Role of  BjuA03.BNT1  in enhancing clubroot resistance in resynthesized  Brassica juncea  L.

doi:10.21203/rs.3.rs-3610592/v1

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Role of BjuA03.BNT1 in enhancing clubroot resistance in resynthesized Brassica juncea L.

https://doi.org/10.21203/rs.3.rs-3610592/v1

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In recent years, clubroot disease has emerged as a significant impediment to rapeseed production. Breeding new varieties resistant to clubroot is the most effective method for disease management. However, the pool of clubroot-resistant germplasm of rapeseed remains notably limited. In order to address this challenge, we synthesized the clubroot-resistant mustard, CT19, via remote hybridization, and subsequently an F₂ segregating population was created by intercrossing CT19 with a clubroot-susceptible germplasm CS15. A major-effect QTL qCRa3-1 on chromosome A03 was identified through QTL scanning, demonstrating a substantial association with clubroot resistance. Transcriptome analyses of CT19 and CS15 revealed that the mechanisms conferring resistance to Plasmodiophora brassica likely involved the regulation of flavonoid metabolism, fatty acid metabolism, and sulfur metabolism. By combining the results from transcriptome analyses, QTL mapping and gene sequencing, a candidate gene BjuA03.BNT1, encoding TIR-NBS-LRR protein, was obtained within the region of qCRa3-1. Intriguingly, an insertion of a base T in the CDS of BjuA03.BNT1 in CT19 was discovered, which caused an alteration within the conserved domain TIR. Overexpression of BjuA03.BNT1 of CT19 notably enhanced the resistance to clubroot while overexpression of BjuA03.BNT1 of CS15 augmented susceptibility to the disease in Arabidopsis. Our investigations revealed that BjuA03.BNT1 regulated the resistance to clubroot by modulating fatty acid synthesis and the structure of cell wall. The one base insertion in the CDS of BjuA03.BNT1 serves as a pivotal determinant in conferring resistance to clubroot. These findings bear significant implications for molecular breeding aimed at enhancing clubroot resistance in rapeseed.

Brassica juncea

clubroot

Plasmodiophora brassica

QTL

BjuA03.BNT1

BjuA03.BNT1 regulates the resistance to clubroot by modulating fatty acid synthesis and the structure of cell wall in Brassica juncea, and a T base insertion in the CDS of BjuA03.BNT1 serves as a pivotal determinant in conferring resistance to clubroot.

Clubroot disease, caused by the soilborne pathogen Plasmodiophora brassicae, is one of the most devastating diseases of cruciferous crops, including Brassica napus, Brassica juncea, Brassica oleracea and Brassica rapa (Guo et al. 2022). This disease has led to substantial economic losses on a global scale, impacting regions across Asia, Europe, Australia, and the Americas (Vañó et al. 2023). The incidence rate of clubroot disease in rapeseed field reaches approximately 17% during the seedling stage and maintains an average of 15% during the adult stage, resulting in yield losses of 20–30% on average and surpassing 60% in severely affected fields (Ochoa et al. 2023). The pervasive distribution of clubroot poses a formidable and contemporary challenge to rapeseed cultivation.

A number of genes or quantitative trait loci (QTL) associated with clubroot-resistance have been identified in Cruciferae crops. In B. rapa, more than 28 QTLs associated with clubroot resistance have been localized on five chromosomes, namely A01, A02, A03, A06, and A08 (Sakamoto et al. 2008; Chen et al. 2013; Hatakeyama et al. 2013; Chu et al. 2014; Huang et al. 2017; Yu et al. 2017; Pang et al. 2018; Huang 2019; Laila et al. 2019; Zhu et al. 2019; Cui et al. 2020; Karim et al. 2020; Paul et al. 2021; Wang et al. 2022). Among these QTLs, 12 QTLs (Crr3, Rcr1, Rcr2, Rcr4, Rcr5, PbBa3.1, CRk, CRd, CRb, CRa, CRaki, and PbBp3.3) are located on chromosome A03. These loci are interlinked, suggesting that clubroot-resistance loci originating from cabbage are relatively homogeneous. Among these QTLs, only the genes at the CRa and Crr1 loci have been cloned (Takken and Tameling 2009; Chen et al. 2013), both of which have the TIR-NBS-LRR (Toll-Interleukin-1 Receptor-Nucleotide-Binding Site-Leucine-Rich-Repeat) structural domain, a characteristic feature of resistance genes (R genes). The majority of the proteins encoded by R genes contain conserved Nucleotide-Binding Site (NBS) and Leucine-Rich-Repeat (LRR) sequences. Additionally, some NBS-LRR-type R proteins in dicotyledonous plants have a TIR (Toll-Interleukin-1 Receptor) motif. It has been shown that R genes with the TIR-NBS-LRR structure play pivotal roles in modulating plant immune responses against a broad spectrum of pathogenic microorganisms, thereby enhancing plant resistance to these pathogens (Hejna et al. 2019; Schulze et al. 2022; Ge et al. 2023; Wang et al. 2023). In B. oleracea, a fewer number of clubroot-resistance genes have been identified in the C genome (Benoit S. Landry and 1992; Voorrips et al. 1997; Rocherieux et al. 2004; Moriguchi et al. 2010; Nagaoka et al. 2010; Lee et al. 2016; Dakouri et al. 2018; Pang et al. 2018; Farid et al. 2020). Only one disease resistance gene, Rcr6, has been identified in B. juncea (Chang et al. 2019).

While several loci/genes conferring resistance to clubroot have been discerned across various cultivars, resulting in the breeding of a number of clubroot-resistant rapeseed cultivars utilizing these resistance loci, the resistance exhibited by existing rapeseed cultivars appears to be notably uniform. The resistance of these cultivars gradually deteriorates, especially after prolonged cultivation of a single resistant cultivar or reliance on a cultivar possessing a solitary resistance locus within a specific region (LeBoldus et al. 2012). Notably, in Canada, the early resistant varieties have succumbed to a loss of resistance against clubroot disease due to the homogeneity of resistance loci and continual cultivation of a single variety, thereby fostering the evolution of more virulent strains of P. brassicae (Askarian et al. 2021). Therefore, in order to alleviate the damage of rapeseed by clubroot disease and address the challenge of resistance loss, it becomes imperative to explore new resistance genes within closely related species. Consequently, in this study, we conducted a distant hybridization, crossing clubroot-resistant cabbage with clubroot-resistant black mustard, subsequently synthesizing a high-generation clubroot disease-resistant B. juncea CT19 through embryo rescue and doubling of colchicine. In order to mine the resistance gene of CT19 in B. juncea and comprehend the mechanism underlying its resistance to clubroot, a F₂ segregating population was established by intercrossing clubroot-resistant germplasm CT19 and CS15, which is susceptible to clubroot. This endeavor led to the identification of a major effect QTL qCRa3-1 on chromosome A03, and the identification of the pivotal candidate gene BjuA03.BNT1, which was significantly associated with resistance to clubroot. Our study offers a solid theoretical foundation and genetic reservoirs pivotal for the development of new rapeseed varieties characterized by high yield, superior quality, and robust, broad-spectrum resistance against clubroot.

Plant materials and growth condition

This study utilized inbred lines of clubroot-resistant mustard (CT19), clubroot-susceptible mustard (CS15), and an F₂ segregating population composed of 172 lines, which was derived from the cross between CT19 and CS15. The plants for experiments with two parental lines were grown in a greenhouse environment under controlled conditions, maintaining a temperature of 25°C for 16 hours during the day and 20°C for 8 hours during the night.

Microstructural examination of clubroot-resistant and susceptible parental varieties

At the seventh leaf stage, the root tissues of CT19 and CS15 were sampled at day 7, 14, and 21 after inoculation with the P4 physiological strain of P. brassicae. The samples were fixed in 2.5% glutaraldehyde solution for two hours and subsequently embedded in a pure acetone/embedding solution mixture (2:1). The fixed samples were sliced using a Leica microtome, and then the slices were stained with toluidine blue. The microstructure of the slices was examined using a ZEISS fluorescence microscope.

Identification of resistance to P lasmodiophora brassicae

The seeds of CT19, CS15, and 172 lines of the F₂ populations were germinated on moist filter paper. After four days, the seedlings exhibiting uniform growth were selected and transplanted into nutrient soil (substrate: perlite: vermiculite, 3:1:1). Upon reaching the five-leaf stage, the seedlings were injected at the base of the plant roots with a 5 ml spore suspension of the P4 physiological strain of P. brassicae with a concentration of 1 × 10⁷ spores/ml. At 40 days after inoculation of P. brassicae, tumor formation in the roots was observed to identify the resistance phenotype.

Simplified genome sequencing and data analysis

Genomic DNA of CT19, CS15, and 172 lines of the F₂ populations was extracted using the CTAB method. Subsequently, the DNA was fragmented using restriction endonucleases MseⅠ and SacⅠ, followed by ligation of specific adapters to the enzymatically cleaved fragments. The purified products were amplified and then subjected to electrophoresis using Bio-Rad Certified Megabase Agarose low pressure overnight electrophoresis. The DNA library was obtained by collecting the 300–400 bp fragments. Library quality control Agilent 2100 was utilized to evaluate the insert size to ensure that there was no header contamination. Paired-end 150 bp (PE150) sequencing was performed using the Illumina HiSeq platform.

The simplified genome sequencing data obtained in the previous step were analyzed as follows:

(1) Quality control and statistics of sequencing data: The fastp software was used to perform quality control on the raw data, by which the joints, low-quality bases and reads smaller than 50 bp in the reads were removed, and then the distribution of the base mass number and GC content of the sequencing data were counted to ensure its quality and suitability for subsequent analyses.

(2) Bioinformatics analysis of sequencing data: Firstly, the MEM algorithm of the BWA (Burrow-Wheeler Aligner, version 0.7.15-r1140) software was used to compare the quality-controlled data to the reference genome of mustard (Braju_tum_V2.0, http://brassicadb.cn/#/Download/). The resulting SAM files were converted into BAM files and sorted using samtools (version 1.3.1). The sorted bam files were used to compute sequencing coverage and depth. Coverage refers to the proportion of bases covered by the sequencing data in the reference genome to the entire reference genome, while depth denotes the number of times each base in the reference genome is covered, generally measured by the number of reads. The higher the coverage, the more variants can be detected, and the higher the depth, the higher the accuracy of the variant loci. Variant detection was executed using the HaplotypeCaller module and GenotypeGVCFs module of the GETK software (Genome Analysis ToolKit, version 3.7), and the results included SNPs and InDel. The detected variants were subsequently annotated by the ANNOVAR software (version 2016 Feb1), and their effects on gene function were predicted.

(3) Construction of genetic linkage mapping: Firstly, the parental genotypes were inferred based on the linkage between the markers of the progeny. Subsequently, the genotypes of the progeny were designated as A (CT19) and B (CS15). The truthfulness of the parents was verified based on the actual genotypes of the resistant and susceptible parents. Partial correction of some genotypes was achieved through the application of the Hidden Markov Model (HMM). Finally, the genotypes of the progeny were evaluated by using the MSTMap software to assess the recombination rate among markers, and the genetic map distance among markers was calculated using Kosambi.

Transcriptome sequencing and candidate gene analysis

At the three leaf stage, roots of the clubroot-resistant material CT19 and clubroot-susceptible material CS15 were sampled at 7, 14, and 21 days after inoculation with the P4 physiological strain of P. brassicae and promptly snap-frozen in liquid nitrogen. Three biological replicates were used. The RNA of these 18 samples was extracted with the RNA prep Pure Plant Kit (CHIGEN) and subsequently sent to Shanghai Meiji Biotechnology Company for transcriptome sequencing. Paired-end 150 bp (PE150) sequencing was performed using the Illumina HiSeq platform.

The raw reads of these 18 samples were filtered and aligned to the reference genome of mustard (http://39.100.233.196:82/download_genome/Brassica_Genome_data/Braju_tum_V2.0) using HISAT2 v2.1.0. The gene expression levels were expressed as FPKM (fragments per kilobase per million reads). Differential expression analysis was performed with DESeq2 v1.10.1. Genes were identified as differentially expressed genes (DEGs) if they exhibited a |log2 FC| ≥ 1 and an adjusted p-value (FDR) < 0.01. KEGG enrichment analysis was performed on the DEGs using hypergeometric distribution phyper and the ClusterProfiler package in R.

Amplification of the candidate gene

The candidate gene sequence of CT19 and CS15 was amplified using the DNA of CT19 and CS15, respectively. The coding sequence (CDS) was amplified using the cDNA of the two parents. The open reading frames (ORFs) were predicted using the ORF Finder tool online (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The CDS sequences were translated into proteins by the DNAMAN5.0 software.

Vector construction and genetic transformation in Arabidopsis

To overexpress the candidate gene, the CDS of the gene was constructed into a PC1300-DsRed vector containing the 2*CaMV35S promoter by homologous recombination. The Agrobacterium tumefaciens strain GV3101, carrying the recombinant vector, was utilized for genetically transforming Arabidopsis plants through the floral dip method. Subsequently, the seeds of the T₀-generation plants were collected. The seeds with the DsRed fluorescent protein were screened to ascertain positive transformations by the LUYOR-3415RG dual-wavelength excitation light source. Then, positive seeds were further verified by Hygromycin medium and the T₁-generation seeds were obtained, which were grown until pure and positive T₃ generation lines were obtained. The T₃ generation was subsequently employed for phenotypic identification and gene expression profiling.

Creation of mustard with resistance to clubroot

To augment the genetic reservoir of clubroot resistance, a new germplasm CT19 with resistance to clubroot was created by distant hybridization between a clubroot-resistant cabbage and black mustard. Morphologically, CT19 exhibits fundamental mustard characteristics, featuring green leaves, non-stem-holding moss leaves, and predominantly lobed leaves with distinct petioles and small siliques, (Figs. S1A-C). The results showed that the mustard CT19 was successfully created by remote hybridization. At the five leaf stage, CT19 and CS15 were inoculated with 5 ml spore suspension of the P4 physiological strain of P. brassicae (1×10⁸ spores/ml). It was found that CT19 did not exhibit any clubroot phenotypes after inoculation with P. brassicae (Figs. 1A, B), whereas CS15 displayed evident clubroot phenotypes at 14 days post-inoculation with P. brassicae (Fig. 1C). These results indicated that CT19 exhibited strong resistance to clubroot, and CS15 was extremely sensitive to clubroot.

Ultrastructural examination of resistant and susceptible germplasm after inoculation with P. brassicae

P. brassicae infestation in plants progresses through three distinct phases. The first phase is root hair/cortex infestation, which lasts from the beginning of infestation to the seventh day after inoculation with P. brassicae. The second phase is cortex infestation, which generally lasts from the seventh day to the fourteenth day after infestation. The third phase is root nodule development period, which is from the fourteenth day of infection to the final root rot and disintegration. In order to have a more in-depth understanding of the P. brassicae infestation process in both the resistant (CT19) and susceptible (CS15) clubroot materials, semi-thin sections of the roots were observed at 7, 14, and 21 days following P. brassicae inoculation. There was no significant difference in cell morphology between the infected and normal CS15 at 7 days after inoculation. However, enlarged cells and sporangium clusters were observed in CS15 at 14 and 20 days after inoculation with P. brassicae. (Figs. 2A1-F1). There were no significantly enlarged cells in the resistant CT19 at any stage of inoculation, indicating that the infection of CT19 did not progress to the stage of cortical infection (Figs. 2A-F).

In order to further explore the difference of cell wall structure between resistant- and susceptible-germplasm in response to P. brassicae infestation, ultrathin sections of resistant and susceptible materials were observed. Relative to the control that was not inoculated with P. brassicae, the cell walls of the susceptible CS15 were significantly thinner at 7 and 14 days after P. brassicae inoculation (Figs. 3A1-D3), while a significant thickening was observed at 21 days post-inoculation (Figs. 3E1-F3). Contrastingly, in CT19, the resistant germplasm demonstrated a diametrically opposite pattern, with significantly thickened cell walls at 7 and 14 days after inoculation, followed by significant thinning at 21 days (Figs. 4A1-F3). These observations suggest that the regulation of cell wall thickness during P. brassicae infestation might be a key element in conferring resistance to clubroot disease.

Localization of clubroot resistant loci

To unravel the genetic basis of resistance in the clubroot-resistant mustard CT19 and decipher its underlying resistance mechanisms, an F₂ population was constructed with CT19 and the susceptible CS15 (Fig. 1) as the parental lines. The F₂ population consisting of 172 plants underwent simplified genome sequencing, and the sequences were aligned to the reference genome of mustard (Braju_tum_V2.0, http://brassicadb.cn/#/Download/). A total of 111,124 variant loci were obtained, including 97,876 SNPs and 13,248 InDel (Figs. 5A, B). The average genome coverage and coverage depth of the samples were determined as 4.27% and 19.92%, respectively (Fig. 5C). Subsequently, these 172 F_2:3 lines were characterized for clubroot resistance using the P4 physiological strain of P. brassicae. The result showed that 103 and 63 lines were resistant and susceptible to clubroot, respectively. Combining the variation information and clubroot resistance phenotypes of the F₂ population, a high-density genetic linkage map was constructed. The length of the linkage groups on each chromosome ranged from 39.022 to 114.001 Mb, the total number of markers per linkage group ranged from 2,806 to 14,949, and the average distance between neighboring markers ranged from 0.009 cM to 0.027 cM (Figs. 5D, E). Notably, the genetic map demonstrated sound collinearity with the reference genome (Fig. 5F), implying that this genetic linkage map has high precision and can be used for QTL localization.

Combining with the constructed genetic map of the F₂ population, QTL mapping of clubroot resistance trait in the 172 F₂ populations was analyzed using the composite interval mapping method by the QTL Cartographer software. A major QTL, qCRa3-1, was found in the 25.09 Mb − 25.54 Mb region of chromosome A03, which was significantly associated with clubroot disease resistance, accounting for 74.30% of the phenotype contribution rate (Fig. 6). Additionally, another QTL with a 6% phenotypic contribution was obtained in the range of 55.69 Mb-56.04 Mb on chromosome B02 (Fig. 6). A QTL with a contribution rate of 2.5% was obtained on chromosome A05, with a physical location between 35.28 Mb and 35.84 Mb (Fig. 6). The high level of phenotypic variance explained by qCRa3-1 underlines its robustness and potential significance for the identification of key candidate genes in further studies.

Transcriptome analysis of resistant and susceptible parents post P. brassicae inoculation

In order to further investigate the molecular mechanism of resistant and susceptible parents in response to clubroot disease infestation, root transcriptome sequencing was conducted at 7, 14, and 21 days after P. brassicae inoculation. A total of 8,434, 10,103, and 9,339 differentially expressed genes (DEGs) were obtained between resistant and susceptible parents, respectively. Among these DGEs, 3,817 overlapped across all three periods. Further examination of DEGs in the clubroot-resistant CT19 revealed 803 and 4,715 DEGs at 7 days after P. brassicae inoculation relative to 14 and 21 days, respectively (Fig. S3B). The highest number of DEGs (10,866) was found at 14 and 20 days after P. brassicae inoculation. The number of DEGs in the susceptible material at 7 days after P. brassicae inoculation was 1,134 and 2,473 relative to 14 and 20 days, respectively (Fig. S3C). The number of DEGs between 14 and 20 days after P. brassicae inoculation in the susceptible material was 3,251, lower than that in the resistant parent. These observations suggest that the 14-day post-inoculation period may be pivotal for the expression of disease resistant genes.

KEGG enrichment analysis of DEGs between resistant and susceptible parents at 7 days after P. brassica inoculation revealed significant enrichment in pathways like flavonoid metabolism, fatty acid metabolism, sulfur metabolism, and amino acid metabolism (Fig. 7A). The DEGs enriched at 14 days after P. brassica inoculation partially overlapped with those at 7 days after inoculation, mainly involved in flavonoid metabolism, fatty acid metabolism, and sulfur metabolism, as well as some specific metabolic pathways such as galactose metabolism (Fig. 7B). The DEGs at 21 days after P. brassica inoculation were also enriched in pathways involved in flavonoid metabolism, fatty acid metabolism, and sulfur metabolism. However, unique pathways such as photosynthesis, zeatin synthesis, and pyruvate metabolism and arachidonic acid metabolism were also evident compared with those of the previous two periods (Fig. 7C). These findings indicate that the clubroot-resistant material responds to P. brassicae infestation primarily through flavonoid metabolism, fatty acid metabolism, and sulfur metabolism.

Screening of clubroot resistance candidate genes

To identify potential candidate genes responsible for clubroot resistance, an integrated analysis of QTL localization and transcriptome data was performed. A total of 22 genes in the significant disease resistance locus qCRa3-1 on chromosome A03 displayed noteworthy expression changes at 7, 14, and 21 days after P. brassica inoculation (Fig. 8). Among these genes, two genes, BjuVA03G52300 and BjuVA03G52310, contain a TNL (Toll/Interleukin1 receptor-nucleotide binding site leucine-rich repeat, TIR-NBS-LRR) structure. Specifically, BjuVA03G52300 exhibited significantly higher expression in clubroot disease-resistant material CT19 compared to the susceptible material CS15 (Fig. 8). The sequence of this gene between the resistant and susceptible parents was also analyzed, and it was found that there was a one-base T insertion in the CDS sequence of BjuVA03G52300-R in the disease-resistant parent CT19, leading to a frameshift mutation and altering the conserved TIR domain of BjuVA03G52300 (Fig. 9). Therefore, BjuVA03G52300, herein referred to as BjuA03.BNT1, emerged as a key candidate gene for clubroot resistance.

Functional validation of the clubroot resistance candidate gene BjuA03.BNT1

The CDS sequence of BjuA03.BNT1 from the resistant and susceptible parents was cloned into 2*CaMV35S overexpression vectors and transformed into wild-type Arabidopsis. Notably, Arabidopsis lines overexpressing BjuA03.BNT1 from the resistant material CT19 (BjuA03.BNT1-R-OE) exhibited significantly increased resistance to clubroot compared with that of the wild type. Conversely, overexpression of BjuA03.BNT1 from the susceptible material CS15 (BjuA03.BNT1-S-OE) in Arabidopsis led to enhanced susceptibility to clubroot (Fig. 10). These findings strongly indicate the regulatory role of BjuA03.BNT1 in conferring resistance to clubroot. Moreover, the observed sequence disparity of BjuA03.BNT1 in the two parental lines demonstrates its differential function in mediating resistance to clubroot.

Numerous Brassica species hold considerable economic significance and occupy strategic positions, such as B. rape, B. napus, cabbage, and radish. In recent years, clubroot disease has significantly impacted various Brassica species (Ando et al. 2023). Over 20 countries globally have reported incidents of clubroot disease, resulting in annual yield losses of approximately 10–15% (Pageau et al. 2006; Javed et al. 2023). Currently, various methods have been developed to control clubroot disease, such as chemical control, biological control, and field management. However, these measures are labor-intensive and potentially ecologically damaging, rendering them unsustainable. Hence, breeding resistant varieties stands as the most effective strategy. Therefore, exploring resistance genes to clubroot disease and developing resistant germplasms assumes pivotal importance (Guo et al. 2022). In this study, the clubroot-resistant B.jucu CT19, which is derived from the crossing between clubroot-resistant cabbage and black mustard, was used as the foundation to create germplasm for breeding clubroot-resistant varieties. Upon assessment of resistance to clubroot disease, CT19 exhibited an extraordinarily resistant phenotype compared to CS15 (Fig. 1). Furthermore, to advance practical application in breeding, we localized the clubroot-resistant gene in CT19 through simplified genome sequencing of 172 F₂ populations. We successfully identified a significant QTL on chromosomes A03, A05, and B02, respectively (Fig. 6). Notably, prior studies have pinpointed most clubroot disease resistance loci such as CRa, CRk, CRq, CRd, CR6b, Rcr1, Rcr2, Rcr4, Rcr5, and Rcr3 predominantly on chromosome A03 (Hasan et al. 2021). The QTL identified in this study, qCRa3-1, was located on chromosome A03 between 25,096,779 bp and 25,547,141 bp, overlapping partially with Rcr4, CRb, Rcr2, and CRa. We identified 22 candidate genes within the qCRa3-1 region that exhibited differential expression in the resistant and susceptible parents post-inoculation with P. brassica by combining with the QTL locus and transcriptome data (Fig. 8). Among these, BjuA03.BNT1 stands out as a disease resistance gene with the TNL structure. Notably, this gene exhibits sequence disparity between the two parental lines. Its homologous counterpart, At5g11250 (AtBNT1) of BjuA03.BNT1 in Arabidopsis, encodes a TIR-NBS-LRR protein that is crucial in various biotic and/or abiotic stress responses by regulating hormones levels (Sarazin et al. 2015). Our study showed that the overexpression of BjuA03.BNT1 from CT19 augmented resistance to clubroot in Arabidopsis, further underscoring the vital roles of qCRa3-1 and BjuA03.BNT1 in conferring resistance to clubroot disease. Additionally, we identified a significant QTL locus at 55,316,563 − 56,047,364 bp on chromosome B02, marking the first instance of localizing a clubroot resistance locus on chromosome B02. Previously, only one clubroot disease resistance QTL, Rcr6, was identified on chromosome B03 (Chang et al. 2019).

P. brassica infestation undergoes three phases during host invasion: root hair and epidermis invasion, cortex invasion, and root nodulation stage (Liu et al. 2020). Plants employ different strategies to respond to P. brassica invasion at various stages, which is evident from the differing number of DEGs between CT19 and CS15 after inoculation with P. brassica and the enriched pathways identified through DEG analysis, signifying the complexity of the P. brassica infestation process (Fig. 7). The number of DEGs was the highest at 14 days after P. brassica inoculation compared with that at 21 days after P. brassica inoculation, indicating that the activation of clubroot-resistant genes might be triggered during this period. Electron microscopic observation revealed that cell wall thickening occurred in the resistant material during the first and middle stages of P. brassica inoculation (Fig. 4), whereas the cell wall in the susceptible material became thinner after P. brassica inoculation (Fig. 3). The cell wall and cuticle are the first layer of defense for plant cells, and their structural integrity is crucial for pathogen resistance (Hématy et al. 2009). Fatty acids serve as the essential building blocks for synthesizing cell wall barriers like the cuticle and cork. KEGG analysis highlighted the DEGs were enriched in the fatty acid synthesis pathway between the resistant and susceptible materials at 7 and 14 days post-inoculation with P. brassica. Therefore, the invasion of P. brassica can potentially modify the cell wall structure of susceptible plants to promote its infestation. In contrast, clubroot-resistant plants with these disease-resistant genes can recognize the pathogen and activate the corresponding immune response. Moreover, the synthesis of cuticle and cork can be activated with fatty acids as the substrate, providing resistance to P. brassica reinfection. Our study revealed DEGs between resistant and susceptible parents were also enriched in sulfur metabolism at different periods post-inoculation with P. brassica, a pathway known to play a pivotal role in plant defense. In addition, sulfur metabolism contributes significantly to cysteine and glutathione production. Cysteamine is an important component of several disease-fighting proteins, such as cysteine-rich phyto-defensins (Stotz et al. 2009). Sulfur metabolism is also involved in the nodulation process in legumes and has an important influence on plant-microbe interactions and symbioses (Becana et al. 2018).

In conclusion, our study shows that the main effect QTL qCRa3-1 and BjuA03.BNT1 encoding a TIR-NBS-LRR protein played a vital role in the resistance of plants to clubroot by regulating fatty acid synthesis and the structure of cell walls. Notably, the singular base insertion in the CDS of BjuA03.BNT1 is identified as the determining factor governing its capacity to confer resistance against clubroot.

Acknowledgements

We are grateful to all colleagues in our laboratory, and thank to XLD and SXF for their guidance and help in the study.

Author contributions statement

ZH, KQL, KW, YJS, FHL, XRL, SJB, XLD, and SXF contributed to the conception and design of the study. KW, YJS, FHL, XRL, SJB, BY, and MF contributed to the investigation of the phenotype of the plants and analysis. KQL and KW wrote the manuscript. CYY, AXX, XS, ZYZ, and ZH contributed to revise the manuscript. All authors have read and approved the final manuscript.

Funding

This work was supported by the National Key Research and Development Program (2022YFD1200400), and the Jiangsu Seed Industry Revitalization "the open competition mechanism to select the best candidates" Project (JBGS[2021]061).

Declaration of competing interest

The authors declare that they have no competing interests.

Conflict of interest The authors state that there is no conflict of interest.

Ando S, Otawara S, Tabei Y, Tsushima S (2023) Plasmodiophora brassicae affects host gene expression by secreting the transcription factor-type effector PbZFE1. J Exp Bot https://doi.org/10.1093/jxb/erad377
Askarian H, Akhavan A, González LG, Hwang SF, Strelkov SE (2021) Genetic structure of Plasmodiophora brassicae populations virulent on clubroot resistant Canola (Brassica napus). Plant Dis 105:3694-3704. https://doi.org/10.1094/pdis-09-20-1980-re
Becana M, Wienkoop S, Matamoros MA (2018) Sulfur transport and metabolism in legume root nodules. Front Plant Sci 9:1434. https://doi.org/10.3389/fpls.2018.01434
Benoit S. Landry, Nathalie Hubert, René Crete, Morgan S. Chang, Steven E. Lincoln,(1992) A genetic map for Brassica oleracea based on RFLP markers detected with expressed DNA sequences and mapping of resistance genes to race 2 of Plasmodiophora brassicae (Woronin). Génome 35:409-420. https://doi.org/10.1139/g92-061
Chandna R, Augustine R, Bisht NC (2012) Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR. PLoS One 7:e36918. https://doi.org/10.1371/journal.pone.0036918
Chang A, Lamara M, Wei Y, Hu H, Parkin IAP, Gossen BD, Peng G, Yu F (2019) Clubroot resistance gene Rcr6 in Brassica nigra resides in a genomic region homologous to chromosome A08 in B. rapa. BMC Plant Biol 19:224. https://doi.org/10.1186/s12870-019-1844-5
Chen J, Jing J, Zhan Z, Zhang T, Zhang C, Piao Z (2013) Identification of novel QTLs for isolate-specific partial resistance to Plasmodiophora brassicae in Brassica rapa. PLoS One 8:e85307. https://doi.org/10.1371/journal.pone.0085307
Chu M, Song T, Falk KC, Zhang X, Liu X, Chang A, Lahlali R, McGregor L, Gossen BD, Peng G, Yu F (2014) Fine mapping of Rcr1 and analyses of its effect on transcriptome patterns during infection by Plasmodiophora brassicae. BMC Genomics 15:1166. https://doi.org/10.1186/1471-2164-15-1166
Cui C, Wang JJ, Zhao JH, Fang YY, He XF, Guo HS, Duan CG (2020) A Brassica miRNA regulates plant growth and immunity through distinct modes of action. Mol Plant 13:231-245. https://doi.org/10.1016/j.molp.2019.11.010
Dakouri A, Zhang X, Peng G, Falk KC, Gossen BD, Strelkov SE, Yu F (2018) Analysis of genome-wide variants through bulked segregant RNA sequencing reveals a major gene for resistance to Plasmodiophora brassicae in Brassica oleracea. Sci Rep 8:17657. https://doi.org/10.1038/s41598-018-36187-5
Farid M, Yang RC, Kebede B, Rahman H (2020) Evaluation of Brassica oleracea accessions for resistance to Plasmodiophora brassicae and identification of genomic regions associated with resistance. Genome 63:91-101. https://doi.org/10.1139/gen-2019-0098
Ge W, Lv M, Feng H, Wang X, Zhang B, Li K, Zhang J, Zou J, Ji R (2023) Analysis of the role of BrRPP1 gene in Chinese cabbage infected by Plasmodiophora brassicae. Front Plant Sci 14:1082395. https://doi.org/10.3389/fpls.2023.1082395
Guo Y, Li B, Li M, Zhu H, Yang Q, Liu X, Qu L, Fan L, Wang T (2022) Efficient marker-assisted breeding for clubroot resistance in elite Pol-CMS rapeseed varieties by updating the PbBa8.1 locus. Mol Breed 42:41. https://doi.org/10.1007/s11032-022-01305-9
Hématy K, Cherk C, Somerville S (2009) Host-pathogen warfare at the plant cell wall. Curr Opin Plant Biol 12:406-413. https://doi.org/10.1016/j.pbi.2009.06.007
Hasan J, Megha S, Rahman H (2021) Clubroot in Brassica: recent advances in genomics, breeding, and disease management. Genome 64:735-760. https://doi.org/10.1139/gen-2020-0089
Hatakeyama K, Suwabe K, Tomita RN, Kato T, Nunome T, Fukuoka H, Matsumoto S (2013) Identification and characterization of Crr1a, a gene for resistance to clubroot disease (Plasmodiophora brassicae Woronin) in Brassica rapa L. PLoS One 8:e54745. https://doi.org/10.1371/journal.pone.0054745
Hejna O, Havlickova L, He Z, Bancroft I, Curn V (2019) Analysing the genetic architecture of clubroot resistance variation in Brassica napus by associative transcriptomics. Mol Breed 39:112. https://doi.org/10.1007/s11032-019-1021-4
Huang Z, Peng G, Liu X, Deora A, Falk KC, Gossen BD, McDonald MR, Yu F (2017) Fine mapping of a clubroot resistance gene in Chinese cabbage using SNP markers identified from bulked segregant RNA sequencing. Front Plant Sci 8:1448. https://doi.org/10.3389/fpls.2017.01448
Huang Z, Peng G, Gossen BD, Yu F. (2019) Fine mapping of a clubroot resistance gene from turnip using SNP markers identified from bulked segregant RNA-Seq. Mol Breed 39:131. https://doi.org/10.1007/s11032-019-1038-8
Javed MA, Schwelm A, Zamani-Noor N, Salih R, Silvestre Vañó M, Wu J, González García M, Heick TM, Luo C, Prakash P, Pérez-López E (2023) The clubroot pathogen Plasmodiophora brassicae: A profile update. Mol Plant Pathol 24:89-106. https://doi.org/10.1111/mpp.13283
Karim MM, Dakouri A, Zhang Y, Chen Q, Peng G, Strelkov SE, Gossen BD, Yu F (2020) Two clubroot-resistance genes, Rcr3 and Rcr9wa, mapped in Brassica rapa using bulk segregant RNA sequencing. Int J Mol Sci 21:5033. https://doi.org/10.3390/ijms21145033
Laila R, Park JI, Robin AHK, Natarajan S, Vijayakumar H, Shirasawa K, Isobe S, Kim HT, Nou IS (2019) Mapping of a novel clubroot resistance QTL using ddRAD-seq in Chinese cabbage (Brassica rapa L.). BMC Plant Biol 19:13. https://doi.org/10.1186/s12870-018-1615-8
LeBoldus JM, Manolii VP, Turkington TK, Strelkov SE (2012) Adaptation to brassica host genotypes by a single-spore isolate and population of Plasmodiophora brassicae (clubroot). Plant Dis 96:833-838. https://doi.org/10.1094/pdis-09-11-0807
Lee J, Izzah NK, Choi BS, Joh HJ, Lee SC, Perumal S, Seo J, Ahn K, Jo EJ, Choi GJ, Nou IS, Yu Y, Yang TJ (2016) Genotyping-by-sequencing map permits identification of clubroot resistance QTLs and revision of the reference genome assembly in cabbage (Brassica oleracea L.). DNA Res 23:29-41. https://doi.org/10.1093/dnares/dsv034
Liu L, Qin L, Zhou Z, Hendriks W, Liu S, Wei Y (2020) Refining the life cycle of Plasmodiophora brassicae. Phytopathology 110:1704-1712. https://doi.org/10.1094/phyto-02-20-0029-r
Moriguchi K, Kimizuka-Takagi C, Ishii K, Nomura K (2010) A genetic map based on RAPD, RFLP, isozyme, morphological markers and QTL analysis for clubroot resistance in Brassica oleracea. Breed Sci 49:257-265. https://doi.org/10.1270/jsbbs.49.257
Nagaoka T, Doullah MA, Matsumoto S, Kawasaki S, Ishikawa T, Hori H, Okazaki K (2010) Identification of QTLs that control clubroot resistance in Brassica oleracea and comparative analysis of clubroot resistance genes between B. rapa and B. oleracea. Theor Appl Genet 120:1335-1346. https://doi.org/10.1007/s00122-010-1259-z
Ochoa JC, Mukhopadhyay S, Bieluszewski T, Jędryczka M, Malinowski R, Truman W (2023) Natural variation in Arabidopsis responses to Plasmodiophora brassicae reveals an essential role for resistance to Plasmodiophora brasssicae 1 (RPB1). Plant J https://doi.org/10.1111/tpj.16438
Pageau D, Lajeunesse J, Lafond J (2006) Impact de l'hernie des crucifères [Plasmodiophora brassicae] sur la productivité et la qualité du canola. Can J Plant Pathol 28:137-143. https://doi.org/10.1080/07060660609507280
Pang W, Fu P, Li X, Zhan Z, Yu S, Piao Z (2018) Identification and mapping of the clubroot resistance gene CRd in Chinese cabbage (Brassica rapa ssp. pekinensis). Front Plant Sci 9:653. https://doi.org/10.3389/fpls.2018.00653
Paul P, Chhapekar SS, Rameneni JJ, Oh SH, Dhandapani V, Subburaj S, Shin SY, Ramchiary N, Shin C, Choi SR, Lim YP (2021) MiR1885 regulates disease tolerance genes in Brassica rapa during early infection with Plasmodiophora brassicae. Int J Mol Sci 22 https://doi.org/10.3390/ijms22179433
Rocherieux J, Glory P, Giboulot A, Boury S, Barbeyron G, Thomas G, Manzanares-Dauleux MJ (2004) Isolate-specific and broad-spectrum QTLs are involved in the control of clubroot in Brassica oleracea. Theor Appl Genet 108:1555-1563. https://doi.org/10.1007/s00122-003-1580-x
Sakamoto K, Saito A, Hayashida N, Taguchi G, Matsumoto E (2008) Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Theor Appl Genet 117:759-767. https://doi.org/10.1007/s00122-008-0817-0
Sarazin V, Duclercq J, Mendou B, Aubanelle L, Nicolas V, Aono M, Pilard S, Guerineau F, Sangwan-Norreel B, Sangwan RS (2015) Arabidopsis BNT1, an atypical TIR-NBS-LRR gene, acting as a regulator of the hormonal response to stress. Plant Sci 239:216-229. https://doi.org/10.1016/j.plantsci.2015.07.017
Schulze S, Yu L, Hua C, Zhang L, Kolb D, Weber H, Ehinger A, Saile SC, Stahl M, Franz-Wachtel M, Li L, El Kasmi F, Nürnberger T, Cevik V, Kemmerling B (2022) The Arabidopsis TIR-NBS-LRR protein CSA1 guards BAK1-BIR3 homeostasis and mediates convergence of pattern- and effector-induced immune responses. Cell Host Microbe 30:1717-1731.e1716. https://doi.org/10.1016/j.chom.2022.11.001
Stotz HU, Thomson JG, Wang Y (2009) Plant defensins: defense, development and application. Plant Signal Behav 4:1010-1012. https://doi.org/10.4161/psb.4.11.9755
Takken FL, Tameling WI (2009) To nibble at plant resistance proteins. Science 324:744-746. https://doi.org/10.1126/science.1171666
Vañó MS, Nourimand M, MacLean A, Pérez-López E (2023) Getting to the root of a club understanding developmental manipulation by the clubroot pathogen. Semin Cell Dev Biol 148-149:22-32. https://doi.org/10.1016/j.semcdb.2023.02.005
Voorrips RE, Jongerius MC, Kanne HJ (1997) Mapping of two genes for resistance to clubroot (Plasmodiophora brassicae) in a population of doubled haploid lines of Brassica oleracea by means of RFLP and AFLP markers. Theor Appl Genet 94:75-82. https://doi.org/10.1007/s001220050384
Wang Y, Tang M, Zhang Y, Huang M, Wei L, Lin Y, Xie J, Cheng J, Fu Y, Jiang D, Li B, Yu X (2023) Coordinated regulation of plant defense and autoimmunity by paired trihelix transcription factors ASR3/AITF1 in Arabidopsis. New Phytol 237:914-929. https://doi.org/10.1111/nph.18562
Wang Y, Xiang X, Huang F, Yu W, Zhou X, Li B, Zhang Y, Chen P, Zhang C (2022) Fine mapping of clubroot resistance loci CRA8.1 and candidate gene analysis in Chinese cabbage (Brassica rapa L.). Front Plant Sci 13:898108. https://doi.org/10.3389/fpls.2022.898108
Yu F, Zhang X, Peng G, Falk KC, Strelkov SE, Gossen BD (2017) Genotyping-by-sequencing reveals three QTL for clubroot resistance to six pathotypes of Plasmodiophora brassicae in Brassica rapa. Sci Rep 7:4516. https://doi.org/10.1038/s41598-017-04903-2
Zhu H, Zhai W, Li X, Zhu Y (2019) Two QTLs controlling clubroot resistance identified from bulked segregant sequencing in Pakchoi (Brassica campestris ssp.). Sci Rep 9:9228. https://doi.org/10.1038/s41598-019-44724-z

Supplementalfigures.pdf
Supplemental Fig. S1. Plant morphology of CT19, a resynthesized B. juncea. Significant differences between the two genotypes in the same environment are indicated by aste Supplemental Fig. S2. Venn of DEGs between the resistant and susceptible parents treated with P. brassica. A: Venn of DEGs between the resistant and susceptible parents; B: Venn of DEGs during different stages of the resistant parents; C: Venn of DEGs during different stages of the susceptible parents.
supplementaltables.xlsx
Supplemental Table S1 Statistical table of variation quantity and density of the F₂ population

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Role of BjuA03.BNT1 in enhancing clubroot resistance in resynthesized Brassica juncea L.

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Abstract

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Key message

Introduction

Materials and methods

Plant materials and growth condition

Microstructural examination of clubroot-resistant and susceptible parental varieties

Simplified genome sequencing and data analysis

Transcriptome sequencing and candidate gene analysis

Amplification of the candidate gene

Result

Creation of mustard with resistance to clubroot

Localization of clubroot resistant loci

Screening of clubroot resistance candidate genes

Discussion

Declarations

References

Supplementary Files

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