Generation of novel bpm6 and dmr6 mutants with broad‐spectrum resistance using a modified CRISPR/Cas9 system in Brassica oleracea

Using an optimized CRISPR/Cas9 system to knock out the BTB-POZ and MATH domain gene BoBPM6 and the DOWNY MILDEW RESISTANCE 6 gene in Brassica oleracea resulted in new lines with broad-spectrum disease resistance. Brassica oleracea is an important biennial herbaceous species in the Cruciferae family. With an estimated 3.77 million hectares planted worldwide, these cole crops, for instance, cabbage, broccoli, and cauliflower, constitute significant agricultural resources (Li et al., 2024). In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been extensively applied in major crops, including rice, wheat, and potato. This technology can be utilized to regulate or disrupt gene expression to develop high-quality, disease-resistant, or stress-tolerant crops (Gao, 2021). However, the current low regeneration efficiency of B. oleracea has led to a limited Agrobacterium-mediated transformation efficiency of less than 1.0%, consequently impacting CRISPR/Cas9 based gene editing efficiency, with values as low as 12.9% being observed, compared to 68% in rice (Li et al., 2021; Zhou et al., 2022). This study aimed to enhance the application of CRISPR/Cas9 technology in B. oleracea. We observed that Cas9 from Streptococcus canis (ScCas9) requires a protospacer adjacent motif (PAM) sequence of 5'-NNG-3', after which any base can be selected. However, the editing efficiency is predicted to be greater when the base following the PAM sequence is a “T” (http://crispor.tefor.net/). To test whether a “T” following the PAM sequence for Streptococcus pyogenes Cas9 (SpCas9) can enhance the editing efficiency of CRISPR/Cas9 technology, we developed two separate expression vectors tailored for rice and cabbage to knock out the Phytoene Desaturase genes (OsPDS and BoPDS), leading to an albino phenotype in the edited plants. The sequences with a “T” immediately following the PAM sequence are designated “NGGT”, and others are referred to as “NGGN” (Figure 1A, B). The high-throughput sequencing and analysis (Pinello et al., 2016) results indicated that in rice line Nipponbare, compared with “NGGN,” “NGGT” exhibited an average increase in editing types of 28.6%, and the overall increase in editing efficiency was 13.8% (Figure 1C; Table S1). The results in cabbage inbred line M1-1 indicated that the plants edited with “‘NGGT” exhibited a greater degree of editing, representing a significant improvement in the editing efficiency from 20.4% to 68.7% (Figure 1D; Table S2). These findings suggest that selecting a 5'-NGGT-3' PAM sequence can enhance the diversity of editing types and improve the efficiency of CRISPR/Cas9 technology. Strategies employed for the updating and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) editing system (A) Schematic diagram of the CRISPR/Cas9 construct for PDS editing in rice. NGGN (N1-N3) and NGGT (T1-T3) represent the positions of the target sites. (B) Schematic diagram of the CRISPR/Cas9 construct for PDS editing in cabbage. NGGN (N1–N2) and NGGT (T1–T2) represent the positions of the target sites. (C) The top image shows rice in which the OsPDS gene was knocked out using “NGGN” and “NGGT,” and the unedited green plants are indicated in the red circle. The bottom diagrams display changes in rice editing types (blue line graph) and editing efficiency (yellow bar graph). (D) The top image shows that BoPDS was knocked out using “NGGN” and “NGGT”, with the “MOCK” group representing unedited cabbage plants. The bottom diagrams display changes in editing types (red line graph) and editing efficiency (blue bar graph). (E) Regeneration efficiency using GROWTH-REGULATING FACTOR (GRF)5–GRF-INTERACTING FACTOR (GIF)1–GRF5, GRF4–GIF1–GRF4, GRF5, and GIF1, and the blank control. (F and G) Diagram of BoDMR6 and BoBPM6 gene structures. Light pink and blue blocks represent the gene coding region; dark pink and blue blocks indicate intronic regions; red lines illustrate the single guide RNA (sgRNA) target region (target 1). The sequences of the wild-type (WT) and mutants are outlined below. The target sequence is underlined. The protospacer adjacent motif (PAM) sequence is highlighted in red, insertions in yellow, and deletions in blue. (H and I) Phenotype and disease index of black rot and clubroot in WT and knockout (bodmr6) plants. (J–L) Phenotype and disease index of Fusarium wilt, black rot, and clubroot in WT and knockout (bobpm6) plants. The cabbage sister lines M1-1 and M1-2 with slightly different genetic background were used for bodmr6 and bobpm6 knockout, respectively. For the disease inoculation tests, three knockout lines are selected for both bobpm6 and bodmr6, with 30 seedlings per line (10 seedlings for each of the three replicates). The whole inoculation experiment was repeated three times with similar results. **P < 0.01; ***P < 0.001; ****P < 0.0001 indicate significant differences determined by two-tailed Student's t-test. Scale bars, 10 mm. The lower regeneration efficiency has increased the workload of researchers, and previous studies have confirmed that the overexpression of both GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) proteins, especially the fusion GRF-GIFs, can significantly improve the regeneration efficiency of explants. Through bioinformatic analysis, we identified 19 GRFs and one GIF in the cabbage genome. Among them, two GRF5 proteins, two GRF4 proteins and one GIF1 protein were further studied (Figure S2). GRF and GIF proteins were linked by four alanine residues to form GRF5-GIF1-GRF5 and GRF4-GIF1-GRF4 cassettes (Debernardi et al., 2020). Subsequent experiments were conducted to assess the impact of GRF5-GIF1-GRF5, GRF4-GIF1-GRF4, GRF5, and GIF1 on the regenerative efficiency of B. oleracea (Figure S3). These experiments demonstrated that expression of the GRF5-GIF1-GRF5 fusion significantly increased the average regeneration efficiency by 55.2%, highlighting its potential for enhancing B. oleracea regeneration in Agrobacterium-mediated transformation (Figure 1E; Table S3). Various diseases have a significant impact on crop yields. For example, global cole productions are seriously threatened by three major diseases including Fusarium wilt, black rot and clubroot. Knocking out Susceptibility (S) genes has emerged as a rapid approach for achieving broad-spectrum disease resistance. We discovered a novel differentially expressed gene, BTB/POZ (Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger)-MATH 6 (BPM6), that is induced by Fusarium wilt and black rot of B. oleracea, suggesting that it may be a common S gene induced by various pathogens; this gene is related to fatty acid metabolism according to a previous study (Chen et al., 2013). In addition, Downy Mildew Resistant 6 (DMR6) has been identified as a conserved S gene in previous studies (Thomazella et al., 2021). To test whether BoBPM6 and BoDMR6 can be utilized to create broad-spectrum disease resistance in B. oleracea, we constructed vectors with the optimized system to knock out BoBPM6 and BoDMR6, achieving average transformation efficiencies of 5.5% and 8.2% and editing efficiencies (number of edited plants/number of positive plants) of 62.0% (13/21) and 62.5% (15/24), respectively. Sanger sequencing of randomly selected bodmr6 and bobpm6 mutants that are homozygotes in T1 generation revealed four and three distinct types of edits for both of them (Figure 1F, G). Subsequent inoculation experiments were conducted, with root dipping method for Fusarium wilt, spraying method for black rot and root wounding method for clubroot. The disease index (DI) was calculated as: DI = Σ (the corresponding disease grade × the number of leaves or roots with a particular grade) × 100/(the total number of tested leaves or roots × the highest grade) (Lv et al., 2020; Zhang et al., 2024). Compared to DIs for wild-type (WT) plants, infection of bodmr6 with Fusarium wilt, black rot and clubroot resulted in decreases in the DIs from 79.0 to 78.4, from 79.3 to 55.1 (significant) and from 90.7 to 57.6 (significant), respectively (Figures 1H, I, S1A), and for bobpm6, Fusarium wilt, black rot, and clubroot infection resulted in decreases in the DIs from 65.4 to 14.5 (significant), from 53.8 to 20.9 (significant), and from 63.1 to 55.7, respectively (Figures 1J–L, S1B). In summary, the application of the optimized CRISPR/Cas9 technology to knock out the BoBPM6 and BoDMR6 genes resulted in the creation of new germplasms with broad-spectrum disease resistance. Our findings provide robust support for gene editing and disease resistance breeding techniques in B. oleracea. This work was supported by grants from the National Key R&D Program of China (2023YFE0111400), the Key Technology R&D Program of Jiangsu Province (BE2023366), S&T Program of Hebei Province (22322912D), the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS), the China Agriculture Research System of MOF and MARA (CARS-23), and Wulanchabu City's Challenge and Recruitment Project (2022JB006). The work was also partly supported by the Russian Ministry of Science and Higher Education (No. 075-15-2023-582). The authors declare no conflict of interest. H.L. conceived and designed the study. Y.L.Z. and J.L. performed the experiments. H.L. and Y.L.Z. wrote and revised the manuscript. Y.L., H.M., J.J., Y.W., M.Z., L.Y., Z.F., J.L., C.Z., L.L., M.L., V.T., and Y.Y.Z. analyzed the data and revised the manuscript. All authors have read and approved the final manuscript. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13842/suppinfo Figure S1. Phenotype of the clubroot in wild-type (WT) and knockout (bodmr6 and bobpm6) plants Figure S2. Phylogenetic analysis of homologs of GROWTH-REGULATING FACTOR (GRF) in Brassica oleracea (Bo) and Arabidopsis thaliana (At) Figure S3. Illustration of the GROWTH-REGULATING FACTOR (GRF)5-GRF-INTERACTING FACTOR (GIF)1-GRF5 and GRF4-GIF1-GRF4 fusion proteins and the constructed GRF5 and GIF1 proteins Table S1. The editing types and efficiency in rice determined by high-throughput sequencing Table S2. The editing types and efficiency in cabbage determined by high-throughput sequencing Table S3. The regeneration efficiency data of GROWTH-REGULATING FACTOR (GRF)-INTERACTING FACTOR (GIF)1, GRF5, GRF5-GIF1-GRF5, and GRF4-GIF1-GRF4 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.