Fig 1.
Symptom development and changes in photoassimilate transport routes in oilseed rape roots during P. brassicae infection.
(A) Clubroot phenotypes of the susceptible oilseed rape cultivar ‘Westar’ at different days post-inoculation (dpi). Plants were inoculated with field-collected resting spores of P. brassicae. Scale bar = 5 cm. (B) Cross sections of clubroots corresponding to the tissues in (A), stained with toluidine blue. Red arrows indicate plasmodia of P. brassicae. Scale bar = 50 μm. (C) Changes in assimilate transport routes in the mature zone of the primary root of ‘Westar’ at different infection stages, visualized by CFDA tracer loading. Scale bars = 200 μm. Yellow dashed circles indicate the vascular tissue region, while red dashed circles indicate the cortical region. Regions with fluorescence represent symplastic transport of assimilates, whereas regions without fluorescence represent apoplastic transport.
Fig 2.
BnaA07.SUC2 is involved in sugar acquisition by P. brassicae from the host.
(A) Phylogenetic analysis of 17 differentially expressed oilseed rape SUC genes derived from transcriptome sequencing of P. brassicae-inoculated roots, with 9 SUC genes from Arabidopsis thaliana included as references. (B) Heatmap showing expression profiles of the 17 SUC genes in P. brassicae-infected (Pb) and control (CK) roots. Genes in red boxes were selected as candidates based on their significantly induced expression at 21 and 28 dpi. Data are shown as transcripts per million (TPM) values obtained from the transcriptome dataset. (C) Functional characterization of sucrose transport activity by yeast complementation. The sucrose uptake‑deficient yeast strain SUSY7/ura3 was transformed with empty vector pDR196 (negative control), AtSUC2 (positive control), BnaA07.SUC2, or BnaC07.SUC2. Transformants were spotted on SD/-Ura medium containing sucrose as the sole carbon source. Yeast expressing BnaA07.SUC2 or BnaC07.SUC2 partially restored growth, whereas the empty vector did not. (D) Expression of BnaA07.SUC2 in oilseed rape roots was analyzed by qPCR at 2, 4, 7, 14, 21, and 28 dpi with P. brassicae, using uninoculated roots at the corresponding time points as controls. Data are presented as means ± SD (n = 3). *P < 0.05 (two-way ANOVA followed by Tukey’s test).
Fig 3.
Characterization of BnaA07.SUC2 transport function and localization.
(A) Sucrose uptake assay in Xenopus laevis oocytes expressing BnaA07.SUC2. Uptake was measured at pH 5.5 and pH 7.5 over a 0-8 h period. Water-injected oocytes served as negative control. Data represent means ± SD (n = 3). *P < 0.05 (two‑way ANOVA with Tukey’s test). (B) Current-voltage (I/V) relationship of BnaA07.SUC2‑mediated steady‑state currents. Currents were recorded at pH 5.5 or pH 7.5 using TEVC with voltage steps from +40 mV to –120 mV. Data are shown as means ± SE (n = 6). *P < 0.05 (one‑way ANOVA with Tukey’s test). (C) Subcellular localization of BnaA07.SUC2 in Nicotiana benthamiana epidermal cells. AtPIP2-mCherry was used as a plasma membrane marker. Scale bars = 10 µm. (D) Spatial expression pattern of BnaA07.SUC2 transcripts in oilseed rape roots by FISH. Cross‑sections from P. brassicae-infected (Pb) and control (CK) roots were hybridized with Cy3‑labeled antisense probes. Nuclei were stained with DAPI. Scale bars = 200, 500 µm.
Fig 4.
BnaA07.SUC2 enhances host susceptibility.
(A) Phenotypes of A. thaliana at 28 dpi: wild-type (WT, Col-0), the heterozygous AtSUC2/Atsuc2 mutant, and the AtSUC2/Atsuc2 mutant overexpressing (OE) BnaA07.SUC2 (AtSUC2/Atsuc2/BnaA07.SUC2-OE). Scale bar = 2 cm. (B) Clubroot diameter of A. thaliana lines shown in (A). Data are presented as mean ± SD (n = 6). *P < 0.05 (one-way ANOVA, Dunnett T3’s test). (C) Relative P. brassicae biomass at 28 dpi. P. brassicae biomass was assessed by qPCR and calculated using the 2-ΔΔCT method. Data are presented as mean ± SD (n = 3). *P < 0.05 (one-way ANOVA, Dunnett T3’s). (D) Sequencing chromatograms of the edited sites in two independent knockout lines (BnaA07.suc2-KO) of oilseed rape (Westar). Mutation sites are highlighted in red. (E) Clubroot phenotypes of different oilseed rape lines at 28 dpi. BnaA07.SUC2-OE#1 and BnaA07.suc2-KO#8 lines. Scale bar = 5 cm. (F) Toluidine blue-stained paraffin cross sections showing pathogen proliferation within root cells in (E). Scale bars: 500 μm (whole-section image), 50 μm (inset). Red arrows indicate P. brassicae within root cells. (G)-(I) Disease quantification of BnaA07.SUC2-OE and BnaA07.suc2-KO oilseed rape lines compared with WT at 28 dpi. (G) Clubroot diameter, (H) disease index, and (I) relative P. brassicae biomass. Data are presented as mean ± SD (n = 15 for diameter; n = 3 for disease index and biomass). *P < 0.05 (one-way ANOVA with Dunnett T3’s test).
Fig 5.
BnaA05.MYC2 confers clubroot resistance via repressing BnaA07.SUC2 expression.
(A) A yeast one-hybrid assay showing the binding of BnaA05.MYC2 to the promoter of BnaA07.SUC2. (B) Dual-luciferase reporter assays investigating the regulatory relationship between BnaA05.MYC2 and the BnaA07.SUC2 promoter. 1, 2, and 4 serve as controls. Data are presented as means ± SD (n = 3). *P < 0.05 (one-way ANOVA with Tukey’s test). (C) Electrophoretic mobility shift assays confirming direct binding of BnaA05.MYC2 protein to the BnaA07.SUC2 promoter. Binding was competed by an excess of unlabeled wild-type probe (competitor), but not by mutant probe. Mutated nucleotides in the mutant probe are highlighted in red. “–” and “+” denote absence or presence of the indicated component (D) Clubroot phenotypes of oilseed rape at 28 dpi: WT, BnaA05.MYC2-OE line, and a hybrid line generated by crossing BnaA05.MYC2-OE#1 with BnaA07.SUC2-OE#1 (BnaA05.MYC2-OE/BnaA07.SUC2-OE). Scale bar = 5 cm. (E)-(G) Disease quantification of BnaA05.MYC2-OE oilseed rape lines compared with WT at 28 dpi. (E) Clubroot diameter, (F) disease index, and (G) relative P. brassicae biomass. Data are mean ± SD (n = 12 for diameter; n = 3 for disease index and biomass). *P < 0.05 (one-way ANOVA with Dunnett T3’s test). (H)-(I) Expression levels of (H) BnaA05.MYC2 and (I) BnaA07.SUC2 in two BnaA05.MYC2-OE oilseed rape lines relative to WT. Data are mean ± SD (n = 3). *P < 0.05 (one-way ANOVA with Dunnett T3’s test). (J)-(L) Disease quantification of BnaA05.MYC2-OE#1/BnaA07.SUC2-OE#1 oilseed rape lines compared with BnaA05.MYC2-OE#1 line at 28 dpi. (J) Clubroot diameter, (K) disease index and (L) relative P. brassicae biomass. Data are presented as means ± SD (n = 9 for diameter; n = 3 for disease index and biomass). *P < 0.05 (one-way ANOVA with Dunnett T3’s test).
Fig 6.
BnaA05.MYC2 was regulated by JA signaling during P. brassicae infection.
(A) Heatmap showing the expression profiles of key JA/JA-Ile biosynthetic genes in oilseed rape roots at different time points after infection. For each time point, the two columns represent the control (left) and P. brassicae-inoculated (right) samples. The expression profile of BnaA05.MYC2 (in red) is shown below the heatmap. Data were derived from transcriptome sequencing of P. brassicae-infected oilseed rape roots. (B) Expression of BnaA05.MYC2 in oilseed rape root tissues following MeJA treatment relative to untreated controls at the corresponding time points. Data are presented as mean ± SD (n = 3). *P < 0.05 (two‑way ANOVA with Tukey’s test). (C) qPCR analysis of BnaA05.MYC2 expression in oilseed rape roots at different time points after P. brassicae infection, relative to uninoculated controls at the corresponding time points. Data are presented as mean ± SD (n = 3). *P < 0.05 (two-way ANOVA with Tukey’s test). (D)-(E) Concentrations of (D) JA and (E) JA-Ile in inoculated oilseed rape roots relative to uninoculated controls at the corresponding time points, quantified by UPLC‑MS/MS. Data are presented as mean ± SD (n = 3). *P < 0.05 (two-way ANOVA with Tukey’s test).
Fig 7.
A working model of JA-mediated regulatory of BnaA07.SUC2 during P. brassicae infection.
The model depicts the molecular events at two distinct stages: the early phase (before 14 dpi) and the late phase (after 14 dpi). Early phase (before 14 dpi): Upon infection with P. brassicae, the JA signaling pathway is activated, leading to elevated levels of JA and JA-Ile. This upregulates the expression of BnaA05.MYC2, which in turn represses BnaA07.SUC2 expression. Late phase (after 14 dpi): The JA signaling pathway is suppressed, accompanied by reduced levels of JA and JA-Ile. Consequently, BnaA05.MYC2 expression declines, which attenuates its repressive effect on BnaA07.SUC2. This results in significant upregulation of BnaA07.SUC2, facilitating sugar uptake and promoting the proliferation of P. brassicae. Black flat arrows indicate enhanced inhibitory effects, gray flat arrows represent weakened inhibitory effects, black pointed arrows indicate enhanced promoting effects, and gray pointed arrows represent weakened promoting effects.