So far, the Bt protein in all commercially used Bt insect-resistant crops in the world has accumulated in the cytoplasm. However, studies in recent years have shown that the accumulation of exogenous anti-insect proteins in the cytoplasm may have undesirable effects. For example, the exogenous insect-resistant protein Cry1Ab/c expressed by Huahui No. 1 of transgenic rice in mainland China that has obtained a safety certificate interacts with the endogenous protein Hd3a (Front Bioeng Biotechnol, 2020, 8: 685; Front Plant Sci, 2021, 12: 608721; Front Bioeng Biotechnol, 2021, 9: 759016), which led to undesirable effects on the transformant, which seriously affected the breeding application prospect of the transformant. Recently, Dr. Hua Li from Xiao Guoying's research team from the Institute of Subtropical Agroecology, Chinese Academy of Sciences published articles in the journals Plant Cell, Tissue and Organ Culture and Pest Management Science, respectively, reporting that the new technology of accumulating insect-resistant proteins in the cell wall completely solves the problem of undesirable effects caused by high expression of insect-resistant proteins, which may bring revolutionary changes to the breeding of transgenic insect-resistant crops!
In fact, Xiao Guoying's research team at the Institute of Subtropical Agroecology of the Chinese Academy of Sciences has long noticed that the high expression of insect-resistant proteins in cells may have a negative effect. Cry1C, a Bt insect resistant protein, has the advantages of wide insecticidal spectrum and low semi-lethal dose, and its application prospect is very attractive (Plant Science, 2009, 176: 315-324). More than 10 years ago, they began to develop insect-resistant rice transgenic with Cry1Ca gene, and the expression level of Cry1Ca protein in the leaves of the rice transformant B1C893-6 was as high as 35.07 μg/g, showing good insect resistance. However, the undesirable effects caused by the high expression of exogenous genes were also obvious, which were manifested in shorter plant height, shorter spike length, lower number of grains, lower seed setting rate, and longer growth period (Hybrid Rice, 2014, 29: 67-71). The accumulation of Cry1Ca protein in the leaves of transgenic rice B1C106-3 (Journal of Integrative Agriculture, 2018, 17(3): 493-506) was 11.30 μg/g, which also produced the undesirable effects of decreasing the number of grains, decreasing seed setting rate and decreasing yield. In addition, the content of Cry1Ca protein in the leaves of E1C608-3 (Chinese Journal of Biotechnology, 2019, 39(11): 31-38) was 8.72 μ/g, which also showed undesirable effects such as shorter plant height, shorter panicle, decreased total grain number per panicle and decreased seed setting rate. Only when the average content of Cry1Ca protein in leaves was as low as 1.59 μg/g, no significant undesirable effects were detected in rice transformant E1C4008S-4 (Rice Science, 2020, 7(3): 215-226). If the phenomenon of undesirable effects produced by high expression of Bt protein is widespread, this will lead to large undesired effects of transformants with high expression of insect resistant proteins, low expression of insect resistant proteins of transformants with small undesired effects, and insect resistance and other agronomic traits cannot be taken into account. In addition, if transformants with high insect protein expression are not used in production, it will seriously hinder the implementation of high-dose/shelter strategies for insect-resistant crops, which may lead to the rapid evolution of resistance to insect proteins in target pests, which will greatly shorten the economic lifespan of Bt insect-resistant crops.
In order to solve the problem of undesirable effects caused by the high expression of Cry1Ca protein in cells, they created an insect-resistant fusion protein that can be secreted extracellular and anchored on the cell wall by using the carbohydrate-binding module that can bind carbohydrates such as cellulose and the function of stretching protein signal peptide to guide extracellular secretion, which realizes the directional accumulation of insect-resistant proteins in the plant cell wall and gives the host plant high resistance to the target pest, while avoiding the occurrence of undesirable effects. First, they demonstrated that the extensin signal peptide can guide the secretion of insect-resistant proteins to the extracellular cell using tissue sections, callus smears, paraffin sections, immunofluorescence, and protoplast isolation (Plant Cell, Tissue and Organ Culture, 2023, 154: 527-539). Then, they used this strategy to obtain rice transfused with insect resistance protein genes, and detected that the Cry1Ca protein content in the transformant leaves reached up to 17.5 μg/g, and the lethality rate for lepidopteran pests reached 100%. Most importantly, the homozygous transformants were found to have a higher protein content of Cry1Ca in leaves than in B1C106-3 and E1C608-3 leaves, but did not exhibit the undesirable effects of B1C106-3 and E1C608-3 (Pest Management Science, 2024, 80: 1728-1739). Later, they applied this strategy to maize, and the resulting Cry1Ca-transgenic maize transformant leaves were fed to fall armyworm for 4 days, and the lethality rate reached 100% (Figure 1).
图1 细胞壁积累Cry1Ca抗虫蛋白质的玉米转化体对草地贪夜蛾的抗性细胞壁定向积累抗虫蛋白质技术的建立，能够避免外源Bt蛋白质与细胞内源蛋白质可能存在的相互作用，获得抗虫性和农艺性状俱佳的转化体，突破了所有转基因作物均在细胞质内积累外源蛋白质的技术桎梏，为培育转基因抗虫作物提供了一条全新的技术路线，可能给转基因抗虫作物育种带来一场革命，从而改写转基因抗虫作物育种的历史。 参考文献[1]Fu J and Liu B. Exogenous Cry1Ab/c protein recruits different endogenous proteins for its function in plant growth and development. Frontiers in Bioengineering and Biotechnology, 2020, 8: 685. https://doi.org/10.3389/fbioe.2020.00685[2]Fu J, Liu G and Liu B. Foreign Cry1Ab/c delays flowering in insect-resistant transgenic rice via interaction with Hd3a florigen. Frontiers in Plant Science, 2021, 12: 608721. https://doi.org/10.3389/fpls.2021.608721[3]Fu J, Shi Y, Liu L and Liu B. Cellular localization of exogenous Cry1Ab/c and its interaction with plasma membrane Ca2+-ATPase in transgenic rice. Frontiers in Bioengineering and Biotechnology, 2021, 9: 759016. https://doi.org/10.3389/fbioe.2021.759016[4]Li H, Deng L, Weng L, Li J, Yu J and Xiao G. The carbohydrate‑binding module mediates mCherry protein anchoring on the cell wall in rice. Plant Cell, Tissue and Organ Culture, 2023, 154: 527-539. https://link.springer.com/article/10.1007/s11240-023-02471-0[5]Li H, Deng L, Weng L, Li J, Hu W, Yu J, Xiao Y and Xiao G. Cell wall-localized Bt protein endows rice high resistance to Lepidoptera pests. Pest Management Science, 2024, 80: 1728-1739. https://onlinelibrary.wiley.com/doi/10.1002/ps.7901
[6] Dror Avisar, Haviva Eilenberg, Menachem Keller, Noam Reznik, Michal Segal, Baruch Sneh, Aviah Zilberstein. The Bacillus thuringiensis delta-endotoxin Cry1C as a potential bioinsecticide in plants. Plant Science, 2009, 176: 315-324. http://dx.doi.org/10.1016/j.plantsci.2008.12.010[7]Deng L, Deng X, Wei S, Cao Z, Tang L and Xiao G. Development and identification of herbicide and insect resistant transgenic plant B1C893 in rice. Hybrid Rice, 2014, 29: 67-71. (in Chinese with English abstract) https://doi.org/10.16267/j.cnki.1005-3956.2014.01.022[8]Hu W, Deng X, Deng X, Deng L, Xiao Y, He X, Fu X and Xiao G. Characteristic analysis of tetra-resistant genetically modified rice. Journal of Integrative Agriculture, 2018, 17(3): 493-506. https://doi.org/10.1016/S2095-3119(17)61722-2[9]Zeng Q, Meng Q, Deng L, Li J, Yu J, Weng L and Xiao G. Identification and analysis of important phenotypes E1C608 with glyphosate resistance and lepidopteran resistance in rice. China Biotechnology, 2019, 39(11): 31-38. (in Chinese with English abstract) https://doi.org/10.13523/j.cb.20191104[10]Zeng Q, Deng L, Hu W, He X, Meng Q, Yu J, Li J, Weng L and Xiao G. Verification of glyphosate resistance, lepidopteran resistance and wide compatibility of male sterile line E1C4008S in rice. Rice Science, 2020, 27(3): 215-226. http://dx.doi.org/10.1016/j.rsci.2020.04.004
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