Development of insect-resistant transgenic rice with Cry1C -free endosperm

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1 Research Article Received: 13 October 2008 Revised: 29 December 2008 Accepted: 26 January 2009 Published online in Wiley Interscience: 28 May 2009 (www.interscience.wiley.com) DOI /ps.1788 Development of insect-resistant transgenic rice with Cry1C -free endosperm Rongjian Ye, Haiqun Huang, Zhou Yang, Taiyu Chen, Li Liu, Xianghua Li, Hao Chen and Yongjun Lin Abstract BACKGROUND: Yellow stem borer (Tryporyza incertulas Walker), striped stem borer (Chilosuppressalis Walker) and leaf folder (Cnaphalocrocis medinalis Guenec) are three lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, novel insect-resistant transgenic rice was developed in which Bt protein expression was nearly absent in the endosperm. The resistant gene, cry1c, driven by the rice rbcs promoter (small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase), was introduced into Zhonghua 11 (Oryza sativa L. ssp. japonica) by Agrobacterium-mediated transformation. RESULTS: A total of 83 independent transformants were obtained, 19 of which were characterised as single-copy foreign gene insertion. After preliminary screening of the T 1 families of these 19 transformants in the field, six highly insect-resistant homozygous lines were selected. These six homozygous transgenic lines were field tested for resistance to leaf folders and stem borers, and for their agronomic performance. The Cry1C protein levels in leaves and endosperm were measured by ELISA. Subsequently, the elite transgenic line RJ5 was selected; this line not only possessed high resistance to leaf folders and stem borers, normal agronomic performance, but also Cry1C expression was only 2.6 ng g 1 in the endosperm. CONCLUSION: These results indicated that RJ5 has the potential for widespread utility in rice production. c 2009 Society of Chemical Industry Keywords: cry1c ; rbcs; leaf folders; stem borers; insect resistance; transgenic rice 1 INTRODUCTION Rice is one of the world s most important food crops. Yellow stem borer (Tryporyza incertulas Walker), striped stem borer (Chilo suppressalis Walker) and leaf folder (Cnaphalocrocis medinalis Guenec) are three major pests of rice that cause severe yield losses in most rice-producing countries. For a long time, control of these pests has chiefly depended on the use of large amounts of chemical insecticides, leading to considerable environmental pollution and representing a health hazard to farmers, as well as significantly increasing the costs of rice production. Bacillus thuringiensis (Bt) is an ideal biological insecticide alternative to chemical insecticides because of its safety to the user and environment. Owing to limited field stability, the inability to reach cryptic insects and a narrow spectrum of activity, Bt products currently represent only a small portion of the insecticide market. 1 Plant transgenic approaches provide access to an unlimited gene pool for the transfer of desirable genes between any two species of interest, irrespective of their evolutionary or taxonomic relation. Genes codifying Bt insecticidal crystal proteins can be introduced into plants for insect control. Bt genes were first introduced and expressed in tobacco 2,3 and tomato. 4 Since then, insect-resistant crops containing Bt genes have been developed at very fast pace. In 2006, the estimated global area of Bt crops reached 32.2 million ha. 5 These crops have benefited the growers and environment by greatly reducing the use of chemical insecticides. 1 During the last two decades, considerable research efforts have been invested to introduce insecticidal crystal protein genes into rice by transgenic approaches However, some novel problems have arisen, one of which is promoter usage. Constitutive promoters are widely used in insect-resistant transgenic rice to express Bt genes, such as the 35sCaMV promoter, 9,11 the ubiquitin1 promoter 18,19 and the Actin1 promoter. 8,13 However, compared with the temporal- or spatial-specific expression of the toxin, constitutive expression of foreign proteins in transgenic plants may cause adverse effects, such as the metabolic burden imposed on plants for constant synthesis of foreign gene products, and these may increase the potential risk of resistance of the target insects to Bt. There is also concern about the food safety of genetically modified plants. Therefore, in certain circumstances, it is desirable to use expression-specific promoters which only express the foreign gene in specific plant tissues or organs. 23 Correspondence to: Yongjun Lin, National Key Laboratory of Crop Genetic Improvement andnational CentreofPlant Gene Research, Huazhong Agricultural University, Wuhan , PR China. These authors contributed equally to this work. National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan , PR China 1015 Pest Manag Sci 2009; 65: c 2009 Society of Chemical Industry

2 R Ye et al. The Cry1C toxin is effective against the main lepidopteran pests such as rice stem borers and leaf folders, and does not share a common binding site in brush border membrane vesicles (BBMVs) oftheinsectmidgutwithcry1atoxins. 24 Therefore, it seems that the Cry1C toxin can be a potential alternative to Cry1A toxins. There are few reports on cry1c transgenic plants. 25,26 The authors group has reported the introduction of a codon-optimised cry1c gene, referred to as cry1c, under the control of ubiquitin1 promoter, into the elite indica rice line Minghui 63 via Agrobacterium-mediated transformation, and the transgenic plants showed high resistance to rice stem borers and leaf folders. 19 Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of photosynthesis and the most abundant leaf protein. Plants rely on it for carbon fixation. Rubisco content is thought to be a rate-limiting factor for the light-saturated rate of photosynthesis at present atmospheric carbon dioxide pressures. 27 Rubisco in higher plants possesses an L 8 S 8 structure, with the large subunit (LS) encoded in the chloroplast by rbcl and the small subunit encoded by the nuclear RBCS gene family. Many experiments demonstrate that rbcs is a tissue-specific expression gene, especially in green tissues, and that the expression pattern is regulated by light All of these results indicate that the rbcs promoter might be useful for the expression of target genes in transgenic rice, with particularly high efficiency in green tissues (most notably in leaves). Zheng et al. 32 expressed Cry1Ca in transgenic shallots under the control of a chrysanthemum Rubisco small subunit promoter, and the transgenic plants showed high resistance to beet armyworm. In the present study, a modified novel cry1c gene 19 was driven by the rice rbcs promoter, 31 then introduced into Zhonghua 11 (Oryza sativa L. ssp. japonica)byagrobacterium-mediated transformation. Transgenic plants were examined for both insect resistance and agronomic traits under field conditions, with the goal of obtaining insect-resistant transgenic rice that was Cry1C free in endosperm tissue. 2 MATERIALS AND METHODS 2.1 Transformation construct (P rbcs cry1c ) and transformation The transformation construct is displayed in Fig. 1. Plasmid pcambia1300 was kindly provided by the Centre for the Application of Molecular Biology in International Agriculture, Australia. The cry1c gene 19 was driven by the rice rbcs promoter 31 and was cloned into the polylinker of the plasmid. The hygromycin phosphotransferase (hph) gene was replaced with the phosphinotricin acetyltransferase (bar) gene. A strain of Agrobacterium tumefaciens EHA105, harbouring a disarmed plasmid ptibo542, was used for the transformation experiments. The callus culture and transformation procedures were carried out as described by Hiei et al PCR and southern blot analyses PCR and southern blot analyses were used to determine the presence of the cry1c gene and the copy number in transformed plants. PCR analysis was performed using the primers cry1c -F (5 - ttctactggggaggacatcg-3 )andcry1c -R (5 -cggtatctttgggtgattgg -3 ). A 20 µl mixture of 30 ng of template DNA, 1 buffer (50 mm KCl, 10 mm Tris-HCl, 0.1% Triton X-100), 0.15 mm of dntps, mm of MgCl 2,0.1 µm of each primer and 1 U of Taq DNA polymerase was prepared for the PCR assays. The PCR reaction was performed at 94 C for 5 min, and then for 30 cycles of 94 Cfor1min,57 C for 1 min, 72 C for 1 min, followed by 72 Cfor5min.ThePCR products were then checked by electrophoresis. Plant genomic DNA was extracted by the CTAB method. 34 For southern blot analysis, 5 µg of genomic DNA from each sample was digested with DraI and separated on a 0.8% agarose gel, then transferred to a nylon membrane. The probe was prepared from a PCR-amplified fragment of cry1c. All procedures for the hybridisation were conducted as described by Lin and Zhang Selection of homozygous cry1c plants with single-copy insertion In 2006, T 1 transgenic plant families (with single-copy insertions) derived from fertile T 0 transgenic plants were grown on the experimental farm of Huazhong Agricultural University in Wuhan, China. No insecticide was applied during the entire growth period of the rice. Highly insect-resistant families without significant phenotypic changes were selected on the basis of visual observation. Mature seeds were harvested from individual plants containing single-copy insertion from families with high resistance that did not display significant phenotypic changes. A seed germination assay was carried out to select for homozygous plants containing the cry1c gene. The procedure of the seed germination assay was performed as described by Chen et al Field tests for insect resistance and agronomic performance For the evaluation of insect resistance and agronomic performance, the selected homozygous transgenic lines were planted at the experimental farm of Huazhong Agricultural University in Wuhan, China, in The materials tested were six homozygous cry1c transgenic lines, and Zhonghua 11 as the control (designated RJ1). The seeds were sown in a seedling bed on 27 May, and seedlings were subsequently transplanted to the paddy fields on 28 June. The field layout followed a randomised complete block design with three replications. Each plot consisted of 20 plants in two rows, with distances of 14.0 cm between plants within a row and 18.0 cm between rows. The experimental plots were bordered by four rows of non-transgenic japonica rice plants. Evaluation of insect resistance of transgenic plants in the field was conducted by artificial infestation using yellow stem borer combined with natural infestation of stem borers and leaf folders. 18 No chemical insecticides targeted against lepidopteran pests were 1016 Figure 1. T-DNA region of the transformation construct P rbcs Cry1C.TheCry1C gene was driven by a rice rbcs promoter and terminated by the nopaline synthase (nos) terminator. The bar gene was used as a selectable marker and was under the control of the CaMV35S promoter and tailed by the CaMV35S polya. LB: left border of T-DNA region; RB: right border of T-DNA region. c 2009 Society of Chemical Industry Pest Manag Sci 2009; 65:

3 Insect-resistant transgenic rice with Cry1C -free endosperm applied throughout the growth period. Each rice plant was infested with first-instar larvae of yellow stem borers at the tillering stage. The severity of damage caused by the natural infestation of leaf folders was recorded within 7 days after peak damage appeared. Leaves with visible scrapes or folds were considered to be damaged. Dead hearts caused by stem borers were counted at the late maximum tillering stage, and white heads were counted at the flowering stage. The experimental design for the field testing of the agronomic performance of the transgenic plants was conducted as described above, with the exception that chemical pesticides were applied throughout the growth period for crop protection. At maturity, plant height was measured in the field, and all plants from each plot were harvested for measurements of the panicle length, number of panicles per plant, number of grains per panicle, 1000-grain weight, yield per plant and seed-set rate. 2.5 Quantification of the Cry1C protein The amount of Cry1C protein in leaves was measured at the tillering and filling stages, and the amount of Cry1C protein in the endosperm was measured at the wax ripeness stage, using the enzyme-linked immunosorbent assay (ELISA) kit from Enviro- Logix (Portland, ME). The protein assayprocedures were conducted according to the manufacturer s instructions. 3 RESULTS 3.1 Transformation and DNA assay Transformation of 1377 calli resulted in a total of 87 transformants, of which 83 grew to maturity. PCR analysis identified that 79 of the 83 transformants were transgene positive. Southern blot analysis showed that the copy numbers of the transgenic plants varied from 1 to 5 (Fig. 2). Of the 79 transgenic plants, 19 contained a single copy of the transgene. 3.2 Selection of insect-resistant homozygous transgenic lines Nineteen T 1 single-copy transgenic plant families derived from fertile T 0 transgenic plants were grown in the field. No chemical insecticide was applied during the entire rice growth period. Selection of plants was based on high insect resistance and no significant phenotypic changes; four out of 19 T 1 transgenic families were chosen. To obtain homozygous transgenic plants, the mature seeds from selected T 1 transgenic plants were collected separately and germinatedonfresh1/2 MS medium containing 10 mg L 1 Basta. Homozygous transgenic plants, heterozygous plants and negative plants were differentiated bytheir germination ratios, which were 100, 75 and 0% respectively. Finally, six homozygous transgenic plant lines (designated RJ2, RJ3, RJ4, RJ5, RJ6 and RJ7) were selected for further analyses. 3.3 Evaluation of insect resistance and agronomic performance in the field The selected six homozygous transgenic plant lines (RJ2, RJ3, RJ4, RJ5, RJ6 and RJ7) were field tested for their resistance to leaf folders and stem borers, and also evaluated for agronomic performance. The results of insect resistance are shown in Table 1. It was seen that 37.41% of tillers of the wild-type Zhonghua 11 (RJ1) were damaged to various degrees by leaf folders, with an average of 4.25 leaves affected per tiller, while only slight damage ( leaves per tiller) was observed on the six selected homozygous transgenic lines. As Table 1 shows, Zhonghua 11 (RJ1) was also severely damaged by stem borers, with 36.25% of the plant tillers having dead hearts or white heads, while again Table 1. Resistance of the six homozygous transgenic plant lines against stem borers and leaf folders under field conditions (2007, Wuhan, China) Damage by stem borers a,b (%) Damage by leaf folders a,b Number of tillers affected (%) Number of damaged leaves per tiller RJ1 (CK) (±15.93) (±6.89) 4.25 (±0.55) RJ (±0.00) 1.41 (±0.80) 0.08 (±0.03) RJ (±1.76) 0.52 (±0.50) 0.05 (±0.05) RJ (±1.11) 1.01 (±1.41) 0.07 (±0.07) RJ (±1.73) 0.58 (±0.68) 0.05 (±0.05) RJ (±1.21) 0.33 (±0.57) 0.03 (±0.06) RJ (±0.68) 0.00 (±0.00) 0.00 (±0.00) a Values are given as means (± standard deviation). b Means significantly different from the control (P < 0.01). Figure 2. Southern blot analysis of total genomic DNA of T 0 transgenic plants. The DNA samples were digested with DraI and hybridised with the prepared radioactive probe (no DraI restriction site is present within the T-DNA region). The probe size was 0.8 kb and was a fragment of the Cry1C gene.m:dnamarker;nt:zhonghua11;lanes 1 to 18, transgenic plants. Figure 3. Field performance of transgenic Cry1C lines: A, Zhonghua 11 control; B, a homozygous transgenic line. Plants of the Zhonghua 11 control were severely damaged by the yellow stem borer and leaf folder. Pest Manag Sci 2009; 65: c 2009 Society of Chemical Industry 1017

4 R Ye et al. only slight damage ( %) was observed in the six selected homozygous transgenic plant lines (Fig. 3). The insect resistances of the six cry1c homozygous transgenic lines were very similar, and all were significantly (P < 0.01) superior to that of the wild-type Zhonghua 11 (Table 1). The data collected from the field experiment for agronomic performance, however, showed that only the homozygous transgenic line RJ5 had no significant differences from the wild-type Zhonghua 11 (RJ1). The other five homozygous transgenic lines each had various degrees of differences in agronomic traits, especially in plant height (Table 2). These results indicated that RJ5 was the ideal line for the development of transgenic rice against lepidopteran pests. 3.4 Cry1C protein quantification of the homozygous transgenic lines The Cry1C protein contents of fresh leaves (at the tillering and filling stages) and the endosperm (at the wax ripeness stage) of the homozygous transgenic lines were measured. The Cry1C protein concentrations of the six homozygous transgenic lines ranged from 0.87 µg g 1 leaf fresh weight in line RJ5 to 3.13 µgg 1 in RJ7 at the tillering stage, and the Cry1C protein concentrations ranged from 0.71 to 0.86 µgg 1 leaf fresh weight at the filling stage (Figs 4 and 5). A declining trend of the Cry1C protein concentration was found between the developing vegetative growth and the reproductive growth. In endosperm, the Cry1C expression level was very low. The highest concentration was only µgg 1 endosperm. In RJ5, the target homozygous transgenic line, the Cry1C protein concentration was only µgg 1 endosperm. This result was in accordance with the original intention of designing a transgenic line of rice with low cry1c expression in the endosperm. 4 DISCUSSION In this study, a modified novel cry1c gene 19 was driven by a rice rbcs promoter, 31 and was then introduced into Zhonghua 11 (O. sativa L. ssp. japonica)byagrobacterium-mediated transformation. Based on high insect resistance and no significant phenotypic changes, six homozygous transgenic lines were selected for field testing for resistance to leaf folders and stem borers, and also for agronomic performance. The Cry1C protein levels in leaves and endosperm were measured at the same time. The homozygous transgenic line (RJ5) not only had high resistance to lepidopteran pests and normal agronomic performance, but also the concentration of the Cry1C protein was only 2.6 ng g 1 endosperm, that is, it was nearly absent from the endosperm. These results indicated that RJ5 possessed great potential for use in rice production. Stem borers and leaf folders are two groups of major pests in rice that cause severe yield loss in most rice-producing countries. Cry1Ab,cry1Ac and fusion genecry1ac/cry1ab are the Bt genes most commonly used in transgenic crops, including rice. 6 9,11,13,15,36 These proteins share a common binding site in BBMV, 37 and thus a mutant that is able to overcome one of the cry1a genes is also likely to be resistant to other cry1a genes. Consequently, combinations of cry1a genes with other groups of Bt genes should be explored as a means of preventing or delaying the emergence of pest Table 2. Agronomic traits of the six homozygous transgenic lines and wild-type Zhonghua 11 under field conditions (Wuhan, China, 2007) ab Plant height (cm) Panicles per plant Grains per panicle Seed-set rate (%) 1000-grain weight (g) Yield per plant (g) Panicle length (cm) RJ1 (CK) (±2.66) (±0.31) (±4.80) (±3.74) (±0.35) (±2.45) (±0.24) RJ (±2.66) (±0.89) (±1.31) (±1.68) (±0.18) (±2.06) (±0.23) RJ (±3.00) (±0.98) (±5.49) (±2.06) (±0.19) (±3.11) (±0.86) RJ (±3.16) (±1.51) (±1.89) (±1.13) (±0.22) (±3.10) (±1.40) RJ (±2.48) (±1.98) (±5.79) (±0.48) (±0.41) (±4.10) (±0.26) RJ (±1.32) (±2.27) (±7.03) (±2.53) (±0.06) (±3.08) (±0.30) RJ (±1.47) (±2.56) (±4.19) (±2.55) (±0.29) (±3.78) (±0.38) LSD LSD a Values are given as means (± standard deviation). b Means significantly different from the control (P < 0.05). Means significantly different from the control (P < 0.01) Figure 4. Cry1C protein assay of fresh leaves from six homozygous transgenic lines, as quantified by ELISA. c 2009 Society of Chemical Industry Pest Manag Sci 2009; 65:

5 Insect-resistant transgenic rice with Cry1C -free endosperm Figure 5. Cry1C protein assay of endosperm from six homozygous transgenic lines, as quantified by ELISA. resistance. The Cry1C toxin is effective against stem borers and leaf folders, and does not share a common binding site with Cry1A toxins in BBMV. 24 Therefore, it would appear that the Cry1C toxin could be a potential alternative to Cry1A toxins. Early research in the authors lab indicated that Cry1C toxicity to lepidopterans was higher than Cry2A toxicity, and was equivalent to Cry1Ac and Cry1Ab in the transgenic line Minghui ,19,35,38 The potential of insects to develop resistance to Bt toxins is an increasing concern, especially with the widespread adoption of Bt crops. In fact, a number of resistant insect strains have been selected under laboratory, greenhouse and/or field conditions, 1,21,39,40 indicating the potential for the evolution of resistance. To meet this challenge, several strategies have been proposed to manage insect resistance, such as the highdose/refuge strategy, gene stacking, and temporal- or spatialspecific expression of the toxin. 1,21,39,41 This study used the last strategy, in which a tissue-specific promoter (P rbcs )wasusedto control the expression of Cry1C. The results showed that Cry1C could be expressed at high levels in green tissues and was nearly absent from the endosperm. In comparison with the ubiquitin1 promoter, the expression of P rbcs was3timeshigherinleaves 19 but less than one-thousandth in the endosperm (Yang Zh, unpublished data). All of these results indicated that the rbcs promoter might be very useful for the expression of target genes in transgenic rice research. ACKNOWLEDGEMENTS This research was funded by the National High-Technology Research and Development Programme of China (programme 863), the National Programme on Research and Development of Transgenic Plants and the National Natural Science Foundation of China. REFERENCES 1 Ferre J and Van RJ, Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu Rev Entomol 47: (2002). 2 Barton KA, Whiteley HR and Yang NS,Bacillusthuringiensisδ-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidopteran insects. Plant Physiol 85: (1987). 3 Vaeck M, Reynaerts A, Hofte H, Jansens S, Beukeleer MD, Dean C, et al, Transgenic plants protected from insect attack. Nature 328:33 37 (1987). 4 Fischhoff DA, Bowdish KS, Perlak FJ, Marrone PG, McCoormick SM, Niedermeyer JG, et al, Insect tolerant transgenic tomato plants. 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