Editing of BoMLO12 confers broad-spectrum disease resistance and enhanced yield in Brassica oleracea

Dear Editor, Brassica oleracea, a biennial herbaceous species within the Brassicaceae family, includes a variety of globally cultivated vegetable crops of significant agronomic and economic importance, such as cabbage, broccoli, and kale [1]. However, their production is severely constrained by soil-borne and foliar diseases, such as Fusarium wilt caused by Fusarium oxysporum f. sp. conglutinans, black rot caused by Xanthomonas campestris pv. campestris, and black spot caused by Alternaria brassicicola. Disease-resistant cultivars are typically developed by introducing dominant resistance (R) genes and knocking out susceptibility (S) genes. Notwithstanding these approaches, R genes typically confer resistance solely to specific cognate pathogens, thereby limiting broad-spectrum resistance. At the same time, editing S genes sometimes confers plants with nonspecific, durable, and broad-spectrum disease resistance. Thus, harnessing loss-of-function mutations in S genes has emerged as a promising strategy for breeding disease-resistant crops. The advent of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) systems has revolutionized plant genome engineering, enabling precise genetic modifications and significantly facilitating the development of crop varieties with enhanced disease resistance [2]. According to previous studies, editing the BoBPM6 and BoDMR6 genes resulted in the creation of new germplasms with broad-spectrum disease resistance in cabbage [3]. In addition, MLO is a well-characterized S gene, and the wheat MLO mutant Tamlo-R32 has been demonstrated to exhibit broad-spectrum resistance against powdery mildew while maintaining optimal yield potential [4]. In this study, we conducted functional characterizations of the MLO genes in cabbage (B. oleracea var. capitata). To identify potential target for enhancing disease resistance in B. oleracea, phylogenetic analysis was conducted on 28 identified MLO homologs. Among these, 22 genes containing complete functional motifs were selected for phylogenetic tree construction with Arabidopsis thaliana MLO genes, indicating BoMLO12 (Bo4g188000.1) as a promising candidate. Notably, BoMLO12 showed high sequence similarity to Tamlo-R32 (Fig. 1A). Subsequently, a specific target site within the fourth exon of BoMLO12 was edited using CRISPR/Cas9 technology (Fig. 1B and C). Through Agrobacterium-mediated transformation, a transformation efficiency of 4.6% and an editing efficiency of 42.0% (3/7) were attained. Additionally, Sanger sequencing of T1 generation bomlo12 mutants (bomlo12-1, bomlo12-2, bomlo12-3) revealed six distinct mutation types, including insertions, deletions, and substitutions (Fig. 1C). Based on genetic characterization, inoculation assays were performed using three distinct pathogens: root dipping method for Fusarium wilt, needle puncturing method for black rot, and mycelial plug inoculation method of black spot. Three independent T2 bomlo12 mutant lines were subjected to standardized pathogenicity assays. Comparative disease assessment revealed that bomlo12 mutants exhibited statistically significant reductions in disease index (DI) values compared to wild-type (WT) controls, from 31.4 to 21.8 (significant) for black rot, from 65.4 to 15.3 (significant) for Fusarium wilt, and from 77.8 to 43.3 (significant) for black spot (Fig. 1D–F). Subsequently, we performed quantitative real-time PCR of the BoMLO12 and defense-related genes in both WT and mutant plants before and after inoculation. The results demonstrated that the expression of BoMLO12 gene showed a significant downregulation trend following inoculation. Similarly, the expression of the defense-related gene BoPR10 was significantly downregulated, while both BoPAL and BoNPR1 exhibited significant upregulation, which is consistent with previous research findings [5] (Fig. 1M and P). CRISPR/Cas9-mediated BoMLO12 editing enhances disease resistance and agronomic performance in B. oleracea. (A) Phylogenetic relationship of MLO homologs in B. oleracea and A. thaliana. (B) Schematic of CRISPR-Cas9 vector design targeting BoMLO12. (C) Diagram of BoMLO12 gene structures. Rectangular blocks represent the gene coding region; lines indicate intronic regions; dashed line illustrate the single guide RNA (sgRNA) target region (Target 1). The sequences of the WT and mutants are outlined below. The target sequence is underlined. The protospacer adjacent motif (PAM) sequence is highlighted in red, insertions in yellow, substitution in blue, and deletions marked with horizontal lines. (D–F) Phenotype and disease index of black rot, Fusarium wilt, and black spot in WT and knockout (bomlo12) plants. (G–I) Agronomic analysis of plant expansion and head height in WT and bomlo12 plants across three different stages. (J, K) The head weight of WT and bomlo12 plants. (L) Spread phenotype of WT and bomlo12 plants in harvestng stage. (M) The expression levels of bomlo12 were assessed in WT and knockout plants. (N, O) Investigation of agronomic traits in mutants, including taproot length, maximum outer leaf length, and leaf thickness. (P) The relative expression levels of defense-related genes were analyzed in the mutants before and after pathogen inoculation. For the disease inoculation tests, three knockout lines are selected for bomlo12. The whole inoculation experiment was repeated three times with similar results. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 indicate significant differences determined by two-tailed Student’s t-test. Scale bars: 5 cm. To evaluate the agronomic effects of BoMLO12 knockout, three T2 generation bomlo12 mutant lines and WT plants were grown under controlled conditions. We conducted biweekly field measurements of yield-related traits in both WT and bomlo12 plants across three developmental stages: rosette stage, heading stage, and harvesting stage. During the rosette stage, bomlo12 mutants showed a 10.7%–21.7% increase in plant expansion, with no significant change in head height observed. During the heading stage, compared to WT plants, all bomlo12 mutants (bomlo12-1, bomlo12-2, and bomlo12-3) exhibited significant increases, with plant expansion rising by 32.8%–48.6% and head height by 30.6%–38.2%. During the harvesting stage, bomlo12-1 displayed a significant 34.7% increase in plant expansion, while bomlo12-2 and bomlo12-3 showed significant head height increases of 38.4%–52.3% (Fig. 1G–I, L). Following this, outer leaves were removed to assess the fresh weight of individual heads. Notably, the individual head weight of bomlo12 mutants was ~2-fold greater than that of WT plants (Fig. 1J and K). And the results of root growth (maximum root length) and plant growth parameters (leaf thickness, leaf length, and plant expansion index), also demonstrated statistically significant upward trends (Fig. 1N and O). These findings suggest that BoMLO12 knockout enhances resistance to black rot, Fusarium wilt, and black spot while improving agronomic performance in B. oleracea through increased plant size. In summary, CRISPR/Cas9-mediated editing of the BoMLO12 gene successfully produced novel B. oleracea germplasms, demonstrating enhanced broad-spectrum resistance and significantly improved yield performance. These findings establish a foundation for breeding B. oleracea varieties with increased disease resistance and productivity. This work was supported by grants from the National Key R&D Program of China (2023YFD1201501), the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS), and the China Agriculture Research System of MOF and MARA (CARS-23). H.L. conceived and designed the experiments. Y.Z. and G.H. performed the experiments and data analysis. Y.Z. and G.H. wrote the manuscript. L.Y., Y.Y.Z., M.Z., Y.W, J.J., and X.Y. provided valuable advice on the manuscript. H.L. revised the manuscript. All authors contributed to the article and approved the submitted version. The authors confirm that the data supporting the findings of this study are available within the article. The authors declare no conflicts of interest.