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Molecular characterization, expression analysis and RNAi knock-down of elongation factor 1α and 1γ from Nilaparvata lugens and its yeast-like symbiont

Published online by Cambridge University Press:  04 November 2016

W.X. Wang ,
T.H. Zhu ,
K.L. Li ,
L.F. Chen ,
F.X. Lai  and
Q. Fu
W.X. Wang
Affiliation:
State Key Laboratory of Rice Biology, China National Rice Research Institute, Tiyuchang Road 359, Hangzhou, Zhejiang, 310006, People's Republic of China
T.H. Zhu
Affiliation:
College of Biological and Environmental Engineering, Zhejiang University of Technology, Chaowang Road, Hangzhou, Zhejiang, 310014, People's Republic of China
K.L. Li
Affiliation:
State Key Laboratory of Rice Biology, China National Rice Research Institute, Tiyuchang Road 359, Hangzhou, Zhejiang, 310006, People's Republic of China
L.F. Chen
Affiliation:
State Key Laboratory of Rice Biology, China National Rice Research Institute, Tiyuchang Road 359, Hangzhou, Zhejiang, 310006, People's Republic of China College of Biological and Environmental Engineering, Zhejiang University of Technology, Chaowang Road, Hangzhou, Zhejiang, 310014, People's Republic of China
F.X. Lai*
Affiliation:
State Key Laboratory of Rice Biology, China National Rice Research Institute, Tiyuchang Road 359, Hangzhou, Zhejiang, 310006, People's Republic of China
Q. Fu*
Affiliation:
State Key Laboratory of Rice Biology, China National Rice Research Institute, Tiyuchang Road 359, Hangzhou, Zhejiang, 310006, People's Republic of China
*
*Author for correspondence Phone: Fax: +86-571-63370348 E-mail: [email protected], [email protected]
*Author for correspondence Phone: Fax: +86-571-63370348 E-mail: [email protected], [email protected]
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Abstract

In the present paper, four cDNAs encoding the alpha and gamma subunits of elongation factor 1 (EF-1) were cloned and sequenced from Nilaparvata lugens, named NlEF-1α, NlEF-1γ, and its yeast-like symbiont (YLS), named YsEF-1α and YsEF-1γ, respectively. Comparisons with sequences from other species indicated a greater conservation for EF-1α than for EF-1γ. NlEF-1α has two identical copies. The deduced amino acid sequence homology of NlEF-1α and NlEF-1γ is 96 and 64%, respectively, compared with Homalodisca vitripennis and Locusta migratoria. The deduced amino acid sequence homology of YsEF-1α and YsEF-1γ is 96 and 74%, respectively, compared with Metarhizium anisopliae and Ophiocordyceps sinensis. Reverse transcription-quantitative polymerase chain reaction (RT–qPCR) analysis revealed that the expression level of NlEF-1α and NlEF-1γ mRNA in hemolymph, ovary, fat body and salivary glands were higher than the midgut and leg tissue. YsEF-1α and YsEF-1γ was highly expressed in fat body. The expression level of NlEF-1α was higher than that of NlEF-1γ. Through RNA interference (RNAi) of the two genes, the mortality of nymph reached 92.2% at the 11th day after treatment and the ovarian development was severely hindered. The RT–qPCR analysis verified the correlation between mortality, sterility and the down-regulation of the target genes. The expression and synthesis of vitellogenin (Vg) protein in insects injected with NlEF-1α and NlEF-1γ double-stranded RNA (dsRNA) was significantly lower than control groups. Attempts to knockdown the YsEF-1 genes in the YLS was unsuccessful. However, the phenotype of N. lugens injected with YsEF-1α dsRNA was the same as that injected with NlEF-1α dsRNA, possibly due to the high similarity (up to 71.9%) in the nucleotide sequences between NlEF-1α and YsEF-1α. We demonstrated that partial silencing of NlEF-1α and NlEF-1γ genes caused lethal and sterility effect on N. lugens. NlEF-1γ shares low identity with that of other insects and therefore it could be a potential target for RNAi-based pest management.

Keywords

Nilaparvata lugensyeast-like symbiontselongation factor 1 gammaelongation factor 1 alphaRNAi
Type
Research Papers
Information
Bulletin of Entomological Research , Volume 107 , Issue 3 , June 2017 , pp. 303 - 312
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Copyright
Copyright © Cambridge University Press 2016 

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References

Belles, X. (2010) Beyond Drosophila: RNAi in vivo and functional genomics in insects. Annual Review of Entomology 55, 111–128.Google Scholar
Billaut-Mulot, O., Fernando-Gomez, R., Loyens, M. & Ouaissi, A. (1996) Trypanosoma cruzi elongation factor 1-alpha, nuclear localization in parasites undergoing apoptosis. Gene 174, 19–26.Google Scholar
Chen, C.C., Cheng, L.L., Kuan, C.C. & Hou, R.F. (1981) Studies on the intracellular yeast-like symbiote in the brown planthopper, Nilaparvata-lugens Stal. Histological observations and population-changes of the symbiote. Journal of Applied Entomology 91(1), 321–327.Google Scholar
Chen, E., Proestou, G., Bourbeau, D. & Wang, E. (2000) Rapid up-regulation of peptide elongation factor EF-1alpha protein levels is an immediate early event during oxidative stress-induced apoptosis. Experiment of Cell Research 259(1), 140–148.Google Scholar
Condeelis, J. (1995) Elongation factor 1a, translation and the cytoskeleton. Trends in Biochemical Sciences 20, 169–170.Google Scholar
Danforth, B.N. & Ji, S. (1998) Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects. Molecular Biology and Evolution 15, 225–235.CrossRefGoogle ScholarPubMed
Ejiri, S. (2002) Moonlighting functions of polypeptide elongation factor 1: from actin bundling to zinc finger protein R1-associated nuclear localization. Bioscience Biotechnology and Biochemistry 66(1), 1–21.CrossRefGoogle ScholarPubMed
Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411(6836), 494–498.Google Scholar
Fan, H.W., Noda, H., Xie, H.Q., Suetsugu, Y., Zhu, Q.H., Zhang, C.X. (2015) Genomic analysis of an ascomycete fungus from the rice planthopper reveals how it adapts to an endosymbiotic lifestyle. Genome Biology and Evolution 7(9), 2623–2634.CrossRefGoogle Scholar
Hongoh, Y., Sasaki, T. & Ishikawa, H. (2000) Cloning sequence analysis and expression in Escherichia coli of the gene encoding a uricase from the yeast-like symbiont of the brown planthopper, Nilaparvata lugens. Insect Biochemistry and Molecular Biology 30, 173–182.CrossRefGoogle ScholarPubMed
Hovemann, B., Richter, S., Walldorf, U. & Cziepluch, C. (1988) Two genes encode related cytoplasmic elongation factors 1 alpha (EF-1 alpha) in Drosophila melanogaster with continuous and stage specific expression. Nucleic Acids Research 16, 3175–3194.Google Scholar
Janssen, G.M.C. & Möller, W. (1988) Elongation factor 1βγ from Artemia. Purification and properties of its subunits. European Journal of Biochemistry 171, 119–129.Google Scholar
Kamiie, K., Taira, H., Ooura, H., Kakuta, A., Matsumoto, S., Ejiri, S. & Katsumata, T. (1993) Nucleotide sequence of the cDNA encoding silk gland elongation factor 1 alpha. Nucleic Acids Research 21, 742.CrossRefGoogle ScholarPubMed
Kamiie, K., Nomura, Y., Kobayashi, S., Taira, H., Kobayashi, K., Yamashita, T., Kidou, S. & Ejiri, S. (2002) Cloning and expression of Bombyx mori silk gland elongation factor 1γ in Escherichia coli . Bioscience Biotechnology and Biochemistry 66, 558–565.Google Scholar
Kamiie, K., Yamashita, T., Taira, H., Kidou, S. & Ejiri, S. (2003) Interaction between Elongation Factors 1β and 1γ from Bombyx mori Silk Gland. Bioscience Biotechnology and Biochemistry 67(7), 1522–1529.Google Scholar
Kaur, K.J. & Ruben, L. (1994) Protein translation elongation factor-1α from Trypanosoma brucei binds calmodulin. Journal of Biology Chemistry 269, 23045–23050.Google Scholar
Kidou, S. & Ejiri, S. (1998) Isolation, characterization and mRNA expression of four cDNAs encoding translation elongation factor 1A from rice (Oryza sativa L.). Plant Molecular Biology 36, 137–148.CrossRefGoogle ScholarPubMed
Kidou, S., Tsukamoto, S., Kobayashi, S. & Ejiri, S. (1998) Isolation and characterization of a rice cDNA encoding the γ-subunit of translation elongation factor 1B (eEF1Bγ). FEBS Letters 434(3), 382–386.CrossRefGoogle ScholarPubMed
Lamberti, A., Sanges, C. & Chambery, A. (2011) Analysis of interaction partners for eukaryotic translation elongation factor 1A M-domain by functional proteomics. Biochemistry 93(10), 1738–1746.Google Scholar
Laurie, J.D., Linning, R. & Bakkeren, G. (2008) Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis . Current Genetics 53(1), 49–58.CrossRefGoogle Scholar
Le Sourd, F., Boulben, S., Le Bouffant, R., Cormier, P., Morales, J., Belle, R. & Mulner-Lorillon, O. (2006) eEF1B: at the dawn of the 21st century. Biochimica et Biophysica Acta 1759, 13–31.Google Scholar
Liu, S., Ding, Z., Zhang, C., Yang, B. & Liu, Z. (2014) Gene knockdown by intro-thoracic injection of double-stranded RNA in the brown planthopper, Nilaparvata lugens . Insect Biochemisty Molecular Biology 40, 666–671.CrossRefGoogle Scholar
Liu, S., Liang, Q.M., Zhou, W.W., Jiang, Y.-D., Zhu, Q.Z., Yu, H., Zhang, C.X., Gurr, G.M. & Zhu, Z.R. (2015) RNA interference of NADPH-cytochrome P450 reductase of the rice brown planthopper, Nilaparvata lugens, increases susceptibility to insecticides. Pest Management Science 71, 32–39.Google Scholar
Livak, K.J. & Schmittgen, T.D. (2011) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408.Google Scholar
Lu, F., Qi, G.J., Qin, R.R., Hu, G., Wang, Z., Zhang, X.X., Cheng, X.N. & Zhai, B.P. (2011) The processes of morphological change and grading criteria for ovarian development in the brown planthopper. Chinese Journal of Applied Entomology 48(5), 1394–1400.Google Scholar
Nakayashiki, H., Kadotani, N. & Mayama, S. (2006) Evolution and diversification of RNA silencing proteins in fungi. Journal of Molecular Evolution 63(1), 127–135.Google Scholar
Negrutskii, B.S. & El'skaya, A.V. (1998) Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and possible role in aminoacyl-tRNA channeling. Progress in Nucleic Acid Research and Molecular Biology 60, 47–78.Google Scholar
Noda, H. & Koizumi, Y. (2003) Sterol biosynthesis by symbiotes: cytochrome P450 sterol C-22 desaturase genes from yeastlike symbiotes of rice planthoppers and anobiid beetles. Insect Biochemistry and Molecular Biology 33, 649–658.Google Scholar
Rual, J.F., Klitgord, N. & Achaz, G. (2007) Novel insights into RNAi off-target effects using C. elegans paralogs. BMC Genomics 8, 106.Google Scholar
Shepherd, J.C., Walldorf, U., Hug, P. & Gehring, W.J. (1989) Fruit flies with additional expression of the elongation factor EF-1 alpha live longer. Proceedings of the National Academy of Sciences of the United States of America 86, 7520–7521.Google Scholar
Slobin, L. (1980) The role of eukaryotic elongation factor Tu in protein synthesis. The measurement of the elongation factor Tu content of rabbit reticulocytes and other mammalian cells by a sensitive radioimmunoassay. European Journal of Biochemistry 110, 555–563.Google Scholar
Sogawa, K. (1982) The rice brown planthopper-feeding physiology and host plant interactions. Annual Review of Entomology 27, 49–73.Google Scholar
Suh, S.O., Noda, H. & Blackwell, M. (2001) Insect symbiosis: derivation of yeast-like endosymbionts within an entomopathogenic filamentous lineage. Molecular Biology and Evolution 18, 995–1000.Google Scholar
Taira, H., Kamiie, K., Kakuta, A., Ooura, H., Matsumoto, S., Ejiri, S. & Katsumata, T. (1992) Nucleotide sequence of the cDNA encoding silk gland elongation factor 1 beta’. Nucleic Acids Research 20, 6734.Google Scholar
Vinkenoog, R., Sperança, M.A., van Breemen, O., Ramesar, J., Williamson, D.H., Ross-MacDonald, P.B., Thomas, A.W., Janse, C.J., del Portillo, H.A. & Waters, A.P. (1998) Malaria parasites contain two identical copies of an elongation factor 1 alpha gene. Molecular and Biochemical Parasitology 94(1), 1–12.Google Scholar
Wang, W.X., Li, K.L., Chen, Y., Lai, F.X. & Fu, Q. (2015) Identification and function analysis of enolase gene NlEno1 from Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). Journal of Insect Science 15(1), 66.CrossRefGoogle ScholarPubMed
Webster, G.C. & Webster, S.L. (1983) Decline in synthesis of elongation factor one (EF-1) precedes the decreased synthesis of total protein in aging Drosophila melanogaster . Mechanisms of Ageing and Development 22, 121–128.CrossRefGoogle ScholarPubMed
Xue, J., Zhou, X., Zhang, C.X., Yu, L.L. & Cheng, J.A. (2014) Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation. Genome Biology 15, 521.CrossRefGoogle ScholarPubMed
Zhao, L.N., Qin, Z., Wei, P., Guo, H.S., Dang, X.L., Wang, S.G. & Tang, B. (2012) Elongation Factor 1 beta ’ Gene from Spodoptera exigua: characterization and function identification through RNA Interference. International Journal of Molecular Sciences 13(7), 8126–8141.Google Scholar
Zhou, S., Zhang, J., Fam, M.D., Wyatt, G.R. & Walker, V.K. (2002) Sequences of elongation factors-1α and -1γ and stimulation by juvenile hormone in Locusta migratoria . Insect Biochemistry and Molecular Biology 32, 1567–1576.Google Scholar
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