The CCR genomic sequences in B. napus Arabidopsis thaliana, Brassica rapa, and Brassica oleracea were aligned using ClustalW with the default settings. Phylogenetic trees were constructed using the neighbor-joining method implemented in MEGA 7.0 software. The bootstrap test was executed with 1,000 replications (Larkin et al., 2007).

The BnaC.CCR2.b (BnaC06g40190D) gene was cloned from the cDNA library of B. napus cultivar J964, a Sclerotinia-resistant line, using BnCCR2-F/R primers (Supplementary Table S1). A fragment of BnaC.CCR2.b was then subcloned into PMDC83 using SpeI+AscI double digestion, generating a 35S:BnaC.CCR2.b vector. The 35S:BnaC.CCR2.b vector construct was then introduced into Agrobacterium tumefaciens strain GV3101 for genetic transformation. The B. napus line J9712 (a susceptible line), which was kindly provided by Professor Yongming Zhou (National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University), was used as a receptor. B. napus was transformed using the A. tumefaciens-mediated hypocotyl method. Selected plump seeds were surface sterilized with a 2% NaClO solution and subsequently rinsed with sterile distilled water. The seeds were germinated on half-strength Murashige and Skoog (MS) basal media supplemented with 2% sucrose in darkness. The seedlings were subsequently grown at 25°C in the dark for 7days. Afterward, the hypocotyl (~15mm) was cut, and the explants were floated in infection media [MS media supplemented with 3% sucrose and 100μm acetosyringone (AS); pH 5.8] for 20min. The explants were then transferred to cocultivation media (MS media supplemented with 3% sucrose, 1mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.3mg/l kinetin, 100μm AS, and 8g/l agar; pH 5.8) and incubated for 3days. The explants were subsequently transferred to MS callus induction media supplemented with 3% sucrose, 1mg/l 2,4-D, 0.3mg/l kinetin, 5mg/l AgNO₃, 300mg/l timentin, 25mg/l hygromycin B (Hyg), and 8g/l agar (pH 5.8) and incubated at 25°C for 20days. The explants were then transferred to shoot differentiation media [MS media supplemented with 1% glucose, 100μm AgNO3, 2.0mg/l zeatin, 0.1mg/l indoleacetic acid (IAA), 300mg/l timentin, 25mg/l Hyg, and 8g/l agar; pH 5.8] and incubated until shoot growth was initiated (fresh MS media was replaced every 20days). Healthy green shoots were ultimately transferred to bottles containing root induction media (MS media supplemented with 1% sucrose and 8g/l agar; pH 5.8), after which the plantlets acclimated and became established. Total DNA was extracted from the young leaves of each transgenic plant using the cetyltrimethylammonium bromide (CTAB) method, after which PCR was performed to identify positive transformants using the specific primers 35S-3 and BnCCR2-R (Supplementary Table S1).

Histological staining of lignin was performed using Wiesner reagent (Li et al., 2013). Wiesner stain is known to react with cinnamaldehyde residues in lignin, and the color intensity is consistent with the total lignin content. Whole cross-sections and manually cut cross-sections of stems were obtained at the same position (approximately 30–40cm above the ground) at the initial, full, and final flowering stages. The stem sections were treated with a 1% phloroglucinol alcohol solution for 2min, followed by the application of a drop of concentrated hydrochloric acid (32%). The manually cut cross-sections were viewed and imaged (bright field) using a Zeiss AxioPlan 2 microscope.

Stem tissues of B. napus lines J964 and J902 were collected at the final flowering stage. Stem tissues of the transgenic lines in the T₂ generation were collected at the mature stage, because of the requirement for seed harvesting. Stems at a length of approximately 30cm of six individuals of each accession in each replicate were cut at 20cm above the ground using a sharp knife. The samples were then dried at 60°C, cut into small pieces, ground into powder with a grinder, filtered through an 80-mesh screen (0.15×0.15mm), and then stored in a dry container. The total lignin content was determined by a two-step acid hydrolysis method, with modifications (Xu et al., 2012). The lignin consisted of both acid-insoluble lignin (AIL) and acid-soluble lignin (ASL).

ASL: A 0.3g sample was recorded as W₁. The sample was extracted with benzene-ethanol (67/33, v/v) in a Soxhlet incubator for 4h and then air dried in a fume hood. The sample was hydrolyzed with 10ml of 67% H₂SO₄ (v/v) in a shaker at 30°C for 1.5h. Afterward, 200ml of ddH₂O were added for hydrolysis, which was performed at 120°C for 1h. After hydrolysis, the hydrolysis liquor was transferred to a 250ml volumetric flask and brought up to 250ml (V) with 2.88% sulfuric acid. The absorbance was between 0.2 and 0.7, and the dilution factor was denoted as D. The absorbance of the sample was read at 205nm via UV–Vis spectroscopy, and 2.88% sulfuric acid was used as a blank. The amount of ASL was calculated as follows: ASL (%)=A×D×V/(1,000×K×W₁)×100, where A is the absorption value, D is the dilution ratio of the sample, V is the total volume of the filtrate, and K is the absorptivity constant (110l/g/cm).

ASL: The hydrolysis liquor obtained previously was filtered through a G3 crucible filter, and the total residue was then transferred to a crucible filter. The acid-insoluble residue was dried in an oven at 60°C until a constant weight was achieved. The samples were then removed from the oven and cooled in a dry container. The weight of both the crucible and the dry residue was recorded (W₂). The dried residue was ultimately ashed in a muffle furnace at 200°C for 30min followed by 575°C for 4h. The crucibles and ash were subsequently weighed, and the weight (W₃) was recorded. The AIL of the original sample was calculated as follows: AIL (%)=(W₂−W₃)×100/W1. The total lignin (%)=ASL%+AIL%. The lignin content was determined from 300mg of extract-free dry stems for three biological replicates. The lignin content was calculated as the weight percent of the dry extract-free stems. All experiments were conducted with three replications.

Sclerotinia sclerotiorum isolate SS-1 was maintained and cultured on potato dextrose agar (PDA) media (Wu et al., 2019). Under natural field conditions, stems were inoculated with S. sclerotiorum to evaluate its resistance at the termination of flowering. Approximately 10 stems of each transgenic line (T₂ and T₃) in each replicate were inoculated at a height of 40–50cm above the ground with mycelial agar plugs (5mm diameter). Each plug was affixed with plastic wrap to ensure close contact of the inoculum with the stem surface and to maintain a high humidity. The lesion length along the stems was measured at 7days post-inoculation (dpi). This assessment was conducted for three replications.

To examine the expression level of BnaC.CCR2.b in the transgenic lines, the stem tissues of plants in the T₀ and T₁ generations were collected and then quickly put into liquid nitrogen for RNA extraction. The stem tissues of the plants in the T₁ generation were collected (three biological replicates), and each replicate consisted of three stem tissues from three different plants. qBnaC.CCR2.b-F/R primers (Supplementary Table S1) were used to measure the expression of BnaC.CCR2.b.

To detect whether overexpression of BnaC.CCR2.b in B. napus would alter the expression levels of genes related to lignin synthesis, J9712 plants and overexpression plants (T₂ generation) were selected for inoculation and sampling (three biological replicates). When the plants were at the flower termination stage, three sites on the primary stem were inoculated at three consecutive internodes (approximately 30–60cm above the ground) with 5mm diameter mycelial agar plugs. Epidermal stem tissues extending 10mm beyond the inoculation site and 1mm deep were sampled. Each replicate involved 12 plants per line at three different time points (0, 48, and 72 hpi).

Total RNA was extracted using a Plant Total RNA Extraction Kit (BioTeke, China) following the manufacturer’s instructions and then treated with RNase-free DNase I (Thermo Scientific, United States) to remove genomic DNA contaminants. The RNA was transcribed into cDNA using the HiScript^® QRT Super Mix for qPCR (Vazyme, China). The expression levels of the key lignin synthesis pathway genes BnPAL2, BnPAL1, BnC4H, Bn4CL1, BnHC, BnC3H, BnCCoAOM, BnFAH1, BnCCR1, BnCCR2, BnCOMT, BnCAD4, and BnCAD5 were measured via qRT-PCR. The primers were designed such that the sequences of all copies of each gene were incorporated (Supplementary Table S1). qRT-PCR was performed for three technical replicates. The BnUBC9 gene was chosen as the reference gene. Normalization was then performed, and the relative expression was calculated to analyze the qRT-PCR data.

Expression patterns of BnCCR genes in various tissues of B. napus line J9712 were determined by transcriptome sequencing. Tissue samples were collected at different growth stages. Cotyledon, seedling root, stem, rosette leaf, cauline leaf, shoot apical meristem, flower bud, unpollinated ovary, seed, and silique walls at different stages (14days, 24days, 34days, and 50days after flowering) were collected from five plants in each biological replicate. Three biological replicates were performed. All samples were sequenced using an Illumina HiSeq X Ten sequencer. The sequencing was performed as paired-end reads that were 2× 150bp in length. The original data set was deposited in the NCBI Sequence Read Archive (accession no. PRJNA749379). RNA sequence analysis was performed as described in our previous study (Wu et al., 2016).

In our previous study, we performed dynamic transcriptomic analyses to understand the different defense responses to S. sclerotiorum in a resistant B. napus line (J964) and a susceptible B. napus line (J902) at 24, 48, and 96h post-inoculation (hpi). We found that differences between J964 and J902 were associated with differences in the magnitude of gene expression changes, which were detected mainly at 48 or 96 hpi. At 24 hpi, only 122 genes were slightly upregulated in J964, while 4,129 genes were upregulated in J902 (Wu et al., 2016). GO enrichment analysis showed that half of the top 30 enriched GO terms in the biological process for the 4,129 upregulated genes in J902 belonged to the secondary GO category, response to stimulus (Supplementary Figure S1), suggesting that the defense response was faster in J902 than in J964. However, S. sclerotiorum infected and propagated more easily in J902 than in J964. This finding suggests that in addition to the second line of defense (the plant immune system), the first line of defense (the passive defense line) may also differ between the two lines.

To determine whether the stem lignin differed before inoculation, the stem cross-sections of the two lines were examined carefully. Interestingly, a significant difference was found after the stems were stained with Wiesner reagent to detect lignin. Both lines presented deep magenta staining in the xylem, but J964 presented better xylem development and deeper lignin staining than J902 (Figures 1A,B). In J964, the vascular bundles were tightly arranged and had developed neatly, and the water content of the pith was high (Figure 1C). However, in J902, the vascular bundles were loosely arranged and unevenly developed. Even the xylem of some vascular bundles was degraded, and the pith lost a large amount of water and began to soften (Figure 1D). As expected, the stem lignin content in J964 was significantly higher than that in J902 (Figure 1E). Hence, the better development of xylem and the higher content of lignin in the stems seem to limit the invasion and spread of S. sclerotiorum.

In addition, according to the transcriptome data, genes involved in lignin biosynthesis were induced after S. sclerotiorum infection (Figure 1F). However, no obvious difference in gene expression patterns between the two lines was observed (Figure 1F). These results indicate that lignin synthesis pathway was induced in both lines under the infection of S. sclerotiorum and lignin was deposited proactively in infected tissues, possibly in an attempt to limit pathogen colonization. Therefore, we overexpressed genes involved in lignin biosynthesis in B. napus to determine whether the lignin content in the stems could be increased to improve resistance to S. sclerotiorum. Then, we chose the CCR gene for overexpression in B. napus. This gene encodes the first rate-limiting enzyme that catalyzes the reaction of lignin-specific pathways (Menden et al., 2007).

To better understand the evolutionary history and functions of CCR in Brassicaceae, we searched for all possible copies of CCR in the released reference genome sequence of Darmor-bzh B. napus, with Arabidopsis AtCCR1 (AT1G15950.1) and AtCCR2 (AT1G80820.1) gene sequences used as queries. For both AtCCR1 and AtCCR2, four close homologs were identified in B. napus (Figure 2A). We named these AtCCR1 homologs BnaA.CCR1.a (BnaA06g10620D), BnaA.CCR1.b (BnaA09g56490D), BnaC.CCR1.a (BnaC05g12180D), and BnaC.CCR1.b (BnaC08g38580D), which were 98.2, 97.1, 98.0, and 96.5% similar, respectively, to AtCCR1 at the amino acid level. Similarly, four AtCCR2 homologs were named BnaA.CCR2.a (BnaA02g36250D), BnaA.CCR2.b (BnaA07g35280D), BnaC.CCR2.a (BnaC02g46610D), and BnaC.CCR2.b (BnaC06g40190D), whose sequences were 97.9, 96.4, 97.6, and 95.8% homologous, respectively, to that of AtCCR2. Homology analysis indicated that the BnCCR1 and BnCCR2 genes may have conserved functions similar to those of AtCCR1 and AtCCR2, respectively. In addition, for each BnCCR gene, the closest homologous gene was found in the corresponding progenitor genomes (B. oleracea and B. rapa; Figure 2A), suggesting that BnCCR genes may not have undergone duplication or deletion after B. napus formation due to recent allopolyploidy between ancestors of B. oleracea and B. rapa.

The transcriptome data indicated that BnCCR1 genes were highly expressed in all tissues of B. napus, while the BnCCR2 genes were not expressed or expressed at low levels in most tissues (Figure 2B). However, all four copies of BnCCR2 were induced after S. sclerotiorum infection, while only two copies of BnCCR1 were moderately induced (Figure 1F). These results are consistent with those of previous studies in Arabidopsis showing that AtCCR1 and AtCCR2 are differentially expressed during development and in response to pathogen infection (Lauvergeat et al., 2001). Most BnCCR2 genes were expressed to a much greater degree in the resistant line J964 or resistant bulk (mixed pools of resistant lines) group than in the susceptible line J902 or susceptible bulk (mixed pools of susceptible lines) group at 48 hpi (Figures 2C,D). The most strongly induced BnCCR2 gene was BnaC.CCR2.b, whose expression increased 427.5-fold in the resistant bulk group but only 7.6-fold in the susceptible bulk group (Figure 2D). These findings suggest that BnCCR2 genes, especially BnaC.CCR2.b, are important for Sclerotinia resistance.

To investigate whether overexpression of BnaC.CCR2.b in B. napus can increase resistance to S. sclerotiorum, we introduced the BnaC.CCR2.b gene into B. napus line J9712 (a susceptible line) via a pMDC83 binary expression vector containing a hygromycin B resistance gene and the BnaC.CCR2.b gene under the control of the CaMV 35S promoter (Figure 3A). The 35S:BnaC.CCR2.b vector was subsequently transformed into B. napus via Agrobacterium-mediated transformation. Seven independent overexpression transgenic lines of the T₀ generation were ultimately obtained (named OE-1 to OE-7). We performed qRT-PCR to measure the expression of BnaC.CCR2.b in the transgenic lines and found that its expression in all seven transgenic lines was higher than that in J9712 and empty vector (EV)-transformed control plants (Figure 3B). The expression level of BnaC.CCR2.b in the OE-1/2/5/6 transgenic lines of the T₁ generation was further measured, the results of which were consistent with the results in the T₀ generation (Figure 3C). The OE-1/2/5/6 transgenic lines with high and stable expression of BnaC.CCR2.b were selected for subsequent experiments.

To further confirm whether BnaC.CCR2.b is related to resistance to S. sclerotiorum, we evaluated the Sclerotinia resistance of BnaC.CCR2.b overexpression lines in the T₂ and T₃ generations at the flower-termination stage. The stem inoculation experiment results showed that, compared with those of J9712 and the EV-transformed plants, the stem lesions of the plants of the overexpression lines expanded more slowly (Figure 4A). Statistical analysis showed that, compared with that of the J9712 line, the stem lesion length of the OE-1/2/5/6 transgenic lines in the T₂ and T₃ generations was reduced by 12.2–33.7% and 32.5–37.3%, respectively, at 7 dpi (Figures 4B,C). The lesion expanded further to a large part of the stem of J9712 at 20 dpi, while the lesion was restricted to approximately 6–8cm on the stems of the overexpression plants (Figure 4D). Taken together, the above results indicate that BnaC.CCR2.b positively regulates B. napus resistance to S. sclerotiorum.

To determine whether overexpression of BnaC.CCR2.b in B. napus would change the accumulation of lignin, cross-sections of the stems of overexpression lines (T₂) and control plants at the flower-termination stage were strained with Wiesner reagent to evaluate the lignin deposition. Phloroglucinol-HCl staining of lignin in whole cross-sections showed that the staining was more intense in the overexpression transgenic lines than in the J9712 plants and EV-transformed lines (Figure 5A). Staining of the manually cut cross-sections revealed that, in the overexpression transgenic lines, the number of vascular bundles did not change significantly; however, individual vascular bundles became wider, the xylem layer became thicker, and the staining was more intense in the overexpression lines compared with the J9712 and EV lines (Figure 5B). Chemical analysis indicated that the overexpression transgenic lines had a higher lignin content (21.6–22.2%) than the J9712 plants (19.3%) and EV-transformed lines (19.2%; Figure 5C). Taken together, these results indicate that the overexpression of BnaC.CCR2.b can significantly increase the lignin accumulation in the stems of B. napus, which might thus limit the invasion and spread of S. sclerotiorum.

In addition, the agronomic traits of BnaC.CCR2.b-overexpressing plants in the T₂ generation were investigated, and no significant differences between the transgenic plants and J9712 plants were detected (Supplementary Table S2).

To assess whether overexpression of BnaC.CCR2.b in B. napus alters the expression of genes in the lignin synthesis pathway, the expression levels of key lignin synthesis pathway genes BnPAL2, BnPAL1, BnC4H, Bn4CL1, BnC3H, BnCCoAOM, BnFAH1, BnCCR1, BnCCR2, BnCOMT, BnCAD4, and BnCAD5 were measured via qRT-PCR analyses. The results showed that the expression of most of the lignin synthesis-related genes (BnPAL2, BnC4H, BnC3H, BnCCoAOM, BnCCR1, BnCOMT, and BnCAD5) was upregulated in the stems of OE-6 plants compared with J9712 plants under normal growth conditions (before inoculation, 0 hpi; Figure 6). These results suggested that overexpression of BnaC.CCR2.b in B. napus induced the lignin synthesis pathway, which altered the lignin content in the stems of plants under normal growth conditions.

We further examined the expression of genes related to the lignin synthesis pathway in plants under infection with S. sclerotiorum. The results showed that the expression levels of genes related to lignin synthesis in the OE-6 and J9712 lines changed after S. sclerotiorum infection. In both lines, the differences in the levels of these genes were most pronounced from 48 to 72 hpi, and the number of differentially expressed genes was highest at 72 hpi. However, the expression levels of the Bn4CL1 and BnCCR1 genes appeared to be moderately altered. Overall, BnCAD4 and BnFAH1 were drastically downregulated in both lines from 48 to 72 hpi, while BnPAL2, BnPAL1, and BnC4H were upregulated (Figure 6). It was also found that the expression changes of some lignin biosynthesis-related genes (BnPAL1, BnPAL2, BnC3H, BnCCoAOM, BnFAH1, BnCCR2, and BnCOMT) were more significant in OE-6 than in J9712 (Figure 6), suggesting that the lignin synthesis pathway in the BnaC.CCR2.b overexpression line was induced more drastically. Taken together, these results suggest that BnCCR2 is involved in pathogen-induced lignification, which is also important for increasing resistance to S. sclerotiorum.