Expression of gene for Dioscorea batatas tuber lectin 1 in transgenic tobacco confers resistance to green-peach aphid

Abstract

Dioscorea batatas tuber lectin 1 (DB1) is a storage protein isolated from yam tuber, and is shown to be a mannose-binding lectin. It has 58% amino-acid identity to insecticidal snowdrop bulb lectin GNA. In this study, we demonstrated that Ն1 mg ml Ϫ1 DB1 in an artificial diet significantly decreased the survival and fecundity of green peach aphid, Myzus persicaeca. We produced transgenic tobacco plants expressing cDNA of DB1 under the control of Cauliflower mosaic virus 35S promoter (35S-DB1) or phloem-specific promoter of rice sucrose synthase-1 gene (RSs1-DB1), and evaluate the degree of aphid resistance in whole plant bioassays. The number of survival aphids was reduced to 60% in transgenic lines with 35S-DB1 and RSs1-DB1, which accumulated DB1 at a level of 1.8% and 0.25%, respectively, of total soluble protein. Our results indicate that DB1 can be used to enhance resistance to sap-sucking insects in transgenic crops.Key words: Aphid, Dioscorea batatas tuber lectin 1, insect resistance, transgenic tobacco.Plant Biotechnology 27, 141-145 (2010) Original PaperThis article can be found at http://www.jspcmb.jp/ Materials and methods Artificial diet bioassaysGreen peach aphid (Myzus persicae) was obtained form JT (Japan Tobacco Inc. Japan). Aphids were maintained on mature plants of Nicotiana tabacum cv. Petit Havana SR1 in a controlled environment growth chamber, 25 Ϯ 1 o C, 16L/8D. The artificial diet bioassays for aphids was carried out as described by Powell et al. (1993) using an artificial diet for aphids (Dadd and Mitter 1966). DB1 was incorporated at 2, 1 and 0.1 mg ml Ϫ1 in the artificial diet. Controls were set as treatment with feed using an artificial diet containing 0 mg ml Ϫ1 DB1 or 1 mg ml Ϫ1 BSA, and treatment with no diet (no diet controls). Ten adult apterous aphids were transferred from the host plant to glass petri-dishes. Each petri-dish was sealed with a stretched Parafilm (Pechiney Plastic Packaging Inc., USA), an 500 ml artificial diet was placed on top of the stretched Parafilm, and then the artificial diet was covered with another stretched Parafilm. The diet and Parafilm were changed daily. The bioassays were conducted in an environmental growth chamber (25 Ϯ 1°C, 16L/8D), and the number of surviving adults and newborn nymphs was counted with four sets of replication. A statistical analysis was carried out using the Excel Statistics software. Transformation of tobaccoThe nucleotide sequence for a DB1 isoform, DB1 (Leu86), under the accession no. AB178475 in DDBJ was revealed to be lacking a part of 5Ј signal sequence. PCR for 5Ј RACE was carried out to obtain full length cDNA using yam tuber mRNA (Ohizumi et al. 2009) and the Marathon cDNA Amplification Kit (BD Bioscience Clontech). The nucleotide sequence of full length open reading frame (ORF) of DB1 cDNA was deposited to DDBJ (accession no. AB513659). It encodes 172 aminoacid protein with a predicted targeting-signal sequence to endoplasmic reticulum.The cDNA covering full-length ORF was PCR cloned into pGEM T-vectors (P...

Development of Transgenic Cotton Lines Expressing Allium sativum Agglutinin (ASAL) for Enhanced Resistance against Major Sap-Sucking Pests

“…It was also reported that transgenic crops expressing garlic lectins (ASA and ASAL) could confer resistance against various lepidopteran and homopteran insects, viz., Nilaparvata lugens , Nephotettix virescens , Sogatella furcifera, Spodoptera littoralis , Lipaphis erysimi and Aphis craccivora [8], [17], [29]-[31]. Further, it was reported that the expression of Dioscorea batatas tuber lectin in rice and tobacco reduced the survival of BPH and aphid by 30% and 60%, respectively [32], [33]. Garlic lectins serve as promising candidate molecules for the development of transgenic crops against sucking pests as they are from edible source, and as such may not pose any biosafety problem.…”

Production of transgenic rice plants expressing Dioscorea batatas tuber lectin 1 to confer resistance against brown planthopper

“…The rate of adults emerging from pupae was reduced to 33%, when fed on 0.01% (w/v) DB1 in an artificial diet (Ohizumi et al 2009). We also demonstrated that ≥1 mg/ml DB1 in an artificial diet significantly decreased the survival and fecundity of green peach aphid, Myzus persicaeca (Kato et al 2010). The number of survival aphids was reduced to 60% in transgenic tobacco expressing cDNA of DB1 under the control of Note DOI: 10.5511/plantbiotechnology.12.0726b…”

“…AB513659) and the rice sucrose synthase-1 (RSs1) promoter (accession no. AJ401233; Shi et al 1994) were obtained as described in Kato et al (2010). The terminator of RSs1 was PCR cloned using RSs1ter-1s primer (5′-TAA TGG AGG GGA AAA TAT GCA TCT TCA GCA GG-3′) and RSs1ter-2a-Sac primer (5′-GAGCTCATC TAC TAC AGC AGT AGA AAA GAT GCA ACC-3′; SacI site is underlined) and connected to the 3′end of the DB1 cDNA by blunt ligation.…”

“…Each plant showed 3 to 6 bands of different sizes, indicating integration of 3 to 6 copies of DB1 ( Figure 1B). The DB1 concentration in the total soluble protein in leaves was determined by comparing the intensity of bands which reacted with anti-DB1 polyclonal antiserum with those of series of known amounts of purified DB1, as described previously (Kato et al 2010). An approximately 12-kd band corresponding to mature DB1 monomer was detected at the same position as that of the standard DB1 purified from yam tuber, indicating the proper processing of 16-kd DB1 premature protein in transgenic rice.…”

RNA Interference in Insect Vectors for Plant Viruses

“…For example, M. persicae feeding on the transgenic plants producing the dsRNAs or siRNA of salivary proteins MpC002 , MpPIntO1 , and MpPIntO2 ; receptor of activated kinase C gene MpRack1 [46,61,151]; serine protease ( MySP ) [62]; Acetylcholinesterase 2 gene MpAChE2 ; V-ATPase E ; 40S ribosomal protein S5-like isoform-1 Rps5 ; SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1-like gene SMARCD1 ; tubulin folding cofactor D gene TBCD ; mediator complex subunit 31 Med31 ; ribosomal protein S14 Rps14 [63], hb [64], MpMIF1 [59] and Aquaporin gene MpAQP1 ; sucrase gene MpSUC1 ; and sugar transporter gene MpSt4 [65], resulted in significant reduction in the target gene expression and reduced fecundity. Aside from plant-mediated silencing, several cases in which plants expressing lectins for controlling M. persicae were reported and those include Galanthus nivalis agglutinin, GNA expressing tobacco and potato plants [66,67], Allium sativum leaf lectin, ASAL [68], Allium cepa agglutinin, ACA expressing mustard [69], Pinellia ternate agglutinin, PTA expressing tobacco plants [70,152], Dioscorea batatas tuber lectin 1, DB1 [71], legume lectins: ConA expressing potato plants [72], Jacalins: Helianthus tuberosus agglutinin, HTA expressing tobacco plants [73] and NICTABA -related lectin, AtPP2 expressing in Arabidopsis [74], all showed significant insecticidal activity against M. persicae .…”