Genome editing of <i>RECEPTOR‐LIKE KINASE 902</i> confers resistance to necrotrophic fungal pathogens in <i>Brassica napus</i> without growth penalties

Abstract

Rapeseed (Brassica napus) is a major source of plant edible oil around the world. However, its production has been continuously threatened by the necrotrophic pathogens Sclerotinia sclerotiorum and Botrytis cinera, causing the stem rot disease (SRD) and the grey mould disease (GMD), respectively. Considering a phylogenetically close relationship between S. sclerotiorum and B. cinerea, a similar pathogenic strategy adopted by S. sclerotiorum and B. cinerea and putative genes simultaneously controlling SRD and GMD resistance likely exist in B. napus. To exploit these resistance genes from B. napus, the detached leaves of 230 genome-resequencing rapeseed accessions were respectively inoculated with S. sclerotiorum and B. cinerea mycelial plugs under controlled conditions, and the disease lesion sizes were independently conducted GWAS using 2 779 265 SNPs with a minor allele frequency of ≥0.05 and three different models (Figure 1a, Figures S1 and S2) (Cui et al., 2023; He et al., 2022). The results showed that a significant region on the chromosome A05 was repeatedly identified and strongly associated with both SRD and GMD (Figure 1a, Figure S1). In this region, the two most significant SNPs (SNP_17,088,971 and SNP_17,088,560) located in the third exon of BnaA05g22400D defined two haplotypes (Figure 1b,c). The two haplotypes (Hap_1 and Hap_2) divided 170 accessions into two groups which have a significant difference in both SRD and GMD lesion sizes (Figure 1c). Hap_2 (AG), causing the amino acid changes A344T and D570E in BnaA05g22400D protein, displayed reduced SRD and GMD lesions than that of Hap_1 (GC, Figure 1c). Synteny analysis revealed that the Arabidopsis ortholog of BnaA05g22400D protein is RECEPTOR-LIKE KINASE 902 (RLK902) and their protein sequence identity is 82% (Figures S3a and S4). The deduced protein of BnaA05g22400D contained the typical domains of RLK proteins and was localized at the plasma membrane revealed by transient expression in the protoplasts of Arabidopsis (Figure S3a,b). Thus, we designated BnaA05g22400D as BnaA05.RLK902. BnaA05.RLK902 expressed in most tissues of B. napus and had the highest levels in the bud revealed by quantitative PCR in both susceptible and resistant B. napus lines (Figure S3c). In the leaf, BnaA05.RLK902 expression was strongly induced by S. sclerotiorum or B. cinerea inoculation in the disease-susceptible accession Zhongshuang11 (ZS11), but down-regulated in the resistant line Zhongyou821 (ZY821), suggesting an essential role of BnaA05.RLK902 for S. sclerotiorum or B. cinerea pathogenicity (Figure S3d). To validate whether BnaA05.RLK902 is the target gene, we firstly investigated the effects of RLK902 loss-of-function mutation on SRD and GMD resistance in Arabidopsis. We found that the Arabidopsis mutant rlk902 (SALK No. CS410869) displayed an enhanced resistance to both SRD and GMD compared with the wild type (Figure 1d, Figure S5). Furthermore, we respectively expressed two haplotypes of BnaA05.RLK902 in Arabidopsis wild-type and rlk902 mutant. The results showed that the plants of Arabidopsis mutant rlk902 expressing the Hap_1 of BnaA05.RLK902 restored the SRD or the GMD susceptibility to the wild-type level whereas the Hap_2 of BnaA05.RLK902 failed. The wild-type plants expressing Hap_2 of BnaA05.RLK902 exhibited enhanced SRD and GMD resistance than expressing Hap_1 of BnaA05.RLK902, but the level was still lower than the mutant rlk902 (Figure 1d, Figure S5). These results strongly support that BnaA05.RLK902 negatively regulates plant resistance to SRD and GMD, and genome editing to knock out BnaA05.RLK902 could significantly enhance resistance to both SRD and GMD. Interestingly, RLK902 contributes to resistance against the hemi-biotrophic bacterial pathogen Pseudomonas syringae in Arabidopsis and this resistance is mediated by another RLK protein BSK1 (Zhao et al., 2019) which also confers resistance to the biotrophic powdery mildew pathogen (Shi et al., 2022). Therefore, it appears that RLK902 plays opposite roles in the regulation of plant immunity against necrotrophic and biotrophic pathogens and this phenomenon is commonly observed in plants. To decipher the underlying disease resistance mechanism, we performed transcriptome sequencing of Arabidopsis wild-type and rlk902 plants before or after S. sclerotiorum inoculation. The generated differentially expressed genes (DEGs) between rlk902 and wild-type plants at 24 post-inoculation (hpi) were used for KEGG enrichment analysis, in which the terms ‘response to jasmonic acid’ and ‘defense response to fungus’ are most significantly enriched (Figure 1e). Furthermore, the genes involved in JA biosynthesis, modification and signalling, and the downstream defence responses including biosynthesis of lignin, camalexin and ROS were coordinately up-regulated in the rlk902 mutant than in the wild type (Figure 1f, Table S1). These results supported that the JA-mediated plant immunity was activated in the rlk902 mutant upon pathogen infections. Then, we introduced the mutation jar1-1 (jasmonate resistant 1-1), of which the JA-Ile biosynthesis and JA-Ile-mediated immunity were nearly abolished (Staswick et al., 2002), into the rlk902 mutant and created the double mutant rlk902 jar1-1 for further validating whether JA-mediated immunity was responsible for the enhanced resistance of rlk902. As expected, the double mutant rlk902 jar1-1 restored the susceptibility of either SRD or GMD to the wild-type level (Figure 1d, Figure S5). Taken together, the activation of JA-mediated plant immunity is responsible for the enhanced SRD and GMD resistance of the rlk902 mutant. To confirm whether knocking out BnaA05.RLK902 improves the resistance to SRD and GMD, we used the CRISPR-Cas9 system to knock out BnaA05.RLK902 in ZS11. Two guide sequences (sgRNA1 and sgRNA2) in the first exon were designed as the editing targets (Figure 1g). From the T2 transgenic plants, we obtained two homozygous mutated alleles of BnaA05.RLK902 with frameshift in the coding region resulted from insertion of an additional base A into the sgRNA1 or the sgRNA2 (Figure 1g). Then, the detached leaves were inoculated to test their SRD and GMD resistance and as expected, the SRD and GMD resistance of the BnaA05.rlk902 plants were significantly increased (Figure 1h,i). Furthermore, the key agronomic traits were not significantly altered in the BnaA05.rlk902 plants in the field conditions (Figure 1j,k). Therefore, genome editing of BnaA05.RLK902 could efficiently improve plant resistance both SRD and GMD without growth and development penalties in B. napus. Our study identified a natural variation of RLK902 conferring resistance to SRD and GMD in a natural population of B. napus. Importantly, knocking out RLK902 by genome editing displayed a higher level of disease resistance in B. napus. We also elucidated that JA-mediated immunity underlay this resistance of rlk902 mutant. Therefore, this study provides a valuable gene and materials for genetic improvement of SRD and GMD resistance in B. napus and new insights into understanding the mechanisms of the plant resistance against necrotrophic pathogens. The work was supported by the National Natural Science Foundation of China (U20A2034), National Biotechnology Breeding Program of China (2023ZD04042), China Agriculture Research System of MOF and MARA (CARS-12) and Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2021-OCRI). The authors declare no conflict of interest. L.L. and S.L. designed the study. C.Z., Yi.Z., L.G. and L.Z. performed the experiments. M.X. and C.T. performed bioinformatic analysis. X.C., J.L., Yu.Z. and Yi.Z. measured the phenotypic data. Y.L. maintained population materials. C.Z. and X.C. wrote the manuscript, and L.L., Q.H. and S.L. revised the manuscript. All authors read and approved the final manuscript. Figure S1 Manhattan plot of the GWAS. Three individual models were used here. (a) GWAS of SRD lesion sizes at 36 hpi and 48 hpi. (b) GWAS of GMD lesion sizes at 48 hpi and 60 hpi. Figure S2 The phenotype distribution and QQ plot analysis in GWAS. (a) The phenotype distribution of SRD lesion sizes at 36 hpi, 48 hpi, and GMD lesion sizes at 48 hpi, 60 hpi. (b) QQ plots of the three individual models, including GLM, MLM, and FarmCPU in GWAS of SRD and GMD. Figure S3 Molecular characteristics of RLK902. (a) The conserved domains and colinear analysis of RLK902 between A. thaliana and B. napus. (b) The subcellular localization of BnaA05.RLK902 in the chloroplasts of A. thaliana. (c) The expression levels of BnaA05.RLK902 in various tissues of susceptible (S) and resistant (R) B. napus lines. (d) The expression of BnaA05.RLK902 responding to pathogen infection in leaf of susceptible (S) and resistant (R) B. napus lines. Figure S4 The protein sequence alignment of AtRLK902 and BnaA05.RLK902. Figure S5 Disease symptoms on the inoculated leaves at 48 hpi to S. sclerotiorum (a) and 60 hpi to B. cinerea (b). Scale bar (a, b) = 1 cm. Table S1 The FPKM value of genes involved in JA pathway and defence response in WT and rlk902 mutant. 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