Journal Article

Creating artificial miR2118a/b to boost yield and broad-spectrum resistance in soybean via CRISPR/Cas9-targeted mutation

Shan, Zhihui, Yang, Zhonglu, Hao, Qingnan, Chen, Haifeng, Chen, Limiao, Hu, Yanfeng, Peng, Lihua, Huang, Yi, Zhou, Xinan, Chen, Shuilian, Ouyang, Wenqi, Cao, Dong, Wang, Xinyang, Zhang, Chanjuan, Xu, Dami, Guo, Wei, Tran, Lam-Son Phan, Xu, Juan, Xia, Rui, Hewezi, Tarek, Chen, Peike, Yang, Hongli, Pan, Mengyue, Yuan, Songli

2025

Overview

miR2118 triggers phased small interfering RNA (phasiRNA) biogenesis in plants and has multifaceted roles in plant development and disease resistance.Targeting the passenger strands (miR2118a/b-5p) of soybean miR2118a/b via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) successfully created transgene-free amiR2118a/b mutants.Artificial miR2118a/b (amiR2118a/b) double mutants showed an altered secondary structure of pre-amiR2118a/b, and suppressed biogenesis of mature miR2118a/b and phasiRNAs; and upregulated expression of growth-related and defense-related genes under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, respectively.Two transgene-free amir2118a/b double mutants exhibited enhanced resistance to Pseudomonas syringae pv. glycinea, soybean cyst nematode, and root-knot nematode, and achieved increased yield under pathogen-free field conditions.A strategy is provided for generating artificial miRNAs (amiRNAs) to improve crops via the CRISPR/Cas system by mutating miRNAs* in crops. While regulatory functions of mature miRNAs are well established, the functions of miRNAs* and their potential for genetic engineering in crop improvement remain underexplored. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) to generate artificial miR2118a/b (amiR2118a/b) by editing miR2118a/b-5p and obtained several amir2118a/b mutants in soybean (Glycine max). miR2118a/b-5p modifications altered the secondary structure of precursor amiR2118a/b (pre-amiR2118a/b) and reduced mature miR2118a/b levels. These amir2118a/b mutants retained the ability to initiate biogenesis of phased small interfering RNAs (phasiRNAs), albeit with a reduced abundance compared with wild-type (WT) plants. Furthermore, these mutants upregulated the expression of genes related to growth and defense under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, respectively. Notably, two transgene-free amir2118 mutants exhibited enhanced resistance to Psg, soybean cyst nematode (SCN), and root-knot nematode (RKN), and achieved increased yield under pathogen-free field conditions. This study provides a strategy to generate artificial miRNAs (amiRNAs) for crop improvement through the CRISPR/Cas system by mutating miRNAs* in crops. [Display omitted] This work presents a convenient and reliable strategy to generate artificial miRNAs (amiRNAs) for soybean improvement through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) system by mutating miRNAs* in soybean. The current technology readiness level (TRL) of the described technology is 4-5. At TRL 4, data from laboratory and controlled environments confirm core mechanisms, that is, amiR2118a/b mutants show altered pre-amiR2118a/b secondary structure, reduced mature miR2118a/b and phased small interfering RNAs (phasiRNAs) levels, upregulated expression of growth-related and defense-related genes under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, respectively, and enhanced resistance to Psg, soybean cyst nematode (SCN, races 3/5) and root-knot nematode (RKN) in greenhouses without growth inhibition. At TRL 5, field trials conducted in Beijing and Hanchuan from 2023 to 2024 show that the mutants achieved a consistent yield increase of 7.4–8.7%; pot experiments in greenhouses using naturally SCN-infested soil collected from Daqing confirmed that the SCN population density in the mutants decreased by 22.19–32.68% without yield loss, realizing the connection between laboratory and real agricultural scenarios. However, when this technology is applied on a large scale, it may face several challenges, including genotype dependence, mechanistic gaps, field-scale pathogen resistance verification, and integration of regulatory and breeding processes. These challenges need to be addressed by expanding genotype and crop testing, filling mechanistic gaps, conducting advanced field verification, and optimizing regulatory and breeding processes. This study uses clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) to mutate miR2118a/b-5p, generating amiR2118a/b mutants, altering pre-amiR2118a/b structures, reducing the levels of mature miR2118a/b and phased small interfering RNAs (phasiRNAs), upregulating growth/defense-related genes under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, and enhancing soybean field yield and broad-spectrum resistance, thus offering a novel crop improvement approach.

• Publisher: Elsevier Ltd
• Subject: artificial miRNAs / biogenesis / biotechnology / broad-spectrum resistance / Glycine max / Heterodera glycines / Internal Medicine / microRNA / miR2118 / miR2118-5p / mutation / phasiRNA / Pseudomonas syringae pv. glycinea / root-knot nematodes / soybean / soybeans / yield