringent selection pressure, molecular characterization and stress-specific bio-efficacy studies. We identified 8 superior events with 50–100% resistance against CLCuD, while reduction in the root-knot nematode multiplication factor in the range of 35–75% confirmed resistance to RKN. These transgenic cotton events were also detrimental to the growth and development of CLH, as only 43.3–62.5% of nymphs could survive. Based on the corroborating evidences obtained by all the bioefficacy analyses, 3 events viz., L-75-1, E-27-11, E-27-7 were found to be consistent in tackling the target pests. To the best of our knowledge, this report is the first of its kind demonstrating the possibility of combinatorial management of pests/diseases in cotton using MIGS approach. These identified events demonstrate immense utility of the strategy towards combinatorial stress management in cotton improvement programs.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Abdurakhmonov IY, Ayubov MS, Ubaydullaeva KA, Buriev ZT, Shermatov SE, Ruziboev HS, Pepper AE (2016) RNA interference for functional genomics and improvement of cotton (Gossypium sp.). Front Plant Sci 7:202. https://doi.org/10.3389/fpls.2016.00202
Agrawal A, Rajamani V, Reddy VS, Mukherjee SK, Bhatnagar RK (2015) Transgenic plants over-expressing insect-specific microRNA acquire insecticidal activity against Helicoverpa armigera: an alternative to Bt-toxin technology. Transgenic Res 24:791–801. https://doi.org/10.1007/s11248-015-9880-x
Akhtar S, Tahir MN, Amin I, Mansoor S (2021) Amplicon-based RNAi construct targeting beta-C1 gene gives enhanced resistance against cotton leaf curl disease. 3 Biotech 11:1–10. https://doi.org/10.1007/s13205-021-02816-6
Akmal M, Baig MS, Khan JA (2017) Suppression of cotton leaf curl disease symptoms in Gossypium hirsutum through over expression of host-encoded miRNAs. J Biotechnol 263:21–29. https://doi.org/10.1016/j.jbiotec.2017.10.003
Allen E, Howell MD (2010) miRNAs in the biogenesis of trans-acting siRNAs in higher plants. Semin Cell Dev Biol 21:798–804. https://doi.org/10.1016/j.semcdb.2010.03.008
Axtell MJ, Bowman JL (2008) Evolution of plant microRNAs and their targets. Trends Plant Sci 13:343–349. https://doi.org/10.1016/j.tplants.2008.03.009
Baig MS, Akhtar S, Khan JA (2021) Engineering tolerance to CLCuD in transgenic Gossypium hirsutum cv. HS6 expressing Cotton leaf curl Multan virus-C4 intron hairpin. Sci Rep 11:1–11. https://doi.org/10.1038/s41598-021-93502-3
Banakar P, Hada A, Papolu PK, Rao U (2020) Simultaneous RNAi knockdown of three FMRFamide-like peptide genes, Mi-flp1, Mi-flp12, and Mi-flp18 provides resistance to root-knot nematode Meloidogyne incognita. Front Microbiol 11:2690. https://doi.org/10.3389/fmicb.2020.573916
Bhardwaj A, Thapliyal S, Dahiya Y, Babu K (2018) FLP-18 functions through the G-protein-coupled receptors NPR-1 and NPR-4 to modulate reversal length in Caenorhabditis elegans. J Neurosci 38:4641–4654. https://doi.org/10.1523/JNEUROSCI.1955-17.2018
Biswas KK, Bhattacharyya UK, Palchoudhury S, Balram N, Kumar A, Arora R, Sain SK, Kumar P, Khetarpal RK, Sanyal A, Mandal PK (2020) Dominance of recombinant cotton leaf curl Multan-Rajasthan virus associated with cotton leaf curl disease outbreak in northwest India. PLoS ONE 15:e0231886. https://doi.org/10.1371/journal.pone.0231886
Bonfim K, Faria JC, Nogueira EO, Mendes ÉA, Aragão FJ (2007) RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol Plant-Microbe Interact 20:717–726. https://doi.org/10.1094/MPMI-20-6-0717
Carbonell A, Lisón P, Daròs JA (2019) Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance. Plant J 100:720–737. https://doi.org/10.1111/tpj.14466
Catarino R, Ceddia G, Areal FJ, Park J (2015) The impact of secondary pests on Bacillus thuringiensis (Bt) crops. Plant Biotechnol J 13:601–612. https://doi.org/10.1111/pbi.12363
Chaudhary S, Dutta TK, Tyagi N, Shivakumara TN, Papolu PK, Chobhe KA, Rao U (2019) Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant. Transgenic Res 28:327–340. https://doi.org/10.1007/s11248-019-00126-5
de Felippes FF (2019) Gene regulation mediated by microRNA-triggered secondary small RNAs in plants. Plants 8:112. https://doi.org/10.3390/plants8050112
de Felippes FF, Wang JW, Weigel D (2012) MIGS: miRNA-induced gene silencing. Plant J 70:541–547. https://doi.org/10.1111/j.1365-313X.2011.04896.x
Felippes FF, Weigel D (2009) Triggering the formation of tasiRNAs in Arabidopsis thaliana: the role of microRNA miR173. EMBO Rep 10:264–270. https://doi.org/10.1038/embor.2008.247
Guo H, Song X, Wang G, Yang K, Wang Y, Niu L, Chen X, Fang R (2014) Plant-generated artificial small RNAs mediated aphid resistance. PLoS ONE 9:e97410. https://doi.org/10.1371/journal.pone.0097410
Hada A, Kumari C, Phani V, Singh D, Chinnusamy V, Rao U (2020) Host-induced silencing of FMRFamide-like peptide genes, flp-1 and flp-12, in rice impairs reproductive fitness of the root-knot nematode Meloidogyne graminicola. Front Plant Sci 11:894. https://doi.org/10.3389/fpls.2020.00894
Hada A, Singh D, Papolu PK, Banakar P, Raj A, Rao U (2021a) 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. https://doi.org/10.1007/s00299-021-02767-5
Hada A, Patil BL, Bajpai A, Kesiraju K, Dinesh-Kumar S, Paraselli B, Sreevathsa R, Rao U (2021b) Micro RNA-induced gene silencing strategy for the delivery of siRNAs targeting Meloidogyne incognita in a model plant Nicotiana benthamiana. Pest Manag Sci 77:3396–3405. https://doi.org/10.1002/ps.6384
Han Y, Zhang B, Qin X, Li M, Guo Y (2015) Investigation of a miRNA-induced gene silencing technique in petunia reveals alterations in miR173 precursor processing and the accumulation of secondary siRNAs from endogenous genes. PLoS ONE 10:e0144909. https://doi.org/10.1371/journal.pone.0144909
Hashmi JA, Zafar Y, Arshad M, Mansoor S, Asad S (2011) Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences. Virus Genes 42:286–296. https://doi.org/10.1007/s11262-011-0569-9
Imin N, Mohd-Radzman NA, Ogilvie HA, Djordjevic MA (2013) The peptide-encoding CEP1 gene modulates lateral root and nodule numbers in Medicago truncatula. J Exp Bot 64:5395–5409. https://doi.org/10.1093/jxb/ert369
Ismayil A, Haxim Y, Wang Y, Li H, Qian L, Han T, Chen T, Jia Q, Yihao Liu A, Zhu S, Deng H (2018) Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase. PLoS Pathog 14:e1007282. https://doi.org/10.1371/journal.ppat.1007282
Jacobs TB, Lawler NJ, LaFayette PR, Vodkin LO, Parrott WA (2016) Simple gene silencing using the trans-acting siRNA pathway. Plant Biotechnol J 14:117–127. https://doi.org/10.1111/pbi.12362
Jones JT, Haegeman A, Danchin EGJ, Gaur HS, Helder J, Jones MG, Kikuchi T, Manzanilla-López R, Palomares-Rius JE, Wesemael WM, Perry RN (2013) Top 10 plant-parasitic nematodes in molecular plant pathology. Mol Plant Pathol 14:946–961. https://doi.org/10.1111/mpp.12057
Karthik K, Nandiganti M, Thangaraj A, Singh S, Mishra P, Rathinam M, Sharma M, Singh NK, Dash PK, Sreevathsa R (2020) Transgenic cotton (Gossypium hirsutum L.) to combat weed vagaries: utility of an apical meristem-targeted in planta transformation strategy to introgress a modified CP4-EPSPS gene for glyphosate tolerance. Front Plant Sci 11:768. https://doi.org/10.3389/fpls.2020.00768
Karthik K, Negi J, Rathinam M, Saini N, Sreevathsa R (2021) Exploitation of Novel Bt ICPs for the Management of Helicoverpa armigera (Hübner) in Cotton (Gossypium hirsutum L.): a transgenic approach. Front Microbiol 12:794. https://doi.org/10.3389/fmicb.2021.661212
Kaur R, Choudhury A, Chauhan S, Ghosh A, Tiwari R, Rajam MV (2021) RNA interference and crop protection against biotic stresses. Physiol Mol Biol Plants 27:2357–2377. https://doi.org/10.1007/s12298-021-01064-5
Kesiraju K, Sreevathsa R (2017) Apical meristem-targeted in planta transformation strategy: an overview on its utility in crop improvement. Agri Res Technol Open Access J 8:555734. https://doi.org/10.19080/ARTOAJ.2017.08.555734
Kesiraju K, Mishra P, Bajpai A, Sharma M, Rao U, Sreevathsa R (2020) Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker. Physiol Mol Biol Plants 26:2319–2327. https://doi.org/10.1007/s12298-020-00887-y
Khatoon S, Kumar A, Sarin NB, Khan JA (2016) RNAi-mediated resistance against cotton leaf curl disease in elite Indian cotton (Gossypium hirsutum) cultivar Narasimha. Virus Genes 52:530–537. https://doi.org/10.1007/s11262-016-1328-8
Kim DS, Zhang J (2022) Strategies to improve the efficiency of RNAi-mediated crop protection for pest control. Entomologia Generalis. https://doi.org/10.1127/entomologia/2022/1638
Kimber MJ, McKinney S, McMaster S, Day TA, Fleming CC, Maule AG (2007) flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference. FASEB J 21:1233–1243. https://doi.org/10.1096/fj.06-7343com
Kumari C, Dutta TK, Chaudhary S, Banakar P, Papolu PK, Rao U (2017) Molecular characterization of FMRFamide-like peptides in Meloidogyne graminicola and analysis of their knockdown effect on nematode infectivity. Gene 619:50–60. https://doi.org/10.1016/j.gene.2017.03.042
Liang S, Luo J, Alariqi M, Xu Z, Wang A, Zafar MN, Ren J, Wang F, Liu X, Xin Y, Xu H (2021) Silencing of a LIM gene in cotton exhibits enhanced resistance against Apolygus lucorum. J Cell Physiol 236:5921–5936. https://doi.org/10.1002/jcp.30281
Lisei-de-Sá ME, Rodrigues-Silva PL, Morgante CV, de Melo BP, Lourenço-Tessutti IT, Arraes F, Grossi-de-Sa MF (2021) Pyramiding dsRNAs increases phytonematode tolerance in cotton plants. Planta 254:1–14. https://doi.org/10.1007/s00425-021-03776-0
Lu Y, Wyckhuys KA, Yang L, Liu B, Zeng J, Jiang Y, Desneux N, Zhang W, Wu K (2021) Bt cotton area contraction drives regional pest resurgence, crop loss and pesticide use. Plant Biotechnol J. https://doi.org/10.1111/pbi.13721
MahaLakshmi MS, Prasad NVVSD (2020) Insecticide resistance in field population of Cotton Leaf Hopper, Amrasca devastans (Dist.) in Guntur, Andhra Pradesh, India. Int J Curr Microbiol App Sci 9:3006–3011. https://doi.org/10.20546/ijcmas.2020.906.361
Muralimohan N, Saini RP, Kesiraju K, Pattanayak D, Ananda Kumar P, Kasturi K, Sreevathsa R (2020) Molecular stacking of two codon-modified genes encoding Bt insecticidal proteins, Cry1AcF and Cry2Aa for management of resistance development in Helicoverpa armigera. J Plant Biochem Biotechnol 29:518–527. https://doi.org/10.1007/s13562-020-00569-6
Mutlu AS, Gao SM, Zhang H, Wang MC (2020) Olfactory specificity regulates lipid metabolism through neuroendocrine signaling in Caenorhabditis elegans. Nature Commun 11:1–15. https://doi.org/10.1038/s41467-020-15296-8
Naranjo SE (2011) Impacts of Bt transgenic cotton on integrated pest management. J Agric Food Chem 59:5842–5851. https://doi.org/10.1021/jf102939c
Ni M, Ma W, Wang X, Gao M, Dai Y, Wei X, Zhang L, Peng Y, Chen S, Ding L, Tian Y (2017) Next-generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance. Plant Biotechnol J 15:1204–1213. https://doi.org/10.1111/pbi.12709
Niu J, Taning CNT, Christiaens O, Smagghe G, Wang JJ (2018) Rethink RNAi in insect pest control: challenges and perspectives. Adv Insect Physiol 55:1–17. https://doi.org/10.1016/bs.aiip.2018.07.003
Noman A, Bashir R, Aqeel M, Anwer S, Iftikhar W, Zainab M, Zafar S, Khan S, Islam W, Adnan M (2016) Success of transgenic cotton (Gossypium hirsutum L.): fiction or reality? Cogent Food Agric 2:1207844. https://doi.org/10.1080/23311932.2016.1207844
Papolu PK, Gantasala NP, Kamaraju D, Banakar P, Sreevathsa R, Rao U (2013) Utility of host delivered RNAi of two FMRF amide like peptides, flp-14 and flp-18, for the management of root knot nematode Meloidogyne Incognita. PLoS ONE 8:e80603. https://doi.org/10.1371/journal.pone.0080603
Park J, Choi W, Dar AR, Butcher RA, Kim K (2019) Neuropeptide signaling regulates pheromone-mediated gene expression of a chemoreceptor gene in C. elegans. Mol Cells 42:28
Patil BL, Fauquet CM (2021) Ecology of plant infecting viruses, with special reference to Gemini viruses. In: Hurst C (ed) Studies in viral ecology. Wiley, Hobeken, pp 183–229. https://doi.org/10.1002/9781119608370.ch6
Patil BL, Ogwok E, Wagaba H, Mohammed IU, Yadav JS, Bagewadi B, Taylor NJ, Kreuze JF, Maruthi MN, Alicai T, Fauquet CM (2011) RNAi-mediated resistance to diverse isolates belonging to two virus species involved in Cassava brown streak disease. Mol Plant Pathol 12:31–41. https://doi.org/10.1111/j.1364-3703.2010.00650.x
Patil BL, Bagewadi B, Yadav JS, Fauquet CM (2016) Mapping and identification of cassava mosaic geminivirus DNA-A and DNA-B genome sequences for efficient siRNA expression and RNAi based virus resistance by transient agro-infiltration studies. Virus Res 213:109–115. https://doi.org/10.1016/j.virusres.2015.11.011
Patil BL, Chakraborty S, Czosnek H, Fiallo-Olivé E, Gilbertson RL, Legg J, Mansoor S, Navas-Castillo J, Naqvi RZ, Rahman SU, Zerbini FM (2021) Plant resistance to geminiviruses. Elsevier Limited. In: Bamford DH, Zuckerman M (eds) Encyclopedia of virology, 4th edn., Academic Press, Oxford, pp 554-566. https://doi.org/10.1016/B978-0-12-809633-8.21565-3
Paul S, Das S (2021) Natural insecticidal proteins, the promising bio-control compounds for future crop protection. The Nucleus 64:7–20. https://doi.org/10.1007/s13237-020-00316-1
Ray P, Sahu D, Aminedi R, Chandran D (2022) Concepts and considerations for enhancing RNAi efficiency in phytopathogenic fungi for RNAi-based crop protection using nanocarrier-mediated dsRNA delivery systems. Front Fungal Biol 3:977502. https://doi.org/10.3389/ffunb.2022.977502
Senthil-Kumar M, Mysore KS (2014) Tobacco rattle virus–based virus-induced gene silencing in Nicotiana benthamiana. Nat Protoc 9:1549–1562. https://doi.org/10.1038/nprot.2014.092
Singh S, Gupta M, Pandher S, Kaur G, Rathore P, Palli SR (2018) Selection of housekeeping genes and demonstration of RNAi in cotton leafhopper, Amrasca biguttula biguttula (Ishida). PLoS ONE 13:e0191116. https://doi.org/10.1371/journal.pone.0191116
Ullah F, Gul H, Tariq K, Hafeez M, Desneux N, Gao X, Song D (2022) RNA interference-mediated silencing of ecdysone receptor (EcR) gene causes lethal and sublethal effects on melon aphid Aphis gossypii. Entomologia Generalis 42:791–797. https://doi.org/10.1127/entomologia/2022/1434
Vajhala CS, Sadumpati VK, Nunna HR, Puligundla SK, Vudem DR, Khareedu VR (2013) Development of transgenic cotton lines expressing Allium sativum agglutinin (ASAL) for enhanced resistance against major sap-sucking pests. PLoS ONE 8:e72542. https://doi.org/10.1371/journal.pone.0072542
Vellichirammal NN, Gupta P, Hall TA, Brisson JA (2017) Ecdysone signaling underlies the pea aphid transgenerational wing polyphenism. PNAS 114:1419–1423. https://doi.org/10.1073/pnas.1617640114
Wagaba H, Patil BL, Mukasa S, Alicai T, Fauquet CM, Taylor NJ (2016) Artificial microRNA-derived resistance to Cassava brown streak disease. J Virol Methods 231:38–43. https://doi.org/10.1016/j.jviromet.2016.02.004
Xu QY, Deng P, Zhang Q, Li A, Fu KY, Guo WC, Li GQ (2020) Ecdysone receptor isoforms play distinct roles in larval–pupal–adult transition in Leptinotarsa decemlineata. Insect Sci 27:487–499. https://doi.org/10.1111/1744-7917.12662
Yan S, Yin MZ, Shen J (2022) Nanoparticle-based nontransformative RNA insecticides for sustainable pest control: mechanisms, current status and challenges. Entomologia Generalis. https://doi.org/10.1127/entomologia/2022/1618
Yasmeen A, Kiani S, Butt A, Rao AQ, Akram F, Ahmad A, Nasir IA, Husnain T, Mansoor S, Amin I, Aftab S (2016) Amplicon-based RNA interference targeting V2 gene of cotton leaf curl KokhranVirus-Burewala strain can provide resistance in transgenic cotton plants. Mol Biotechnol 58:807–820. https://doi.org/10.1007/s12033-016-9980-8
Yoshikawa M, Peragine A, Park MY, Poethig RS (2005) A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev 19:2164–2175
Zheng X, Yang L, Li Q, Ji L, Tang A, Zang L, Deng K, Zhou J, Zhang Y (2018) MIGS as a simple and efficient method for gene silencing in rice. Front Plant Sci 9:662. https://doi.org/10.3389/fpls.2018.00662
Acknowledgements
The authors thank Dr. Subba Reddy Palli, University of Kentucky for kindly sharing cotton leaf hopper gene sequences of Sec23 and EcR. The authors also acknowledge and thank Mr. Manoj Kumar for the maintenance and management of transgenic plants in the net house.
Funding
This work was supported by the grant received from Biotechnology Industry Research Assistance Council (BIRAC) [No. BT/SBIRI-1238/SBIRI-24/14 BIPP].
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Karthik, K., Hada, A., Bajpai, A. et al. A novel tasi RNA-based micro RNA-induced gene silencing strategy to tackle multiple pests and pathogens in cotton (Gossypium hirsutum L.). Planta 257, 20 (2023). https://doi.org/10.1007/s00425-022-04055-2
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DOI: https://doi.org/10.1007/s00425-022-04055-2