Abstract
Main conclusion
In silico identified Gossypium hirsutum ghr-miR166b shows multi-compatible targets in mitochondrial ATP synthase of Bemisia tabaci. Its overexpression in planta has the potential to act as a biopesticide in reducing B. tabaci population, and consequently the spread of whitefly-transmitted plant viruses.
Whiteflies (B. tabaci) are hemipterous insects that act as a vector to transmit plant viruses causing enormous losses to the plants. In the present study, G. hirsutum-encoded miRNAs targeting expressed sequence tags (ESTs) of B. tabaci, based on sequence complimentarity and miRNA-target mRNA thermodynamics, were in silico identified. Out of 108 G. hirsutum miRNAs, 55 targeted the protein encoding ESTs. Among them, ghr-miR166b was selected owing to its intrinsic affinity for ATP synthase. Its functional role was validated following expression of ghr-MIR166b (precursor) sequence in G. hirsutum cv. HS6 plants through Agrobacterium-mediated transformation. Total of seven independent transformed (T0) G. hirsutum lines were obtained. The transcript level of ghr-MIR166b in the transgenic lines was observed to be 2.0- to 17-fold higher as compared to non-transformed plants. Northern-blot analysis of small RNAs isolated from the transgenic plants confirmed the presence of the ghr-miR166b. After feeding on the leaves of transgenic line (HS6-166-30) having highest level of ghr-miR166b expression, B. tabaci population was reduced up to 91% as compared to non-transformed leaves. Further, in the whole plant assay, a maximum of 78% B. tabaci mortality was observed in the same line, while there was an increase in B. tabaci population on the non-transformed plants. Our results revealed that ghr-miR166b supposedly targeting ATP synthase gene of B. tabaci, and subsequently its overexpression in planta has potential to act as biopesticide for reducing B. tabaci population and consequently spread of whitefly transmitted viruses.
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Abbreviations
- siRNA:
-
Small interfering RNA
- RNAi:
-
RNA interference
- EST:
-
Expressed sequence tag
- ghr-miR:
-
Mature Gossypium hirsutum miRNA
- ghr-MIR:
-
Precursor Gossypium hirsutum miRNA
- nt:
-
Nucleotide
References
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 5:791–801
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
Baig MS, Khan JA (2013) Identification of Gossypium hirsutum miRNA targets in the genome of Cotton leaf curl Multan virus and betasatellite. Indian J Biotechnol 12:336–342
Baig MS, Shweta, Khan JA (2011) Computational analysis of cotton mirnas targeting genome of Cotton leaf curl Multan Virus and associated satellite DNA. J Nat Sci Biol Med 2(3):33
Baldrich P, Segundo BS (2016) MicroRNAs in rice innate immunity. Rice (NY) 9:6. https://doi.org/10.1186/s12284-016-0078-5
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233
Bellés X (2010) Beyond Drosophila: RNAi in vivo and functional genomics in insects. Annu Rev Entomol 55:111–128
Candas M, Loseva O, Oppert B, Kosaraju P, Bulla LA Jr (2003) Insect resistance to Bacillus thuringiensis: alterations in the indian meal moth larval gut proteome. Mol Cell Proteom 2(1):19–28
Duan CG, Wang CH, Guo HS (2012) Application of RNA silencing to plant disease resistance. BMC Silenc 3:5. https://doi.org/10.1186/1758-907X-3-5
Dutt U (2007) Mealy bug infestation in Punjab: Bt cotton falls flat. Environment News Service, 21 August. http://countercurrents.org. Accessed 18 July 2017
Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS (2003) MicroRNA targets in Drosophila. Genome Biol 4:P8. https://doi.org/10.1186/gb-2003-4-11-p8
EPPO (2014) PQR database. European and Mediterranean Plant Protection Organization, Paris, France. http://www.eppo.int/DATABASES/pqr/pqr.htm. Accessed 27 Aug 2017
Fleming RA, Retnakaran A (1985) Evaluating single treatment data using Abbot’s formula with reference to insecticide. J Econ Entomol 78:1179–1181
Ghanim M, Kontsedalov S, Czosnek H (2007) Tissue-specific gene silencing by RNA interference in the whitefly Bemisia tabaci (Gennadius). Insect Biochem Mol Biol 37:732–738
Ghosh S, Kakumani PK, Kumar A, Malhotra P, Mukherjee SK, Bhatnagar RK (2014) Genome wide screening of RNAi factors of Sf21 cells reveal several novel pathway associated proteins. BMC Genom 15:775. https://doi.org/10.1186/1471-2164-15-775
Gruber A (2007) Bioinformatics: Methods express. In: Dear PH (ed) Expressed sequence tags, 1st edn. Scion Press, Oxford, pp 141–168
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
Hoagland DR, Arnon DI (1950) The water culture method for growing plants without soil. Calif Agric Exp Stn Circ 347:1–32
Horsch RB, Fry JE, Hoffmann N, Eicholz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227(4691):1229–1231
Hu J, Xia Y (2016) F1-ATP synthase α-subunit: a potential target for RNAi-mediated pest management of Locusta migratoria manilensis. Pest Manag Sci 72:1433–1439
Javaid S, Amin I, Jander G, Mukhtar Z, Saeed NA, Mansoor S (2016) A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters. Sci Rep 6:34706. https://doi.org/10.1038/srep34706
Jones SG, Grocock RJ, Dongen S, Bateman A, Enright AJ (2006) miRBase:microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34:D140–D144
Khan ZA, Abdin MZ, Khan JA (2015) Functional characterization of a strong bi-directional constitutive plant promoter isolated from Cotton Leaf Curl Burewala Virus. PLoS One 10(3):e0121656. https://doi.org/10.1371/journal.pone.0121656
Kumar A, Snehi SK, Raj SK, Kumar J, Khan JA (2011) Association of Cotton leaf curl Burewala virus and its satellite molecules with leaf distortion symptoms of cotton in India. New Dis Rep 24:18
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948
Li Y, Lu YG, Shi Y, Wu L, Xu YJ, Huang F, Guo XY, Zhang Y, Fan J, Zhao JQ, Zhang HY, Xu PZ, Zhou JM, Wu XJ, Wang PR, Wang WM (2014) Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae. Plant Physiol 164(2):1077–1092
Malik HJ, Raza A, Amin I, Scheffler JA, Scheffler BE, Brown JK, Mansoor S (2017) RNAi-mediated mortality of the whitefly through transgenic expression of double-stranded RNA homologous to acetylcholinesterase and ecdysone receptor in tobacco plants. Sci Rep 6:38469. https://doi.org/10.1038/srep38469
Nagata AK, Lima MF, Gilbertson RL (2016) A review of geminivirus (begomovirus) diseases in vegetables and other crops in Brazil: current status and approaches for management. Hortic Brasil 34:008–018
Naqvi AR, Choudhury NR, Mukherjee SK, Haq QM (2011) In silico analysis reveals that several tomato microRNA/microRNA* sequences exhibit propensity to bind to tomato leaf curl virus (ToLCV) associated genomes and most of their encoded open reading frames (ORFs). Plant Physiol Biochem 49:13–17
Naranjo SE (2011) Impacts of Bt transgenic cotton on integrated pest management. J Agric Food Chem 59:5842–5851
Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O, Jones JD (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312(5772):436–439
Naveen NC, Chaubey R, Kumar D, Rebijith KB, Rajagopal R, Subrahmanyam B, Subramanian S (2017) Insecticide resistance status in the whitefly, Bemisia tabaci genetic groups Asia-I, Asia-II-1 and Asia-II-7 on the Indian subcontinent. Sci Rep 7:40634. https://doi.org/10.1038/srep40634
Orom UA, Nielsen FC, Lund AH (2008) MicroRNA-10a binds the 5′ UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell 30:460–471
Pérez-Quintero ÁL, Neme R, Zapata A, López C (2010) Plant microRNAs and their role in defense against viruses: a bioinformatics approach. BMC Plant Biol 10(1):138
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29(9):e45
Rana VS, Popli S, Saurav GK, Raina HS, Chaubey R, Ramamurthy VV, Rajagopal R (2016) A Bemisia tabaci midgut protein interacts with begomoviruses and plays a role in virus transmission. Cell Microbiol 18:663–678
Raza A, Malik HJ, Shafiq M, Amin I, Scheffler JA, Scheffler BE, Mansoor S (2016) RNA interference based approach to down regulate osmoregulators of whitefly (Bemisia tabaci): potential technology for the control of whitefly. PLoS One 11(4):e0153883. https://doi.org/10.1371/journal.pone.0153883
Sambrook J, Russell D (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schneider D (2000) Using Drosophila as a model insect. Nat Rev Genet 1:218–226
Shah MM, Zhang S, Liu T (2015) Whitefly, host plant and parasitoid: a review on their interactions. Asian J Appl Sci Eng 4:48–61
Shukla AK, Upadhyay SK, Mishra M, Saurabh S, Singh R, Singh H, Thakur N, Rai P, Pande P, Hans AL, Srivastava S, Rajapure V, Yadav SK, Singh MK, Kumar KJ, Chandrashekar PC, Verma AP, Singh KN, Nair SB, Wahajuddin M, Singh S, Sharma S, Omkar RS, Upadhyay SA, Ranade RT, Singh PK (2016) Expression of an insecticidal fern protein in cotton protects against whitefly. Nat Biotechnol 34:1046–1051. https://doi.org/10.1038/nbt.3665
Shweta, Khan JA (2014) In silico prediction of cotton (Gossypium hirsutum) encoded microRNAs targets in the genome of Cotton leaf curl Allahabad virus. Bioinformation 10(5):251–255
Shweta, Akhter Y, Khan JA (2018) Genome wide identification of cotton (Gossypium hirsutum )-encoded microRNA targets against Cotton leaf curl Burewala virus. Gene 638:60–65
Sun Y, Yang P, Zhang Y, Bao X, Li J, Hou W, Yao X, Han J, Zhang H (2011) A genome-wide RNAi screen identifies genes regulating the formation of P bodies in C. elegans and their functions in NMD and RNAi. Protein Cell 2(11):918–939
Talamillo A, Fernández-Moreno MA, Martínez-Azorín F, Bornstein B, Ochoa P, Garesse R (2004) Expression of the Drosophila melanogaster ATP synthase a subunit gene is regulated by a transcriptional element containing GAF and Adf-1 binding sites. Eur J Biochem 271:4003–4013
Thakur N, Upadhyay SK, Verma PC, Chandrashekar K, Tuli R, Singh PK (2014) Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene. PLoS One 9(3):e87235. https://doi.org/10.1371/journal.pone.0087235
Thornton B, Basu C (2011) Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ 39:145–154
Upadhyay SK, Chandrashekar K, Thakur N, Verma PC, Borgio JF, Singh PK, Tuli R (2011) RNA interference for the control of whiteflies (Bemisia tabaci) by oral route. J Biosci 36:153–161
Upadhyay SK, Dixit S, Sharma S, Singh H, Kumar J, Verma PC, Chandrashekar K (2013) siRNA machinery in whitefly (Bemisia tabaci). PLoS One 8(12):e83692. https://doi.org/10.1371/journal.pone.0083692pmid:24391810
Vasudevan S (2012) Posttranscriptional upregulation by microRNAs. Wiley Interdiscip Rev RNA 3:311–330
Vaucheret H, Chupeau Y (2012) Ingested plant miRNAs regulate gene expression in animals. Cell Res 22(1):3–5
Vyas M, Raza A, Ali MY, Ashraf MA, Mansoor S, Shahid AA, Brown JK (2017) Knock down of whitefly gut gene expression and mortality by orally delivered gut gene-specific dsRNAs. PLoS One 12(1):e0168921. https://doi.org/10.1371/journal.pone.0168921
Wamiq G, Akhtar S, Khan ZA, Alam P, Khan JA (2016) Development of an efficient method for regeneration and Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L.) cv. HS6. Indian J Biotechnol 15:39–47
Wang H, Zhang C, Dou Y, Yu B, Liu Y, Moss TM, Lu G, Wachholtz M, Bradshaw JD, Twigg P, Scully E, Palmer N (2017) Insect and plant-derived miRNAs in greenbug (Schizaphis graminum) and yellow sugarcane aphid (Sipha flava) revealed by deep sequencing. Gene 599:68–77
Weiberg A, Bellinger M, Jin H (2015) Conversations between kingdoms: small RNAs. Curr Opin Biotechnol 32C:207–215
Wu XM, Yang CQ, Mao YB, Wang LJ, Shangguan XX, Chen XY (2016) Targeting insect mitochondrial complex I for plant protection. Plant Biotechnol J 14:1925–1935
Wuchty S, Fontana W, Hofacker IL, Schuste P (1999) Complete suboptimal folding of RNA and the stability of secondary structures. Biopolymers 49:145–165
Xu P, Zhang Y, Kang L, Roossinck Mysore KS (2006) Computational estimation and experimental verification of off-target silencing during posttranscriptional gene silencing in plants. Plant Physiol 142:429–440
Xue W, Wang Z, Du M, Liu Y, Liu JY (2013) Genome-wide analysis of small RNAs reveals eight fiber elongation-related and 257 novel microRNAs in elongating cotton fiber cells. BMC Genom 14:629. https://doi.org/10.1186/1471-2164-14-629
Yang L, Huang H (2014) Roles of small RNAs in plant disease resistance. J Integr Plant Biol 56:962–970
Zhang Z, Yu J, Li D, Zhang Z, Liu F, Zhou X, Wang T, Ling Y, Su Z (2010) PMRD: plant microRNA database. Nucl Acids Res 38:D806–D813
Zhang L, Hou D, Chen X, Li D, Zhu L, Zhang Y, Li J, Bian Z, Liang X, Cai X, Yin Y, Wang C, Zhang T, Zhu D, Zhang D, Xu J, Chen Q, Ba Y, Liu J, Wang Q, Chen J, Wang J, Wang M, Zhang Q, Zhang J, Zen K, Zhang CY (2012) Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res 22:107–126
Acknowledgements
University Grants Commission (Government of India) is acknowledged for MANF to GW for pursuing Ph.D. and UGC-Major Research Project (MRP43-478/2014(SR) to JAK. We are thankful to Dr. Zainul A. Khan for cloning pBI121-ghr-MIR166b construct; Dr. D.K. Monga (Central Institute of Cotton Research, Sirsa) for providing seeds of G. hirsutum cv. HS6.
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Wamiq, G., Khan, J.A. Overexpression of ghr-miR166b generates resistance against Bemisia tabaci infestation in Gossypium hirsutum plants. Planta 247, 1175–1189 (2018). https://doi.org/10.1007/s00425-018-2852-7
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DOI: https://doi.org/10.1007/s00425-018-2852-7