利用促生枯草芽孢杆菌表达病原相关分子模式：一种同时提高植物免疫和生长的有效策略

doi:10.1016/j.jia.2024.04.034

摘要/Abstract

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.

Key words: plant growth promotion , immunity induction , beneficial microbe , microbial pattern , disease resistance

. 利用促生枯草芽孢杆菌表达病原相关分子模式：一种同时提高植物免疫和生长的有效策略[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.04.034.

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

参考文献

Ahmed A S, Sánchez C P, Candela M E. 2000. Evaluation of induction of systemic resistance in pepper plants (Capsicum annuum) to Phytophthora capsici using Trichoderma harzianum and its relation with capsidiol accumulation, European Journal of Plant Pathology, 106, 817-824.

Alves M S, Dadalto S P, Gonçalves A B, De Souza G B, Barros V A, Fietto L G. 2014. Transcription factor functional protein-protein interactions in plant defense responses, Proteomes, 2, 85-106.

Belkhadir Y, Yang L, Hetzel J, Dangl J L, Chory J. 2014. The growth–defense pivot: crisis management in plants mediated by LRR-RK surface receptors. Trends in Biochemical Sciences, 39, 447-456.

Blake C, Christensen M N, Kovács Á T. 2021. Molecular aspects of plant growth promotion and protection by Bacillus subtilis. Molecular Plant-Microbe Interactions, 34, 15-25.

Blaker N, Hewitt J. 1987. A comparison of resistance to Phytophthora parasitica in tomato. Phytopathology, 77, 1113-1116.

Cao Y, Zhang Z, Ling N, Yuan Y, Zheng X, Shen B, Shen Q. 2011. Bacillus subtilis SQR9 can control Fusarium wilt in cucumber by colonizing plant roots. Biology and Fertility of Soils, 47, 495-506.

Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, Liu Y, Feng J, Chen H, He Y. 2023. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Molecular Plant, 16, 1733-1742.

Chisholm S T, Coaker G, Day B, Staskawicz B J. 2006. Host-microbe interactions: shaping the evolution of the plant immune response. Cell, 124, 803-814.

Cosgrove D J. 2005. Growth of the plant cell wall. Nature Reviews Molecular Cell Biology, 6, 850-861.

Deng Y, Chen H, Li C, Xu J, Qi Q, Xu Y, Zhu Y, Zheng J, Peng D, Ruan L. 2019. Endophyte Bacillus subtilis evades plant defense by producing lantibiotic subtilomycin to mask self-produced flagellin. Communications Biology, 2, 368.

Elkoca E, Kantar F, Sahin F. 2007. Influence of nitrogen fixing and phosphorus solubilizing bacteria on the nodulation, plant growth, and yield of chickpea. Journal of Plant Nutrition, 31, 157-171.

Felix G, Duran J D, Volko S, Boller T. 1999. Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. The Plant Journal, 18, 265-276.

Ferrari S, Savatin D V, Sicilia F, Gramegna G, Cervone F, Lorenzo G D. 2013. Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development. Frontiers in Plant Science, 4, 49.

Gao S, Wu H, Wang W, Yang Y, Xie S, Xie Y, Gao X. 2013. Efficient colonization and harpins mediated enhancement in growth and biocontrol of wilt disease in tomato by Bacillus subtilis. Letters in Applied Microbiology, 57, 526-533.

Gomez-Gomez L, Felix G, Boller T. 1999. A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana. The Plant Journal, 18, 277–284.

Gómez-Gómez L, Boller T. 2000. FLS2: an LRR receptor–like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Molecular Cell, 5, 1003-1011.

Granke L L, Quesada-Ocampo L, Lamour K, Hausbeck M K. 2012. Advances in research on Phytophthora capsici on vegetable crops in the United States. Plant Disease, 96, 1588-1600.

Hashem A, Tabassum B, Abd_Allah E F. 2019. Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi Journal of Biological Sciences, 26, 1291-1297.

Huang Y, Cui J, Li M, Yang R, Hu Y, Yu X, Chen Y, Wu Q, Yao H, Yu G. 2023. Conservation and divergence of flg22, pep1 and nlp20 in activation of immune response and inhibition of root development. Plant Science, 331, 111686.

Huot B, Yao J, Montgomery B L, He S Y. 2014. Growth–defense tradeoffs in plants: a balancing act to optimize fitness. Molecular Plant, 7, 1267-1287.

Jelenska J, Davern S M, Standaert R F, Mirzadeh S, Greenberg J T. 2017. Flagellin peptide flg22 gains access to long-distance trafficking in Arabidopsis via its receptor, FLS2. Journal of Experimental Botany, 68, 1769-1783.

Kiełbowicz-Matuk A. 2012. Involvement of plant C2H2-type zinc finger transcription factors in stress responses. Plant Science, 185, 78-85.

Kovács Á T. 2019. Bacillus subtilis. Trends in Microbiology, 27, 724-725.

Lugtenberg B, Kamilova F. 2009. Plant-growth-promoting rhizobacteria. Annual Review of Microbiology, 63, 541-556.

Meng Y, Zhang Q, Ding W, Shan W. 2014. Phytophthora parasitica: a model oomycete plant pathogen. Mycology, 5, 43-51.

Miao G, ZHOU J, WANG E, Qian C, Jing X, SUN J. 2015. Multiphasic characterization of a plant growth promoting bacterial strain, Burkholderia sp. 7016 and its effect on tomato growth in the field. Journal of Integrative Agriculture, 14, 1855-1863.

Mishina T E, Zeier J. Pathogen-associated molecular pattern recognition rather than development of tissue necrosis contributes to bacterial induction of systemic acquired resistance in Arabidopsis. Plant Journal, 2007, 50, 500-513.

Nakano M, Marahiel M, Zuber P. 1988 Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. Journal of Bacteriology, 170, 5662-5668.

Ouyang X, Chen J, Sun Z, Wang R, Wu X, Li B, Song C, Liu P, Zhang M. 2023. Ubiquitin E3 ligase activity of Ralstonia solanacearum effector RipAW is not essential for induction of plant defense in Nicotiana benthamiana. Frontiers in Microbiology, 14, 1201444.

Ren P, Xie Y, Zhang Y, Wu H, Gao X. 2014. Effect and mechanism of controlling TMV disease on tobacco by surfactin produced by Bacillus subtilis OKB105. Chinese Journal of Biological Control, 30, 216.

Saeid A, Prochownik E, Dobrowolska-Iwanek J. 2018. Phosphorus solubilization by Bacillus species. Molecules, 23, 2897.

Savary S, Willocquet L, Pethybridge S J, Esker P, McRoberts N, Nelson A. 2019. The global burden of pathogens and pests on major food crops. Nature Ecology & Evolution, 3, 430-439.

Spizizen J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proceedings of the National Academy of Sciences of the United States of America, 44, 1072-1078.

Sun W, Dunning F, Pfund C, Weingarten R, Bent A F. 2006. Within-species flagellin polymorphism in Xanthomonas campestris pv campestris and its impact on elicitation of Arabidopsis FLAGELLIN SENSING2–dependent defenses. The Plant Cell, 18, 764-779.

van Dijl J, Hecker M. 2013. Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microbial Cell Factories, 12, 1-6.

van Wees S C, de Swart E A, van Pelt J A, van Loon L C, Pieterse C M. 2000. Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America. 97, 8711-8716.

Wang S, Wu H, Zhan J, Xia Y, Gao S, Wang W, Xue P, Gao X. 2011. The role of synergistic action and molecular mechanism in the effect of genetically engineered strain Bacillus subtilis OKBHF in enhancing tomato growth and Cucumber mosaic virus resistance. BioControl, 56, 113-121.

Wang W, Feng B, Zhou J M, Tang D. 2020. Plant immune signaling: Advancing on two frontiers. Journal of Integrative Plant Biology, 62, 2-24.

Wang Y, Pruitt R, Nürnberger T, Wang Y. 2022. Evasion of plant immunity by microbial pathogens. Nature Reviews Microbiology, 20, 449-464.

Wei Y, Caceres‐Moreno C, Jimenez‐Gongora T, Wang K, Sang Y, Lozano‐Duran R, Macho A P. 2018. The Ralstonia solanacearum csp22 peptide, but not flagellin‐derived peptides, is perceived by plants from the Solanaceae family. Plant Biotechnology Journal, 16, 1349-1362.

Xie S, Wu H, Zang H, Wu L, Zhu Q, Gao X. 2014. Plant growth promotion by spermidine-producing Bacillus subtilis OKB105. Molecular Plant-Microbe Interactions, 27, 655-663.

Xie S, Wu H, Chen L, Zang H, Xie Y, Gao X. 2015. Transcriptome profiling of Bacillus subtilis OKB105 in response to rice seedlings. BMC Microbiology, 15, 1-14.

Xue J, Gong B, Yao X, Huang X, Li J. 2020. BAK1‐mediated phosphorylation of canonical G protein alpha during flagellin signaling in Arabidopsis. Journal of Integrative Plant Biology, 62, 690-701.

Yang B, Yang S, Zheng W, Wang Y. 2022. Plant immunity inducers: From discovery to agricultural application. Stress Biology, 2, 5.

Zhang L, Gleason C. 2020. Enhancing potato resistance against root-knot nematodes using a plant-defence elicitor delivered by bacteria. Nature Plants, 6, 625-629.

Zhang N, Wu K, He X, Li S, Zhang Z, Shen B, Yang X, Zhang R, Huang Q, Shen Q. 2011. A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11. Plant and Soil, 344, 87-97.

Zhu F, Cao M, Zhang Q, Mohan R, Schar J, Mitchell M, Chen H, Liu F, Wang D, Fu Z Q. 2023. Join the green team: inducers of plant immunity in the plant disease sustainable control toolbox. Journal of Advanced Research, S2090-1232(23)00122-4.

Zipfel C. 2014. Plant pattern-recognition receptors. Trends in Immunology, 35, 345-351.