Simultaneously enhancing plant growth and immunity through the application of engineered Bacillus subtilis expressing a microbial pattern

doi:10.1016/j.jia.2024.04.034

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Simultaneously enhancing plant growth and immunity through the application of engineered Bacillus subtilis expressing a microbial pattern

Shuangxi Zhang^1*, Xinlin Wei^1*, Hejing Shen¹, Qinhu Wang², Yi Qiang¹, Langjun Cui¹, Hongxing Xu¹, Yuyan An^1#, Meixiang Zhang^1#

1 College of Life Sciences, National Engineering Laboratory for Endangered Medicinal Resource Development in Northwest China, Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry of Ministry of Education, Shaanxi Normal University, Xi’an 710119, China

2 College of Plant Protection, State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, Northwest A&F University, Yangling 712100, China

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摘要植物病害严重威胁农业生产，导致作物产量和品质下降，同时化学防治方法带来了环境污染、食品安全问题以及病原菌耐药性变异等挑战。植物免疫诱导剂作为一种激活植物自身防御机制的替代方案，正在受到越来越多的关注。然而，植物生长与免疫之间存在一种动态平衡，提高植物的抗病性往往以牺牲生长和产量为代价。因此，增强植物抗病性的同时促进其生长，是农业生产中追求的理想目标。为了实现这一目标，科学家们已经进行了大量的研究工作。其中，有益微生物如枯草芽孢杆菌(Bacillus subtilis)和植物免疫诱导剂如病原相关分子模式(pathogen-associated molecular patterns, PAMPs)等，因其潜在的促进生长或诱导免疫的作用而备受关注。然而，如何平衡植物免疫和生长发育，仍然是一个科学难题。

在本研究中，我们对具有促进植物生长活性的枯草芽孢杆菌OKB105进行了基因工程改造，使其能够分泌病原相关分子模式flg22，以期实现同时提高植物免疫和生长的双重效果。通过将编码flg22的基因经过密码子优化后重组到pBE-S载体中，并转化到OKB105中，我们成功获得了能够分泌flg22的重组枯草芽孢杆菌OKB105 (flg22)。与含有空载体的对照菌株OKB105 (EV)相比，OKB105 (flg22)在促进植物生长方面表现出了相似的活性，这表明改造后的菌株保持了其原有的生长促进功能。更重要的是，与OKB105 (EV)相比，OKB105 (flg22)显著增强了本氏烟(Nicotiana benthamiana)对丁香假单胞菌(Pseudomonas syringae) DC3000 ∆hopQ1-1和寄生疫霉菌(Phytophthora parasitica)的抗性，证实了其在提高植物抗病性方面的有效性。此外，根部灌溉OKB105 (flg22)还能显著提高植物对地上部病原菌的抗性，表明该菌株能够诱导植物产生系统获得性抗性(systemic acquired resistance, SAR)。我们还发现，除了烟草，OKB105 (flg22)也能增强番茄和辣椒等其他可识别flg22的作物的抗病性，显示出其广泛的适用性。通过RNA-Seq和qRT-PCR分析，我们进一步证实了OKB105 (flg22)能够有效诱导模式触发免疫(pattern-triggered immunity, PTI)途径中防御相关基因的表达，从而提高植物的抗病性。

综上所述，通过基因工程改造的枯草芽孢杆菌OKB105 (flg22)，不仅保持了其原有的促进植物生长的活性，而且显著提升了植物对多种病原菌的抗性。这种策略通过利用植物促生菌表达病原相关分子模式，成功实现了在不牺牲植物生长的前提下增强其免疫，为农业生产中病害的有效防治开辟了新的途径，在未来植物病害防控中具有广阔应用前景。

Abstract Simultaneously enhancing plant growth and disease resistance is an ideal goal in Agriculture. Significant efforts have been made to promote plant growth or immunity through the use of biological reagents, such as the application of beneficial microbes and plant immunity inducers. However, balancing plant immunity and growth remains a challenging task. In this study, we engineered the plant growth-promoting bacterium Bacillus subtilis OKB105 to express a secreted microbial pattern, flg22, and accessed its activity in enhancing both plant growth and disease resistance. The OKB105 (flg22) strain exhibited plant growth-promoting activity similar to the OKB105 strain containing an empty vector, OKB105 (EV). Furthermore, the OKB105 (flg22) strain significantly enhanced plant resistance against two distinct pathogens, Pseudomonas syringae DC3000 ΔhopQ1-1 and Phytophthora parasitica, compared to OKB105 (EV), confirming that the engineered OKB105 (flg22) effectively enhances plant disease resistance. Interestingly, root irrigation with OKB105 (flg22) also markedly boosted the plant’s aboveground resistance to pathogens compared to OKB105 (EV). We further demonstrated that OKB105 (flg22) can be applied to confer resistance to pathogens in other plants that recognize flg22. Finally, RNA-Seq and qRT-PCR analyses illustrated that OKB105 (flg22) effectively induced the expression of defense-related genes in pattern-triggered immunity. Our results prove that employing an engineered beneficial microbe expressing a microbial pattern is a promising strategy for simultaneously enhancing plant growth and immunity.

Keywords: plant growth promotion immunity induction beneficial microbe microbial pattern disease resistance

Online: 24 May 2024

Fund: This work was supported by the Technology Innovation Leading Program of Shaanxi (2023QYPY2-01), the National Natural Science Foundation of China (32072399, 32302296), and the Fundamental Research Funds for the Central Universities (GK202201017, GK202207024).

About author: Shuangxi Zhang, E-mail: sxzhang2022@snnu.edu.cn; Xinlin Wei, E-mail: xinlinwei@snnu.edu.cn; #Correspondence Yuyan An, E-mail：anyuyan@snnu.edu.cn; Meixiang Zhang, E-mail: meixiangzhang@snnu.edu.cn *These authors contributed equally to this work.

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Shuangxi Zhang, Xinlin Wei, Hejing Shen, Qinhu Wang, Yi Qiang, Langjun Cui, Hongxing Xu, Yuyan An, Meixiang Zhang. 2024. Simultaneously enhancing plant growth and immunity through the application of engineered Bacillus subtilis expressing a microbial pattern. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.04.034

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