Plant materials and growth
The B. napus accessions in this study were planted in Zhijiang (30° 48’ N, 111° 77’ E) and Taiping (29° 71’ N, 118° 29’ E), spanning three growing seasons (2016–2018). The edited B. napus plants were planted in Hubei (30° 54’ N, 113° 81’ E) and Gansu (38° 43’ N and 100° 81’ E) in 2023. All B. napus materials were maintained by self-pollination. Crop management of field experiments for agronomic trait tests followed the standard protocol of the China National Rapeseed Variety Field Test.
All A. thaliana and Nicotiana benthamiana plants were grown under controlled conditions at 21–24°C and with a 16 h photoperiod. The Arabidopsis materials used in this study include pmr4-1 (gsl5)23, sid2-1 (ref. 39), jar1-1 (ref. 40), ein2-1 (ref. 41), ald1 (ref. 42), pad4 (ref. 43) and the transgenic line NahG44. The homozygosity of T-DNA insertion mutants was genotyped by PCR with both gene-specific and T-DNA border primers. The point mutation lines were genotyped by sequencing the PCR products after amplification with gene-specific primers. The resultant homozygous double mutants were used for the clubroot resistance test. The primers used in this study are listed in Supplementary Table 5.
Phenotyping for field traits
All field experiments for phenotyping the field traits were carried out in a randomized block design with three replicates. Each replicate contained at least 60 plants with a space of about 30 cm between rows, and each row was 2 m in length with a space between plants of approximately 10 cm. The seeds of each accession were sown in late September (26–30), which is the beginning of the growing season for winter canola plantation areas in China. In early December (about 70 days after sowing), the roots of the susceptible control (Westar) became severely clubbed, and the growth of the plants, as well as the P. brassicae infection, was almost terminated in the clubroot disease fields owing to the lower temperature in winter (usually below 8 °C). All tested B. napus accessions in the clubroot disease fields were harvested to score clubroot disease severity. Disease scales 0, 1, 2 and 3 were used to score each plant of each replicate of 244 B. napus accessions and to generate the DI to quantify the disease severity of each accession based on a previously described method7: 0, no clubs; 1, a few small clubs present on less than one-third of the lateral roots or on the main root; 2, moderate clubs present on one-third to two-thirds of the lateral roots or the main root; and 3, severe clubs on the main root or present on more than two-thirds of the lateral roots. In early May of the next year—the end of the growing season for winter canola plantation areas in China—the B. napus plants were harvested for scoring of the key agronomic traits.
To evaluate the key agronomic traits, each replicate comprised 15 rows with 20 plants in each row. The sampling method depended on specific traits: (1) plant height (the length (cm) from the cotyledon node to the tip of a plant); (2) branch number (the number of primary branches per plant); (3) the 1,000-seed weight (in g) per plant; (4) silique number per plant (all siliques in each plant); (5) seed number per silique (the average seed number of the bottom 20 siliques from the main inflorescence of each plant); (6) yield (the seed weight of all plants within each block); and (7) seed oil content. Data from the first five key agronomic traits were determined based on three replicates, each of which comprised ten randomized plants. A Foss NIR Systems 5000 Near-Infrared Reflectance Spectroscope was used to measure the oil content of seeds collected at the maturity period45.
GWAS and favorable allele identification
A total of 244 B. napus accessions were genome-resequenced with at least tenfold genome coverage, and a total of 2,797,642 filtered SNPs with a missing rate of ≤0.1 and minor allele frequency of ≥0.05 were obtained20. The clubroot DI of each B. napus accession was investigated in three consecutive years (2016–2018) and used to generate the best linear unbiased prediction using the R script lme4 (https://cran.r-project.org/web/packages/lme4) and lsmeans21. Three models, including a general linear model, mixed linear model and fixed and random model Circulating Probability Unification (FarmCPU), were used for genome-wide association analysis. The significant P value threshold of the GWAS was set to 1.8 × 10−8 (−log10P = 7.74, calculated as 0.05 / total number of SNPs). Haplotype block estimation was based on the confidence interval method46. The linkage disequilibrium of the whole genome and the significantly associated regions were analyzed using PopLDdecay and LDBlockShow software47,48.
The SNPs of the gene-related regions were extracted from the genomic variation files. A phylogenetic tree was constructed using PHYLIP (v.3.696) with the neighbor-joining method and default parameters and was visualized by FigTree (v.1.4.3)49. PCA analysis was performed using PLINK (v.1.90b4.6), and the first two principal components of the PCA analysis were illustrated by the ggplot2 package in R (v.4.1)50. Population structure was analyzed by ADMIXTURE (v.1.3.0) with the following parameters: the number of subgroups, K, ranged from two to eight, and the cross-validation error was calculated for each K value51.
Gene complementation and expression, RNA interference assay and genome editing
For complementation experiments with the gsl5 mutant, a full-length genomic DNA fragment of GSL5 including approximately 3.0 kb of the upstream sequence of the start codon and the open reading frame was amplified from Arabidopsis, B. napus, B. rapa, B. oleracea and R. sativus using gene-specific primers (Supplementary Table 5). All fragments were respectively cloned into the pBI121 vector, generating the recombinant plasmids proAtGSL5Col-0:AtGSL5Col-0 (GSL5), proBnaA09.GSL5Westar:BnaA09.GSL5Westar(Hap_1), proBnaA09.GSL5ZS11:BnaA09.GSL5ZS11(Hap_2), proBnaC09.GSL5ZS11:BnaC09.GSL5ZS11 (BnaC09.GSL5), proBraGSL5Chiifu:BraGSL5Chiifu (BraGSL5), proBolGSL5ZG11:BolGSL5ZG11 (BolGSL5) and proRsaGSL5MTH:RsaGSL5MTH (RsaGSL5) for Agrobacterium-mediated transfection of Arabidopsis gsl5 plants.
To overexpress AtGSL5 in Arabidopsis, the GSL5 genomic region was cloned into pBI121 driven by the cauliflower mosaic virus 35S promoter, generating the recombinant plasmid 35S:AtGSL5 (GSL5-OE) for Agrobacterium-mediated transfection of wild-type plants.
For the RNA interference assay, a 385 bp coding sequence of PbPDIa was cloned into a pBI121-RNAi vector driven by the cauliflower mosaic virus 35S promoter, generating the recombinant plasmid for Agrobacterium-mediated transfection of wild-type plants. The transgenic plants produced a hairpin containing a 385 bp double-stranded RNA and generated endogenous small RNA that was able to knock down the PbPDIa expression of P. brassicae52.
For genome editing, two single-guide RNA (sgRNA) sequences, sgRNA1 and sgRNA2, from the first exon of GSL5 were designed as the editing targets to knock out the GSL5 from A. thaliana, B. napus, B. rapa and B. oleracea (Supplementary Table 5). All sgRNAs were designed using CRISPR-P (v.2.0) (http://crispr.hzau.edu.cn/CRISPR2)53. A previously developed multiplex genome editing vector, pYLCRISPR-Cas9-DB54, was used to knock out GSL5.
All fused plasmids were confirmed by sequencing and introduced into Agrobacterium tumefaciens strain GV3101 for the following transformation of Arabidopsis, B. napus, B. rapa and B. oleracea, using previously described methods55. All transgenic plants were genotyped by PCR amplification or DNA sequencing with the specific primers listed in Supplementary Table 5. The homozygous gsl5 mutants of A. thaliana, B. napus, B. rapa and B. oleracea were used for further phenotyping.
Pathogen maintenance and inoculation
P. brassicae isolates were collected from diseased plants of different Brassica crops across China and have been previously identified for the pathotypes17. The clubbed roots were harvested, cleaned and stored in −20 °C freezers. To isolate the resting spores, the clubbed roots were cut into small pieces and smashed in distilled water with a blender. The suspensions were filtered with six layers of gauze and adjusted to 1.0 × 107 resting spores per ml. To evaluate clubroot resistance under controlled conditions (21–24 °C, 16 h light, 8 h darkness), 2-week-old seedlings of Arabidopsis or 10-day-old seedlings of B. napus, B. rapa and B. oleracea were inoculated with 1 ml of resting spore suspension (1.0 × 107 spores per ml) per seedling. After 24–30 days, the inoculated plants were harvested for scoring based on disease scale and severity as described previously7.
Subcellular localization
For subcellular localization, the coding sequence of PbPDIa, after removing the signal peptide sequence, was cloned into pBI121-mCherry. Then, pBI121-mCherry-PbPDIa was transformed into A. tumefaciens GV3101 and injected into the leaves of 4-week-old N. benthamiana plants for transient expression. The empty vector pBI121-mCherry was used as a control. The mCherry signals were detected with a confocal microscope (Carl Zeiss) with an excitation wavelength of 561 nm and an emission wavelength of 560–620 nm.
Microscopy analysis
Fluorescent probe-based confocal microscopy was used to visualize the zoosporangia of P. brassicae during the primary infection, and the fluorescent probe HCS LipidTox Green neutral lipid stain (HLG; Thermo Fisher Scientific) was used to label the zoosporangia6. To detect the fluorescence of HLG, an excitation wavelength of 488 nm and an emission wavelength of 500 to 540 nm were used.
Histological technique was used to detect P. brassicae parasites during secondary infection. P. brassicae-inoculated roots of Arabidopsis were harvested at 12 dpi and 21 dpi, and the main roots adjacent to the hypocotyl were sampled for histological analysis as described previously56. In brief, tissues were fixed overnight in a freshly prepared solution of 2% glutaraldehyde, dehydrated through a graded ethanol series, embedded in a mold in melted paraffin and trimmed to produce hemi-sections. The sections were then placed on a slide, stained with 0.05% toluidine blue O and subjected to microscopic analysis.
Transcriptome profiling and quantitative real-time PCR analysis
The roots of Arabidopsis plants with or without P. brassicae inoculation were harvested at 12 dpi and 21 dpi with three replicates and immediately frozen in liquid nitrogen. High-quality RNA was extracted using the Fast Pure Plant Total RNA Isolation Kit (VAZYME). cDNA library construction and sequencing, data quality control and gene expression calculation were performed at Novogene. In brief, clean reads were obtained by filtering raw reads and then mapped to the Arabidopsis Col-0 reference genome using HISAT2 software57,58. The number of fragments per kilobase per million mapped fragments was calculated and used to estimate gene expression levels59. Pearson’s correlation coefficient was calculated with the R script cor (https://search.r-project.org/R/refmans/stats/html/cor.html) to evaluate the correlation between biological replicates60. DESeq software was used to identify the differentially expressed genes with the thresholds |log2(fold change)| > 1 and adjusted P < 0.05 (ref. 61). A Venn diagram of differentially expressed genes between groups was plotted on the website http://jvenn.toulouse.inra.fr/app/example.html62.
For quantitative PCR with reverse transcription (RT–qPCR), total RNA was used for first-strand cDNA synthesis with the Hifair III 1st Strand cDNA Synthesis Kit (gDNA digester plus) following the manufacturer’s instructions (YEASEN). Quantitative PCR was performed with a CFX Connect Real-time PCR system (Bio-Rad), using Hieff UNICON Universal Blue qPCR SYBR Master Mix (YEASEN). Ubiquitin 5 (UBQ5) and Actin 7 were used as the internal control in Arabidopsis and B. napus, respectively. The relative expression level of genes was achieved using the 2(-ΔΔCT) method63. The primers for RT–qPCR are listed in Supplementary Table 5.
MeJA treatments
MeJA powder (Macklin) was dissolved in dimethylsulfoxide to prepare a 1 M stock solution. Different concentrations of working solution were prepared by diluting the stock solution of MeJA with distilled water and used to water the Arabidopsis or B. napus plants at 5 dpi and 9 dpi. The treated plants were collected at 30 dpi for scoring the disease severity.
Measurement of plant hormones, ACC, callose, lignin and reactive oxygen species
The roots of Arabidopsis plants with or without P. brassicae inoculation were harvested at 12 dpi and immediately frozen in liquid nitrogen with three biological replicates. Extraction and quantification were performed at RUIYUAN BIOTECHNOLOGY. In brief, the frozen plant tissues were ground and extracted with specialized buffers, followed by successive incubation, centrifugation, purification and condensation for the preparations. Quantification analysis was performed by ultra-performance liquid chromatography–electrospray tandem mass spectrometry using a high-performance liquid chromatograph (Agilent 1290) and a mass spectrometer (AB Qtrap 6500).
Expression and purification of GSL5 and PbPDIa
GSL5 and PbPDIa were cloned into the engineered pMlink vector with a Flag or His tag, respectively, and then transfected into Expi293F cells (A14528, ThermoFisher, cat. no. 100044202, cGMP bank) for 60 h incubation. After harvest and washing with PBS, the transfected cells were homogenized in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl), disrupted with high pressure and incubated in 1% N-dodecyl-β-d-maltoside (DDM) for 2 h at 4 °C. After centrifugation, the supernatant was incubated with anti-Flag M2 or Ni2+ affinity resin for 1 h. The resin was eluted for Flag-tagged GSL5 with lysis buffer containing 150 μg ml−1 Flag peptide and 0.02% DDM or for His-tagged PbPDIa, with the lysis buffer containing 250 mM imidazole and 0.02% DDM. The eluted samples were concentrated to 1 ml immediately before gel filtration chromatography (Superose 6 10/300, GE Healthcare).
Pull-down assays and protein stability assay
The purified Flag-tagged GSL5, His-tagged PbPDIa and their mixture samples were incubated with anti-Flag M2 affinity resin for 1 h. The resin was washed with washing buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.02% DDM and then eluted with washing buffer containing 150 μg ml−1 Flag peptide. The proteins were detected through immunoblots with antibodies against His or Flag.
Two equal portions of Flag-tagged GSL5 proteins were treated with the lysis buffer without or with PbPDIa at 25 °C. Samples were collected every 4 h and detected for protein stability by immunoblot with Flag antibodies.
In vitro PDI activity assay
An in vitro PDI activity assay using reduced ribonuclease A (rRNase A) as a substrate was carried out as previously described64. RNase A (50 μg) was unfolded in unfolding buffer (100 mM Tris-HCl pH 8.0, 0.3 M DTT, 6 M guanidine-HCl) for 1 h at 37 °C and desalted with a 0.1% acetic acid-equilibrated G25 column. The resultant rRNase A fractions were collected and quantified using a molar extinction coefficient at 280 nm. The PDI activity assay was carried out in refolding buffer (36 μM rRNase A, 100 mM Tris-HCl buffer pH 8.5, 1 mM glutathione, 0.2 mM oxidized glutathione, 16 μM PbPDIa). RNase activity was measured by the A260 absorption value resulting from the cleavage of total RNAs. The relative activity was calculated as (ktreat − kRNA) / (kRNase A − kRNA) × 100%.
Assessment of PbPDIa secretory activity
Assessment of PbPDIa secretory activity was performed with the Yeast Signal Trap Assay Kit (COOLABER). In brief, the DNA sequence of PbPDIa encoding the signal peptide was amplified, cloned into the pSUC2 vector and transformed into the sucrase-deficient yeast strain YTK12. YPRAA medium (a medium containing 1% yeast extract, 2% peptone, 2% raffinose, 2 µg ml−1 antimycin A and 2% agar) was used to evaluate secretory activity, as only the strain containing the functional signal peptide can grow on the YPRAA medium. Furthermore, 2,3,5-triphenyltetrazolium chloride was also included as per the user guide and was reduced by secreted sucrase into insoluble, red-colored 1,3,5-triphenylformazan.
Cell death assay
Cell death was assessed by Evans blue (Merck) staining and by measuring cytoplasmic ion leakage from plant tissues28,29,30. The roots of Arabidopsis plants with or without P. brassicae inoculation were collected at 12 dpi and placed in a 2% Evans blue solution for 30 min of staining. The plants were then rinsed in distilled water until the roots of the solvent-treated plants could not be stained. The Evans blue-stained plants were imaged under a stereomicroscope SZX16 (Olympus).
To measure the cytoplasmic ion leakage, P. brassicae-infected roots of Arabidopsis were collected at 12 dpi for a 3 h inoculation in ultrapure water at room temperature and the conductivity of the bathing solution was measured with a conductivity meter (STARTER 300C; OHAUS), referred to as value C1; the conductivity of the ultrapure water is value C0. The bathing solution containing the root tissues was incubated at 95 °C for 30 min, and conductivity was determined after cooling to room temperature, referred to as value C2. The relative cytoplastic ion leakage was calculated with the following formula: (C1 − C0) / (C2 − C0) × 100%.
Statistical analysis
All statistical analyses were performed using Graphpad Prism (v.7.0; https://www.graphpad.com) or R (v.4.1.2). Detailed information, including the testing model, sample size, replicates and P values, is provided in the individual figures and figure legends.
Inclusion and ethics
The data presented in this study were derived exclusively from Arabidopsis and Brassica crops, without involving any animal experiments. The transgenic planting and artificial inoculation of P. brassicae are subject to strict regulation. All experimental data are included in the Data availability section.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.