Isolation and functional analysis of OsAOS1 promoter for resistance to Nilaparvata lugens Stål infestation in rice.

Insect pests have a great impact on the yield and quality of crops. Insecticide applications are an effective method of pest control, however, they also have adverse effects on the environment. Using insect-inducible promoters to drive insect-resistant genes in transgenic crops is a potential sustainable pest management strategy, but insect-inducible promoters have been rarely reported. In this study, we found rice allene oxide synthase gene (AOS, LOC_Os03g12500) can be highly upregulated following brown planthopper (Nilaparvata lugens Stål, BPH) infestation. Then, we amplified the promoter of OsAOS1 and the ß- glucuronidase reporter gene was used to analyze the expression pattern of the promoter. Through a series of 5' truncated assays, three positive regulatory regions in response to BPH infestation in the promoter were identified. The transgenic plants, P1R123-min 35S and P1TR1-min 35S promoter-driven snowdrop lectin (Galanthus nivalis agglutinin, GNA) gene, demonstrated the highest expression levels of GNA and lowest BPH survival. Our work identified a BPH-inducible promoter and three positive regions within it. Transgenic rice with GNA driven by OsAOS1 promoter and positive regions exhibited an expected lethal effect on BPH. This study proved the application potential of BPH-inducible promoter and provided a novel path for the selection of insect-resistant tools in the future.

Candidate detoxification-related genes in brown planthopper, Nilaparvata lugens, in response to ß-asarone based on transcriptomic analysis.

Nilaparvata lugens(Stål) is a serious pest of rice and has evolved different levels of resistance against most chemical pesticides. ß-asarone is the main bioactive insecticidal compound of Acorus calamus L. that shows strong insecticidal activity against pests. In this study, we conducted a bioassay experiment to determine the contact toxicity of ß-asarone to N. lugens nymphs. The LD30 sublethal dose was 0.106 µg per nymph, with 95% confidence limits of 0.070-0.140 µg. We applied the LD30 concentration of ß-asarone to nymphs for 24 h or 72 h and then performed a transcriptome sequence analysis by referencing the N. lugens genome to characterize the variation. The transcriptomic analysis showed that several GO terms and KEGG pathways presented significant changes. Individually, 126 differentially expressed genes (DEGs), including 72 upregulated and 54 downregulated genes, were identified at 24 h, and 1771 DEGs, including 882 upregulated and 889 downregulated genes, were identified at 72 h. From the DEGs, we identified a total of 40 detoxification-related genes, including eighteen Cytochrome P450 monooxygenase genes (P450s), three Glutathione S-transferase genes, one Carboxylesterase gene, twelve UDP-glucosyltransferases and six ATP-binding cassette genes. We selected the eighteen P450s for subsequent verification by quantitative PCR. These findings indicated that ß-asarone presented strong contact toxicity to N. lugens nymphs and induced obvious variation of detoxification-related genes that may be involved in the response to ß-asarone.

Monitoring reduced accumulation and risk evaluation of Cry1Ca during food chain transfer from genetically engineered plant to endoparasitoid.

BACKGROUND: The potential risk of insecticidal proteins produced by genetically engineered (GE) plants to nontarget organisms have long been an ecotoxicological concern. Apanteles chilonis, an important endoparasitoid of rice pest Chilo suppressalis, potentially is exposed to Bacillus thuringiensis (Bt) endotoxins through a food chain of transgenic Bt rice - C. suppressalis - A. chilonis, and thus, a rigorous risk assessment is urgently needed. Here, we combined a tri-trophic bioassay system with high-dose exposure approach using C. suppressalis hemolymph as the carrier of insecticidal protein to evaluate the biosafety of Cry1Ca to A. chilonis. RESULTS: Cry1Ca protein could be transmitted and retained along the food chain and remains bioactive in the hemolymph of C. suppressalis during the pre-adult duration of A. chilonis. No significant differences in pre-adult period, male and female longevity, adult fecundity and weight, emergence rate nor sex ratio were observed when A. chilonis parasitized C. suppressalis feeding on cry1Ca rice compared with control treatment. However, the pupal period and weight were significantly prolonged and decreased. When A. chilonis parasitized C. suppressalis injected with a high dosage of Cry1Ca protein, no adverse effects on the life-history parameters, peroxidase (POD), superoxide dismutase (SOD) or glutathione reductase (GR) of A. chilonis were observed, demonstrating that the host quality mediates adverse effects during the food chain. CONCLUSIONS: We confirmed that Cry1Ca posed no ecological risk to the nontarget endoparasitoid A. chilonis. This study may serve as an example for future risk assessment of transgenic crops to nontarget endoparasitoids. © 2023 Society of Chemical Industry.

Cloning and functional identification of a Chilo suppressalis-inducible promoter of rice gene, OsHPL2.

BACKGROUND: Promoters play a key role in driving insect-resistant genes during breeding of transgenic plants. In current transgenic procedures for breeding rice resistance to striped stem borer (Chilo suppressalis Walker, SSB), the constitutive promoter is used to drive the insect-resistant gene. To reduce the burden of constitutive promoters on plant growth, isolation and identification of insect-inducible promoters are particularly important. However, few promoters are induced specifically by insect feeding. RESULTS: We found rice hydroperoxide lyase gene (OsHPL2) (LOC_Os02g12680) was upregulated after feeding by SSB. We subsequently cloned the promoter of OsHPL2 and analysed its expression pattern using the ß-glucuronidase (GUS) reporter gene. Histochemical assays and quantitative analyses of GUS activity confirmed that P HPL2 :GUS was activated by SSB, but did not respond to brown planthopper (Nilaparvata lugens Stål, BPH) infestation, mechanical wounding or phytohormone treatments. A series of 5' truncated assays were conducted and three positive regulatory regions (-1452 to -1213, -903 to -624, and -376 to -176) induced by SSB infestation were identified. P2R123-min 35S and P2TR2-min 35S promoters linked with cry1C of transgenic plants showed the highest levels of Cry1C protein expression and SSB larval mortality. CONCLUSION: We identified an SSB-inducible promoter and three positive internal regions. Transgenic rice plants with the OsHPL2 promoter and its positive regions driving cry1C exhibited the expected larvicidal effect on SSB. Our study is the first report of an SSB-inducible promoter that could be used as a potential resource for breeding insect-resistant transgenic crops. © 2020 Society of Chemical Industry.

Ultrabithorax is a key regulator for the dimorphism of wings, a main cause for the outbreak of planthoppers in rice.

Rice planthoppers, the most devastating rice pests, occur in two wing forms: the short-wing form for rapid population growth and long-wing form for long-distance migration, which together create the mechanism for outbreak. Here we show that Ultrabithorax (Ubx) is a key regulator for switching between the long- and short-wing forms of rice planthoppers. Ubx is expressed in both forewing and hindwing pads, which is different from the canonical model of Ubx expression. In brown planthoppers, expression of Ubx (NlUbx) is regulated by nutritional status of the rice host. High-quality young plants induce NlUbx expression leading to the short-wing form; low-quality ripe plants reduce NlUbx expression resulting in long-wing form. We also showed that NlUbx is regulated by the insulin receptors NlInR1 and NlInR2. The default expression of NlInR1 inhibits NlUbx resulting in long-wings, while high-quality hosts induce NlInR2 expression, which represses NlInR1 thus promoting NlUbx expression to produce short-wings.