Korean journal of applied entomology (한국응용곤충학회지)

Korean Society of Applied Entomology (한국응용곤충학회)
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A Freeze-drying Formulation and Target Specificity of Double-stranded RNA-expressing Bacteria to Control Insect Pests

Double-stranded RNA 발현 세균의 동결건조 제형화와 적용 대상 해충 선택성

  • Kim, Eunseong (Department of Bioresource Sciences, Andong National University) ;
  • Kim, Yonggyun (Department of Bioresource Sciences, Andong National University)
  • 김은성 (안동대학교 생명자원과학과) ;
  • 김용균 (안동대학교 생명자원과학과)
  • Received : 2016.02.01
  • Accepted : 2016.03.10
  • Published : 2016.06.01
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Abstract

Double-stranded RNA (dsRNA) has been applied to control insect pests by its suppressive activity against specific target genes. Integrin is a heterodimer (${\alpha}$ and ${\beta}$) transmembrane protein and plays a critical role in cell-to-cell or cell-to-extracellular matrix interactions in eukaryotes. Suppression of ${\beta}$ subunit integrin gene expression by its specific dsRNA (= dsINT) induces significant mortality against target insects. Furthermore, a recombinant bacterium expressing dsINT is potent to kill target insects. However, it is necessary to develop a formulation technique of the dsRNA-expressing bacteria to apply the bacterial insecticide against field populations. This study formulated the recombinant bacteria by freeze-drying and tested its control efficacy against target insects. The formulation maintained significant insecticidal activity against last instar larvae of Spodoptera exigua. While a commercial Bacillus thuringiensis (Bt) insecticide exhibited only about 60% insecticidal activity against S. exigua last instar, an addition of the dsINT-expressing bacterial formulation significantly enhanced the Bt insecticidal activity. The dsINT-expressing bacterial formulation exhibited relative selectivity to target insects depending on sequence similarity. These results indicate that a freeze-dried form of dsRNA-expressing bacteria keeps its insecticidal activity.

이중가닥 RNA (double-stranded RNA, dsRNA)는 표적 유전자의 발현을 억제하는 기능으로 해충방제에 응용되었다. 인테그린은 ${\alpha}$와 ${\beta}$ 단위체로 구성된 이량체 막 단백질이다. 진핵생명체에서 인테그린은 세포-세포 및 세포-세포외기질의 상호연결에 중요한 역할을 담당한다. 인테그린 ${\beta}$ 단위체 발현을 억제하는 특정한 dsRNA (= dsINT)는 해당 곤충에 뚜렷한 치사효과를 유발한다. 또한, dsINT를 발현시키는 형질전환된 대장균도 해당 곤충에 뚜렷한 살충력을 가진다. 그러나 이 세균 살충제의 야외 적용을 위해서는 제형화 기술이 필요했다. 본 연구는 dsINT를 발현하는 재조합 세균을 동결 건조시켜 대상 곤충에 대해 살충효능을 검정하였다. 동결 건조된 세균은 파밤나방(Spodoptera exigua) 종령 유충에 높은 섭식독을 일으켰다. 파밤나방에 대해서 Bacillus thuringiensis 상용 살충제 처리는 불과 60%의 살충력을 보이는 반면, 동결 건조된 dsINT 발현 세균과 혼합 처리할 때 살충력은 크게 증가하였다. dsINT 발현 세균은 해당 인테그린 염기서열 유사성에 따라 차이를 보이는 해충 종에 선택적 독성을 나타냈다. 이 결과는 인테그린에 특이적 dsRNA를 발현하는 세균이 동결 건조 제형화 조건하에서도 살충력을 유지한다는 것을 나타냈다.

Keywords

References

  1. Ahn, S.J., Badenes-Perez, F.R., Heckel, D.G., 2011. A host-plant specialist, Helicoverpa assulta, is more tolerant to capsaicin from Capsicum annuum than other noctuid species. J. Insect Physiol. 57, 1212-1219. https://doi.org/10.1016/j.jinsphys.2011.05.015
  2. Aroujo, R.N., Santos, A., Pinto, F.S., Gontijo, N.F., Lehane, M.J., Pereira, M.H., 2006. RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection. Insect Biochem. Mol. Biol. 36, 683-693. https://doi.org/10.1016/j.ibmb.2006.05.012
  3. Baum, J.A., Bogaert, T., Clinton, W., Heck, G.R., Feldmann, P., Ilangan, O., Johnson, S., Plaetinck, G., Munyikwa, T., Pleau, M., Vaughn, T., Roberts, J., 2007. Control of coleopteran insect pests through RNA interference. Nature Biotech. 25, 1322-1326. https://doi.org/10.1038/nbt1359
  4. Bautista, M.A.M., Miyata, T., Miura, K., Tanaka, T., 2009. RNA interference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin. Insect Biochem. Mol. Biol. 39, 38-46. https://doi.org/10.1016/j.ibmb.2008.09.005
  5. Bravo, A., Likitvivatanavong, S, Gill, S., Soberon, M. 2011. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem. Mol. Biol. 41, 423-431. https://doi.org/10.1016/j.ibmb.2011.02.006
  6. Brower, D.L., 2003. Platelets with wings: the maturation of Drosophila integrin biology. Curr. Opin. Cell Biol. 15, 607-613. https://doi.org/10.1016/S0955-0674(03)00102-9
  7. Burridge, K., Fath, K., Kelly, T., Nuckolls, G., Turner, C., 1988. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell Biol. 4, 487-525. https://doi.org/10.1146/annurev.cb.04.110188.002415
  8. Cancino-Rodezno, A., Alexander, C., Villasenor, R., Pacheco, S., Porta, H., Pauchet, Y., Soberon, M., Gill, S.S., Bravo, A., 2010. The mitogen-activated protein kinase p38 is involved in insect defense against Cry toxins from Bacillus thuringiensis. Insect Biochem. Mol. Biol. 40, 58-63. https://doi.org/10.1016/j.ibmb.2009.12.010
  9. Fablet, M., 2014. Host control of insect endogenous retroviruses: small RNA silencing and immune response. Viruses 6, 4447-4464 https://doi.org/10.3390/v6114447
  10. Fire, A., 2005. Nucleic acid structure and intracellular immunity: some recent ideas from the world of RNAi. Q. Rev. Biophys. 38, 303-309 https://doi.org/10.1017/S0033583505004117
  11. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C., 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811. https://doi.org/10.1038/35888
  12. Goh, H.G., Lee, S.G., Lee, B.P., Choi, K.M., Kim, J.H., 1990. Simple mass-rearing of beet armyworm, Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae), on an artificial diet. Kor. J. Appl. Entomol. 29, 180-183.
  13. Griebler, M., Westerlund, S.A., Hoffmann, K.H., Meyerringe-Vos, M., 2008. RNA interference with the allatoregulating neuropeptide genes from the fall armyworm Spodoptera frugiperda and its effects on the JH titer in the hemolymph. J. Insect Physiol. 54, 997-1007. https://doi.org/10.1016/j.jinsphys.2008.04.019
  14. Hughes, A.L., 2001. Evolution of the integrin ${\alpha}$ and ${\beta}$ protein families. J. Mol. Evol. 52, 63-72. https://doi.org/10.1007/s002390010134
  15. Humphries, M.J., Travis, M.A., Clark, K., Mould, A.P., 2004. Mechanisms of integration of cells and extracellular matrices by integrins. Biochem. Soc. Trans. 32, 822-825. https://doi.org/10.1042/BST0320822
  16. Huvenne, H., Smagghe, G., 2010. Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. J. Insect Physiol. 56, 227-235. https://doi.org/10.1016/j.jinsphys.2009.10.004
  17. Hynes, R.O., 2002. Integrins: bidirectional, allosteric signaling machines. Cell 110, 673-687. https://doi.org/10.1016/S0092-8674(02)00971-6
  18. Hynes, R.O., Zhao, Q., 2000. The evolution of cell adhesion. J. Cell Biol. 150, F89-F95. https://doi.org/10.1083/jcb.150.2.F89
  19. Irving, P., Ubeda, J.M., Doucet, D., Troxler, L., Lagueux, M., Zachary, D., Hoffmann, J.A., Hetru, C., Meister, M., 2005. New insights into Drosophila larval haemocyte functions through genome-wide analysis. Cell. Microbiol. 7, 335-350. https://doi.org/10.1111/j.1462-5822.2004.00462.x
  20. Kim, E., Park, Y., Kim, Y., 2015a. A transformed bacterium expressing double-stranded RNA specific to integrin ${\beta}$1 enhances Bt toxin efficacy against a polyphagous insect pest, Spodoptera exigua. PLoS One 10, e0132631. https://doi.org/10.1371/journal.pone.0132631
  21. Kim, Y., Lee, J., Kang, S., Han, S., 1998. Age variation in insecticide susceptibility and biochemical changes of beet armyworm, Spodoptera exigua (Hubner). J. Asia Pac. Entomol. 1, 109-113. https://doi.org/10.1016/S1226-8615(08)60012-6
  22. Kim, Y.H., Soumaila Issa, M., Cooper, A.M., Zhu, K.Y., 2015b. RNA interference: applications and advances in insect toxicology and insect pest management. Pestic. Biochem. Physiol. 120, 109-117. https://doi.org/10.1016/j.pestbp.2015.01.002
  23. Lavine, M.D., Strand, M.R., 2003. Haemocytes from Pseudoplusia includens express multiple ${\alpha}$ and ${\beta}$ integrin subunits. Insect Mol. Biol. 12, 441-452. https://doi.org/10.1046/j.1365-2583.2003.00428.x
  24. Li, X., Zhang, M., Zhang, H., 2011. RNA interference of four genes in adult Bactrocera dorsalis by feeding their dsRNAs. PLoS One 6, e17788. https://doi.org/10.1371/journal.pone.0017788
  25. Loftus, J.C., Smith, J.W., Ginsberg, M.H., 1994. Integrin-mediated cell adhesion: the extracellular face. J. Biol. Chem. 269, 25235-25238.
  26. Macrae, I.J., Zhou, K., Li, F., Repic, A., Brooks, A.N., Cande, W.Z., Adams, P.D., Doudna, J.A., 2006. Structural basis for double-stranded RNA processing by Dicer. Science 311, 195-198. https://doi.org/10.1126/science.1121638
  27. Mamali, I., Lamprou, I., Karagiannis, F., Karakantza, M., Lampropoulou, M., Marmaras, V.J., 2009. A beta integrin subunit regulates bacterial phagocytosis in medfly haemocytes. Dev. Comp. Immunol. 33, 858-866. https://doi.org/10.1016/j.dci.2009.02.004
  28. Mohamed, A.A., Kim, Y., 2011. A target-specific feeding toxicity of ${\beta}$1 integrin dsRNA against diamondback moth, Plutella xylostella. Arch. Insect Biochem. Physiol. 78, 216-230. https://doi.org/10.1002/arch.20455
  29. Park, Y., Ahn, S.J., Vogel, H., Kim, Y., 2014. Integrin ${\beta}$ subunit and its RNA interference in immune and developmental processes of the Oriental tobacco budworm, Helicoverpa assulta. Dev. Comp. Immunol. 47, 59-67. https://doi.org/10.1016/j.dci.2014.06.017
  30. Park, Y., and Kim, Y., 2013. RNA interference of cadherin gene ex pression in Spodoptera exigua reveals its significance as a specific Bt target. J. Invertebr. Pathol. 114, 285-291 https://doi.org/10.1016/j.jip.2013.09.006
  31. SAS Institute, 1989. SAS/STAT User's Guide. SAS Institute, Inc., Cary, NC.
  32. Scott, J.G., Michel, K., Bartholomay, L.C., Siegfired, B.D., Hunter, W.B., Smagghe, G., Zhu, K.Y., Douglas, A.E., 2013. Towards the elements of successful insect RNAi. J. Insect Physiol. 59, 1212-1221. https://doi.org/10.1016/j.jinsphys.2013.08.014
  33. Shih, J.D., Hunter, C.P., 2011. SID-1 is a dsRNA-selective dsRNA-gated channel. RNA 17, 1057-1065. https://doi.org/10.1261/rna.2596511
  34. Surakasi, V.P., Mohamed, A.A.M., Kim, Y. 2011. RNA interference of ${\beta}$1 integrin subunit impairs development and immune responses of the beet armyworm, Spodoptera exigua. J. Insect Physiol. 57, 1537-1544. https://doi.org/10.1016/j.jinsphys.2011.08.006
  35. Tian, H., Peng, H., Yao, Q., Chen, H., Xie, Q. Tang, B., Zhuang, W., 2009. Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a Non-Midgut gene. PLoS One 4, e6225. https://doi.org/10.1371/journal.pone.0006225
  36. Turner, C.T., Davy, M.W., MacDiarmid, R.M., Plummer, K.M., Birch, N.P., Newcomb, R.D., 2006. RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding. Insect Mol. Biol. 15, 383-391. https://doi.org/10.1111/j.1365-2583.2006.00656.x
  37. van der Flier, A., Sonnenberg, A., 2001. Function and interactions of integrins. Cell Tissue Res. 305, 285-298. https://doi.org/10.1007/s004410100417
  38. Wynant, N., Santos, D., Van Wielendaele, P., Vanden Broeck, J., 2014. Scavenger receptor-mediated endocytosis facilitates RNA interference in the desert locust, Schistocerca gregaria. Insect Mol. Biol. 23, 320-329.
  39. Zhou, X.G., Wheeler, M.M., Oi, F.M., Scharf, M.E., 2008. RNA interference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larva resistance to permethrin. Insect Biochem. Mol. Biol. 39, 38-46.
  40. Zhu, F., Palli, R., Ferguson, J., Palli, S.R., 2011. Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata. Pest Manag. Sci. 67, 175-182. https://doi.org/10.1002/ps.2048
  41. Zhuang, S., Kelo, L., Nardi, J.B., Kanost, M.R., 2007a. An integrin-tetraspanin interaction required for cellular innate immune responses of an insect, Manduca sexta. J. Biol Chem. 282, 22563-22572. https://doi.org/10.1074/jbc.M700341200
  42. Zhuang, S., Kelo, L., Nardi, J.B., Kanost, M.R., 2007b. Neuroglian on hemocyte surfaces is involved in homophilic and heterophilic interactions of the innate immune system of Manduca sexta. Dev. Comp. Immunol. 31, 1159-1167. https://doi.org/10.1016/j.dci.2007.03.002
  43. Zhuang, S., Kelo, L., Nardi, J.B., Kanost, M.R., 2008. Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system. Dev. Comp. Immunol. 32, 365-379. https://doi.org/10.1016/j.dci.2007.07.007