Chrysanthemum Morifolium Ramat.) Induced by Sea Anemone Equistatin Overexpression Mahmood Valizadeh, C
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Aphid Resistance in Florist’s Chrysanthemum (Chrysanthemum morifolium Ramat.) Induced by Sea Anemone Equistatin Overexpression Mahmood Valizadeh, C. Deraison, S.K. Kazemitabar, Yvan Rahbé, Maarten A Jongsma To cite this version: Mahmood Valizadeh, C. Deraison, S.K. Kazemitabar, Yvan Rahbé, Maarten A Jongsma. Aphid Re- sistance in Florist’s Chrysanthemum (Chrysanthemum morifolium Ramat.) Induced by Sea Anemone Equistatin Overexpression. African Journal of Biotechnology, Academic Journals, 2013, 12 (50), pp.12(50):6922-6930. 10.5897/AJB2013.12956. hal-00919389 HAL Id: hal-00919389 https://hal.archives-ouvertes.fr/hal-00919389 Submitted on 16 Dec 2013 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Vol. 12(50), pp. 6922-6930, 11 December, 2013 DOI: 10.5897/AJB2013.12956 ISSN 1684-5315 ©2013 Academic Journals African Journal of Biotechnology http://www.academicjournals.org/AJB Full Length Research Paper Aphid resistance in florist's chrysanthemum (Chrysanthemum morifolium Ramat.) induced by sea anemone equistatin overexpression Mahmood Valizadeh1,2, Celine Deraison3, Seyed Kamal Kazemitabar4, Yvan Rahbé3 and Maarten A. Jongsma2* 1Department of Agriculture, Payame Noor University, Kashmar Branch, Kashmar, Iran. 2Plant Research International, Wageningen University and Research Center, P. O. Box 619, 6700 AP Wageningen, Netherlands. 3INRA-INSA de Lyon, UMR BF2I (Biologie Fonctionelle Insectes & Interactions), 20 ave A. Einstein 69621, Villeurbanne Cedex, France. 4Department of Plant Breeding, Sari Natural Resources and Agricultural University, Sari, Mazandaran, Iran. Accepted 7 November, 2013 Florist's chrysanthemum (Chrysanthemum morifolium Ramat.) belongs to the Asteraceae family and represents the second most important floricultural crop in the world. Most genotypes are sensitive to aphids and infestations can lower quality and cause transmission of viruses. The protease inhibitor Sea Anemone Equistatin (SAE) carries three domains responsible for the inhibition of both cysteine and aspartic proteases. Artificial diet bioassays showed that SAE is readily toxic when ingested by the pea aphid, Acyrthosiphon pisum, and the cotton aphid, Aphis gossypii. We transformed chrysanthemum genotype 1581 by Agrobacterium tumefaciens-mediated transformation with the SAE gene under the control of the chrysanthemum RbcS promoter to induce aphid resistance. Non-choice leaf disk and whole plant bioassays were carried out to analyze deleterious effects of SAE on population growth and survival of both Myzus persicae and A. gossypii. After 7 days, M. persicae populations on specific transgenic lines were up to 69% smaller relative to control populations in a whole plant bioassay. The mortality of cotton aphids was 11% on control lines and up to 32% on transgenic lines after 5 days. The results show that SAE may be a promising agent for the control of some aphid species in transgenic plants. Key words: Chrysanthemum morifolium, aphid resistance, RbcS promoter, sea anemone equistatin, agrobacterium transformation. INTRODUCTION Cultivated chrysanthemum (Chrysanthemum morifolium (Anderson, 1987), belong to the Asteraceae family Ramat), also classified as Dendranthema × grandiflora (Salinger, 1991). Commercially, it is known as florist’s *Corresponding author. E-mail: [email protected]. Abbreviations: BAP, 6-Benzylaminopurine; IAA, indole acetic acid; MS, Musrashige and Skoog’s medium; RbcS, ribulose-1, 5- bisphosphate carboxylase; NPTII, neomycin phosphotransferase; SAE, sea anemone equistatin; PCR, polymerase chain reaction; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; LB, luria-bertani; TSWV, tomato spotted wilt virus. Valizadeh et al. 6923 chrysanthemum or autumn queen and predominantly FaNES1-expressing lines significantly repelled the sold as cut flowers in many countries of the world (Erler aphids. Similarly, the expression of heterologous pro- and Siegmund, 1986). After rose, it is globally the most tease inhibitors in plants has been used as an approach important floricultural crop (Visser et al., 2007). The wide to induce plant resistance to insects (Ryan, 1990). spectrum of colors and shapes, excellent vase life, and Protease inhibitors (PIs) are proteins that form complexes their ability to produce desired grades and types at any- with gut proteases and inhibit their proteolytic activity. time during the year promoted their economic impor- Plants utilize PIs for defense to moderate the adverse tance. Control of the main insect pests, various aphids effects from attacking herbivores or pathogens (Christou (chrysanthemum aphid Macrosiphoniella sanborni Gilette, et al., 2006; Ferry et al., 2006; Zavala et al., 2008; peach aphid Myzus persicae Sulzer, pea aphid Jongsma and Beekwilder, 2011). Many host plant-derived Acyrthosiphon pisum Harris, and cotton aphid Aphis inhibitors are unsuitable; however, due to the fact that gossypii Glover), thrips (mainly Frankliniella occidentalis many insects have evolved resistance (Jongsma and Pergande) and spider mites (Tetranychus urticae Koch) Beekwilder 2011). Extensive screening of non-host plant- on vegetative and flowering chrysanthemum is a critical derived inhibitors has resulted in novel candidate proteins problem. Insects often develop resistance against fre- such as SAE that demonstrated promising levels of quently used pesticides, many pesticides are banned in resistance in vitro and in vivo against various insect pests important production areas, and there is a high cost to including aphids (Gruden et al., 1998; Outchkourov et al., the application of pesticides (Visser et al., 2007). 2004a, 2004b; Ceci et al., 2003). Although, desirable traits have been introduced using In contrast to leaf-eating insect pests, aphids are exclu- classical breeding, there are severe limitations to this sively feeding on plant phloem-sap, which usually dis- technique in a hexaploid species like (cultivated) plays a high free/bound amino acid ratio. Consequently, chrysanthemum. As an alternative, traits have been intro- they are thought not to rely on extensive protein digestion duced by means of Agrobacterium-mediated transforma- for their nitrogen supply. Nevertheless, a significant tion (Fukai et al., 1995; Jaime and Teixeira, 2005; Mao et number of PIs was shown to promote deleterious effects al., 2011) including practical characteristics relating to on different aphid species (Rahbé et al., 1995; Casaretto biotic resistance against aphids, beet army worm, botrytis and Corcuera, 1998; Quillien et al., 1998; Ceci et al., and tomato spotted wilt virus (TSWV) (Kim et al., 2011; 2003). Also, a modified Oryza cystatin-I (Oc-IDD86) and Visser et al., 2007). a chicken egg white cystatin were shown to reduce the Compared to conventional breeding, genetic engi- growth and survival of nymphs of M. persicae in artificial neering offers the advantage that a single gene can be diet assays (Cowgill et al., 2002). The cystatin from rice, inserted in the background of an established cultivar, and OC-I, caused growth reductions of up to 40% and it is possible to stack multiple genes for various traits into reduced fecundity in pea aphid (A. pisum), cotton aphid a single cultivar (Visser et al., 2007). The search for plant (A. gossypii) and peach potato aphid (M. persicae) when compounds that are suitable for genetically based aphid fed at levels up to 0.25 mg ml-1 (Rahbé et al., 2003a). control has resulted in a wide variety of candidate Serine PIs also show insecticidal effects towards aphids. phytochemicals. Methyl ketones, sesquiterpene carbo- For example, a systematic study of isoforms of Bowman- xylic acids and acyl-glucose esters are examples of Birk type PIs from pea seeds showed varying anti- compounds from trichomes with toxic properties against metabolic effects, including significant mortality, to pea aphids (Goffreda et al., 1990). Some plant lectins or aphid, which were associated with inhibitory activity agglutinins are toxic to sap-sucking insects (Rahbé et al., towards pea aphid chymotrypsin (Rahbé et al., 2003b). 1995). Galanthus nivalis agglutinin (GNA) (Stoger et al., Ceci et al. (2003) could identify a novel MTI-2 anti- 1999; Nagadhara et al., 2003), Pinellia ternate agglutinin chymotrypsin inhibitor, Chy8, with highest affinity for (PTA) (Yao et al., 2003), concanavalin A (ConA) bovine chymotrypsin that was highly toxic to nymphs of (Gatehouse et al., 1999) and a lectin from Amaranthus the pea aphid A. pisum, and moderately toxic to nymphs caudatus (ACA) (Rahbé et al., 1995) are examples of of A. gossypii and M. persicae. compounds that significantly inhibit the population Equistatin is a protease inhibitor from the sea anemone development of aphids. Wu et al. (2006) overexpressed Actinia equina that consists of three thyroglobulin type I the aca gene (A. caudatus agglutinin) in cotton plants domains. The first N-terminal domain acts as a cysteine under the control of a phloem-specific promoter. Bio- protease inhibitor, and the second as an aspartic assays using cotton aphid showed that