Downregulation of the CsABCC2 gene is associated with Cry1C resistance in the striped stem borer Chilo suppressalis.

Chilo suppressalis is a major target pest of transgenic rice expressing the Bacillus thuringiensis (Bt) Cry1C toxin in China. The evolution of resistance of this pest is a major threat to Bt rice. Since Bt functions by binding to receptors in the midgut (MG) of target insects, identification of Bt functional receptors in C. suppressalis is crucial for evaluating potential resistance mechanisms and developing effective management strategies. ATP-binding cassette (ABC) transporters have been vastly reported to interact with Cry1A toxins, as receptors and their mutations cause insect Bt resistance. However, the role of ABC transporters in Cry1C resistance to C. suppressalis remains unknown. Here, we measured CsABCC2 expression in C. suppressalis Cry1C-resistant (Cry1C-R) and Cry1C-susceptible strains (selected in the laboratory) via quantitative real-time PCR (qRT-PCR); the transcript level of CsABCC2 in the Cry1C-R strain was significantly lower than that in the Cry1C-susceptible strain. Furthermore, silencing CsABCC2 in C. suppressalis via RNA interference (RNAi) significantly decreased Cry1C susceptibility. Overall, CsABCC2 participates in Cry1C mode of action, and reduced expression of CsABCC2 is functionally associated with Cry1C resistance in C. suppressalis.

Plant breeding involving genetic engineering does not result in unacceptable unintended effects in rice relative to conventional cross-breeding.

Advancements in -omics techniques provide powerful tools to assess the potential effects in composition of a plant at the RNA, protein and metabolite levels. These technologies can thus be deployed to assess whether genetic engineering (GE) causes changes in plants that go beyond the changes introduced by conventional plant breeding. Here, we compare the extent of transcriptome and metabolome modification occurring in leaves of four GE rice lines expressing Bacillus thuringiensis genes developed by GE and seven rice lines developed by conventional cross-breeding. The results showed that both types of crop breeding methods can bring changes at transcriptomic and metabolic levels, but the differences were comparable between the two methods, and were less than those between conventional non-GE lines were. Metabolome profiling analysis found several new metabolites in GE rice lines when compared with the closest non-GE parental lines, but these compounds were also found in several of the conventionally bred rice lines. Functional analyses suggest that the differentially expressed genes and metabolites caused by both GE and conventional cross-breeding do not involve detrimental metabolic pathways. The study successfully employed RNA-sequencing and high-performance liquid chromatography mass spectrometry technology to assess the unintended changes in new rice varieties, and the results suggest that GE does not cause unintended effects that go beyond conventional cross-breeding in rice.

Impacts of Bt rice on non-target organisms assessed by the hazard quotient (HQ).

The potential risk of Bt (Bacillus thuringiensis) crops on non-target organisms (NTOs) has drawn a lot of public concerns. Despite a series of risk assessments of Bt crops on NTOs has been conducted, a quantitative approach which could support a precise judgment of their safety is required. In the present work, hazard quotient (HQ) was applied in the safety evaluation of three Bt rice events (Cry1Ab, Cry1C and Cry2Aa rice) on NTOs. Eight NTOs in different functional guilds associated with Bt rice were selected to conduct the tests. The results showed that the HQs of three Bt rice events for eight NTOs were all below the trigger value 1, while the HQ of Cry1Ab rice for one target pest Chilo suppressalis was three times higher than 1. Our results assured the reliability of the HQ and indicated that the three Bt rice events would pose no risks to the eight NTOs. Further testing of three Bt proteins on biological parameters of one NTO Nasonia virtipennis under no observed adverse effect concentration (NOAEC) confirmed the robustness of HQ assessment. We recommend that the HQ could be applied in tier-1 risk assessments of Bt crops on NTOs as a reference data standard, which would provide more clear and credible safety information of transgenic crops for the public and policy makers.

Insect-Resistant Genetically Engineered Crops in China: Development, Application, and Prospects for Use.

With 20% of the world's population but just 7% of the arable land, China has invested heavily in crop biotechnology to increase agricultural productivity. We examine research on insect-resistant genetically engineered (IRGE) crops in China, including strategies to promote their sustainable use. IRGE cotton, rice, and corn lines have been developed and proven efficacious for controlling lepidopteran crop pests. Ecological impact studies have demonstrated conservation of natural enemies of crop pests and halo suppression of crop-pest populations on a local scale. Economic, social, and human health effects are largely positive and, in the case of Bt cotton, have proven sustainable over 20 years of commercial production. Wider adoption of IRGE crops in China is constrained by relatively limited innovation capacity, public misperception, and regulatory inaction, suggesting the need for further financial investment in innovation and greater scientific engagement with the public. The Chinese experience with Bt cotton might inform adoption of other Bt crops in China and other developing countries.

A new method to obtain isofemale lines for monitoring Chilo suppressalis resistance to Bt toxin.

Cost-effective Bt resistance monitoring programmes exist to manage insect resistance to Bt crops. F2 screening is widely used in detecting rare resistant alleles. One way to establish numerous isofemale lines for F2 bioassays is acquiring many field-trapped copulated females. However, it is difficult to obtain sufficient Chilo suppressalis isofemale lines because females have low mating rates and fecundity. We developed a new method to establish C. suppressalis isofemale lines with field-collected egg masses. No significant difference in detection ability was observed in the F2 screen between the old and new methods. Moreover, the new method is economical, convenient and efficient.

Bt rice plants may protect neighbouring non-Bt rice plants against the striped stem borer, Chilo suppressalis.

The area planted with insect-resistant genetically engineered crops expressing Bacillus thuringiensis (Bt) genes has greatly increased in many areas of the world. Given the nearby presence of non-Bt crops (including those planted as refuges) and non-crop habitats, pests targeted by the Bt trait have a choice between Bt and non-Bt crops or weeds, and their host preference may greatly affect insect management and management of pest resistance to Bt proteins. In this study, we examined the oviposition preference of the target pest of Bt rice, Chilo suppressalis, for Bt versus non-Bt rice plants as influenced by previous damage caused by C. suppressalis larvae. The results showed that C. suppressalis females had no oviposition preference for undamaged Bt or non-Bt plants but were repelled by conspecific-damaged plants whether Bt or non-Bt Consequently, C. suppressalis egg masses were more numerous on Bt plants than on neighbouring non-Bt plants both in greenhouse and in field experiments due to the significantly greater caterpillar damage on non-Bt plants. We also found evidence of poorer performance of C. suppressalis larvae on conspecific-damaged rice plants when compared with undamaged plants. GC-MS analyses showed that larval damage induced the release of volatiles that repelled mated C. suppressalis females in wind tunnel experiments. These findings suggest that Bt rice could act as a dead-end trap crop for C. suppressalis and thereby protect adjacent non-Bt rice plants. The results also indicate that the oviposition behaviour of target pest females should be considered in the development of Bt resistance management strategies.

Bt rice could provide ecological resistance against nontarget planthoppers.

Genetically engineered (GE) rice lines expressing Lepidoptera-active insecticidal cry genes from the bacterium Bacillus thuringiensis (Bt) have been developed in China. Field surveys indicated that Bt rice harbours fewer rice planthoppers than non-Bt rice although planthoppers are not sensitive to the produced Bt Cry proteins. The mechanisms underlying this phenomenon remain unknown. Here, we show that the low numbers of planthoppers on Bt rice are associated with reduced caterpillar damage. In laboratory and field-cage experiments, the rice planthopper Nilapavata lugens had no feeding preference for undamaged Bt or non-Bt plants but exhibited a strong preference for caterpillar-damaged plants whether Bt or non-Bt. Under open-field conditions, rice planthoppers were more abundant on caterpillar-damaged non-Bt rice than on neighbouring healthy Bt rice. GC-MS analyses showed that caterpillar damage induced the release of rice plant volatiles known to be attractive to planthoppers, and metabolome analyses revealed increased amino acid contents and reduced sterol contents known to benefit planthopper development. That Lepidoptera-resistant Bt rice is less attractive to this important nontarget pest in the field is therefore a first example of ecological resistance of Bt plants to nontarget pests. Our findings suggest that non-Bt rice refuges established for delaying the development of Bt resistance may also act as a trap crop for N. lugens and possibly other planthoppers.

Safety of Bacillus thuringiensis Cry1C protein for Daphnia magna based on different functional traits.

Cry1C is a Bacillus thuringiensis (Bt) insecticidal protein and it can be produced by transgenic rice lines developed in China. Cladocera species are common aquatic arthropods that may be exposed to insecticidal proteins produced in Bt-transgenic plants through ingestion of pollen or crop residues in water. As the cladoceran Daphnia magna plays an important role in the aquatic food chain, it is important to assess the possible effects of Bt crops to this species. To evaluate the safety of the Cry1C protein for D. magna, individuals were exposed to different concentrations of purified Cry1C protein in M4 medium for 21 days. Potassium dichromate (K2Cr2O7), a known toxicant to D. magna, was added to M4 medium as a positive control treatment, and pure M4 medium was used as a negative control. Our results show that developmental, reproductive, and biochemical parameters of D. magna were not significantly different between Cry1C and negative control treatments but were significantly inhibited by the positive control. We thus conclude that D. magna is insensitive to Cry1C.

Effects of straw leachates from Cry1C-expressing transgenic rice on the development and reproduction of Daphnia magna.

The transgenic rice line T1C-19 provides high resistance to lepidopteran pests because of the synthesis of the Bacillus thuringiensis (Bt) insecticidal protein Cry1C. It thus shows good prospect for commercial planting in China. Species of Cladocera, an order of aquatic arthropods commonly found in aquatic ecosystems such as rice paddies, might be exposed to the insecticidal protein released from Bt-transgenic rice-straw residues. For the study reported herein, we used Daphnia magna (water flea) as a representative of Cladocera to evaluate whether aquatic arthropods are adversely affected when exposed to Bt rice-straw leachates. We exposed D. magna to M4 medium containing various volume percentages of medium that had been incubated with T1C-19 rice straw or rice straw from its non-transformed near-isoline Minghui 63 (MH63) for 21 days. Compared with pure M4 medium (control), the fitness and developmental and reproduction parameters of D. magna decreased significantly when exposed to rice-straw leachates; conversely, no significant differences between the T1C-19 and MH63 rice-straw leachate treatments were observed, indicating that the Bt rice straw leachate did not adversely affect this non-target species.

Does Bt rice pose risks to non-target arthropods? Results of a meta-analysis in China.

Transgenic Bt rice expressing the insecticidal proteins derived from Bacillus thuringiensis Berliner (Bt) has been developed since 1989. Their ecological risks towards non-target organisms have been investigated; however, these studies were conducted individually, yielding uncertainty regarding potential agroecological risks associated with large-scale deployment of Bt rice lines. Here, we developed a meta-analysis of the existing literature to synthesize current knowledge of the impacts of Bt rice on functional arthropod guilds, including herbivores, predators, parasitoids and detritivores in laboratory and field studies. Laboratory results indicate Bt rice did not influence survival rate and developmental duration of herbivores, although exposure to Bt rice led to reduced egg laying, which correctly predicted their reduced abundance in Bt rice agroecosystems. Similarly, consuming prey exposed to Bt protein did not influence survival, development or fecundity of predators, indicating constant abundances of predators in Bt rice fields. Compared to control agroecosystems, parasitoid populations decreased slightly in Bt rice cropping systems, while detritivores increased. We draw two inferences. One, laboratory studies of Bt rice showing effects on ecological functional groups are mainly either consistent with or more conservative than results of field studies, and two, Bt rice will pose negligible risks to the non-target functional guilds in future large-scale Bt rice agroecosystems in China.

Bt rice in China - focusing the nontarget risk assessment.

Bt rice can control yield losses caused by lepidopteran pests but may also harm nontarget species and reduce important ecosystem services. A comprehensive data set on herbivores, natural enemies, and their interactions in Chinese rice fields was compiled. This together with an analysis of the Cry protein content in arthropods collected from Bt rice in China indicated which nontarget species are most exposed to the insecticidal protein and should be the focus of regulatory risk assessment.

Combined transcriptome and metabolome analyses to understand the dynamic responses of rice plants to attack by the rice stem borer Chilo suppressalis (Lepidoptera: Crambidae).

BACKGROUND: Rice (Oryza sativa L.), which is a staple food for more than half of the world's population, is frequently attacked by herbivorous insects, including the rice stem borer, Chilo suppressalis. C. suppressalis substantially reduces rice yields in temperate regions of Asia, but little is known about how rice plants defend themselves against this herbivore at molecular and biochemical level. RESULTS: In the current study, we combined next-generation RNA sequencing and metabolomics techniques to investigate the changes in gene expression and in metabolic processes in rice plants that had been continuously fed by C. suppressalis larvae for different durations (0, 24, 48, 72, and 96 h). Furthermore, the data were validated using quantitative real-time PCR. There were 4,729 genes and 151 metabolites differently regulated when rice plants were damaged by C. suppressalis larvae. Further analyses showed that defense-related phytohormones, transcript factors, shikimate-mediated and terpenoid-related secondary metabolism were activated, whereas the growth-related counterparts were suppressed by C. suppressalis feeding. The activated defense was fueled by catabolism of energy storage compounds such as monosaccharides, which meanwhile resulted in the increased levels of metabolites that were involved in rice plant defense response. Comparable analyses showed a correspondence between transcript patterns and metabolite profiles. CONCLUSION: The current findings greatly enhance our understanding of the mechanisms of induced defense response in rice plants against C. suppressalis infestation at molecular and biochemical levels, and will provide clues for development of insect-resistant rice varieties.

The development and status of Bt rice in China.

Multiple lines of transgenic rice expressing insecticidal genes from the bacterium Bacillus thuringiensis (Bt) have been developed in China, posing the prospect of increases in production with decreased application of pesticides. We explore the issues facing adoption of Bt rice for commercial production in China. A body of safety assessment work on Bt rice has shown that Bt rice poses a negligible risk to the environment and that Bt rice products are as safe as non-Bt control rice products as food. China has a relatively well-developed regulatory system for risk assessment and management of genetically modified (GM) plants; however, decision-making regarding approval of commercial production has become politicized, and two Bt rice lines that otherwise were ready have not been allowed to enter the Chinese agricultural system. We predict that Chinese farmers would value the prospect of increased yield with decreased use of pesticide and would readily adopt production of Bt rice. That Bt rice lines may not be commercialized in the near future we attribute to social pressures, largely due to the low level of understanding and acceptance of GM crops by Chinese consumers. Hence, enhancing communication of GM crop science-related issues to the public is an important, unmet need. While the dynamics of each issue are particular to China, they typify those in many countries where adoption of GM crops has been not been rapid; hence, the assessment of these dynamics might inform resolution of these issues in other countries.

Comparison of susceptibility of Chilo suppressalis and Bombyx mori to five Bacillus thuringiensis proteins.

Transformation of rice with genes encoding insecticidal Cry proteins from Bacillus thuringiensis (Bt) should confer high resistance to target lepidopteran pests, such as Chilo suppressalis, and low toxicity to non-target organisms, such as silkworm Bombyx mori. Five purified Cry proteins that have been used for plant transformation were tested using dietary exposure assays. The susceptibility of C. suppressalis larvae to the five insecticidal proteins in the decreasing order was: Cry1Ca>Cry1Ab>Cry1Ac>Cry2Aa>Cry1Fa. However, the toxicities of the Cry proteins to B. mori were in the order: Cry1Fa>Cry1Ca>Cry2Aa>Cry1Ab>Cry1Ac. The Cry1Ca, Cry1Ab and Cry1Ac proteins exhibited relatively high toxicity to C. suppressalis larvae, with EC50 values of 16.4, 45.8 and 89.6ng/g, respectively. The toxicities of the three Cry proteins to B. mori larvae were 8, 14, and 22times lower, with EC50 values of 138.3, 628.4 and 1939.2ng/g, respectively. The Cry1Fa and Cry2Aa proteins showed high toxicity to B. mori larvae, with EC50 values of 135.7 and 373.9ng/g, respectively, but low toxicity to C. suppressalis larvae, with EC50 values of 6092.1 and 1208.5ng/g, respectively. We thus conclude that Cry1Ab, Cry1Ac and Cry1Ca are appropriate for transforming rice to control lepidopteran rice pests. In contrast, Cry1Fa and Cry2Aa are not appropriate due to their high toxicity to silkworm larvae and low activity against the target pest.

Development of Bt Rice and Bt Maize in China and Their Efficacy in Target Pest Control.

Rice and maize are important cereal crops that serve as staple foods, feed, and industrial material in China. Multiple factors constrain the production of both crops, among which insect pests are an important one. Lepidopteran pests cause enormous yield losses for the crops annually. In order to control these pests, China plays an active role in development and application of genetic engineering (GE) to crops, and dozens of GE rice and GE maize lines expressing insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) have been developed. Many lines have entered environmental release, field testing, and preproduction testing, and laboratory and field experiments have shown that most of the Bt rice and Bt maize lines developed in China exhibited effective control of major target lepidopteran pests on rice (Chilo suppressalis, Scirpophaga incertulas, and Cnaphalocrocismedinalis) and maize (Ostrinia furnacalis), demonstrating bright prospects for application. However, none of these Bt lines has yet been commercially planted through this writing in 2016. Challenges and perspectives for development and application of Bt rice and maize in China are discussed. This article provides a general context for colleagues to learn about research and development of Bt crops in China, and may shed light on future work in this field.

Binding site concentration explains the differential susceptibility of Chilo suppressalis and Sesamia inferens to Cry1A-producing rice.

Chilo suppressalis and Sesamia inferens are two important lepidopteran rice pests that occur concurrently during outbreaks in paddy fields in the main rice-growing areas of China. Previous and current field tests demonstrate that the transgenic rice line Huahui 1 (HH1) producing a Cry1Ab-Cry1Ac hybrid toxin from the bacterium Bacillus thuringiensis reduces egg and larval densities of C. suppressalis but not of S. inferens. This differential susceptibility to HH1 rice correlates with the reduced susceptibility to Cry1Ab and Cry1Ac toxins in S. inferens larvae compared to C. suppressalis larvae. The goal of this study was to identify the mechanism responsible for this differential susceptibility. In saturation binding assays, both Cry1Ab and Cry1Ac toxins bound with high affinity and in a saturable manner to midgut brush border membrane vesicles (BBMV) from C. suppressalis and S. inferens larvae. While binding affinities were similar, a dramatically lower concentration of Cry1A toxin binding sites was detected for S. inferens BBMV than for C. suppressalis BBMV. In contrast, no significant differences between species were detected for Cry1Ca toxin binding to BBMV. Ligand blotting detected BBMV proteins binding Cry1Ac or Cry1Ca toxins, some of them unique to C. suppressalis or S. inferens. These data support that reduced Cry1A binding site concentration is associated with a lower susceptibility to Cry1A toxins and HH1 rice in S. inferens larvae than in C. suppressalis larvae. Moreover, our data support Cry1Ca as a candidate for pyramiding efforts with Cry1A-producing rice to extend the activity range and durability of this technology against rice stem borers.

Establishment and optimization of a regionally applicable maize gene-flow model.

Because of the rapid development of transgenic maize, the potential effect of transgene flow on seed purity has become a major concern in public and scientific communities. Setting a proper isolation distance in field experiments and seed production is a possible solution to meet seed-quality standards and ensure adventitious contamination of products is below a specific threshold. By using a Gaussian plume model as basis and data recorded by meteorological stations as input, we have established a simple regionally applicable maize gene-flow model for prediction of the maximum threshold distances (MTD) at which gene-flow frequency is equal to or lower than a threshold value of 1 or 0.1 % (MTD1%, MTD0.1%). After optimization of the model variables, simulated outcrossing rate was a good fit to data obtained from field experiments (y = 1.156x, R (2) = 0.8913, n = 30, P < P 0.01). In the process of model calibration, it was found that only 15.82 % of the total amount of the pollen released by each plant participated in the dispersal process. The variable "a" for genetic pollen competitiveness between donor and recipient was introduced into our model, for the "Zinuo18" and "Su608" used, "a" was 17.47. Finally, the model was successfully used in the spring maize-growing region of Northeast China. The range of MTD1% and MTD0.1% in this region varied from 10 m to 49 m and from 17 m to 125 m, respectively.

Biosafety management and commercial use of genetically modified crops in China.

As a developing country with relatively limited arable land, China is making great efforts for development and use of genetically modified (GM) crops to boost agricultural productivity. Many GM crop varieties have been developed in China in recent years; in particular, China is playing a leading role in development of insect-resistant GM rice lines. To ensure the safe use of GM crops, biosafety risk assessments are required as an important part of the regulatory oversight of such products. With over 20 years of nationwide promotion of agricultural biotechnology, a relatively well-developed regulatory system for risk assessment and management of GM plants has been developed that establishes a firm basis for safe use of GM crops. So far, a total of seven GM crops involving ten events have been approved for commercial planting, and 5 GM crops with a total of 37 events have been approved for import as processing material in China. However, currently only insect-resistant Bt cotton and disease-resistant papaya have been commercially planted on a large scale. The planting of Bt cotton and disease-resistant papaya have provided efficient protection against cotton bollworms and Papaya ringspot virus (PRSV), respectively. As a consequence, chemical application to these crops has been significantly reduced, enhancing farm income while reducing human and non-target organism exposure to toxic chemicals. This article provides useful information for the colleagues, in particular for them whose mother tongue is not Chinese, to clearly understand the biosafety regulation and commercial use of genetically modified crops in China.

Green algae (Chlorella pyrenoidosa) adsorbs Bacillus thurigiensis (Bt) toxin, Cry1Ca insecticidal protein, without an effect on growth.

The effect of purified Cry1Ca insecticidal protein on the growth of Chlorella pyrenoidosa was studied in a three-generation toxicity test. The C. pyrenoidosa medium with a density of 5.4 × 10(5) cells/mL was subcultured for three generations with added Cry1Ca at 0, 10, 100, and 1000 µg/L, and cell numbers were determined daily. To explore the distribution of Cry1Ca in C. pyrenoidosa and the culture medium, Cry1Ca was added at 1000 µg/L to algae with a high density of 4.8 × 10(6) cells/mL, and Cry1Ca content was determined daily in C. pyrenoidosa and the culture medium by enzyme-linked immunosorbent assays. Our results showed that the growth curves of C. pyrenoidosa exposed to 10, 100, and 1000 µg/L of Cry1Ca almost overlapped with that of the blank control, and there were no statistically significant differences among the four treatments from day 0 to day 7, regardless of generation. Moreover, the Cry1Ca content in the culture medium and in C. pyrenoidosa sharply decreased under exposure of 1000 µg/L Cry1Ca with high initial C. pyrenoidosa cell density. The above results demonstrate that Cry1Ca in water can be rapidly adsorbed and degraded by C. pyrenoidosa, but it has no suppressive or stimulative effect on algae growth.