Table 1

List of genes targeted for TRV-mediated silencing"

Table 2

Primers used in this study"

Table 3

sgRNA sequences"

Fig. 1

Workflow of TRV-mediated gene silencing in Brassica napus A: a 300-500 bp core sequence of the target gene was cloned into the TRV2 vector, the recombinant pTRV2 vector, empty pTRV2 vector, and pTRV1 vector were separately transformed into Agrobacterium tumefaciens GV3101, cultured in LB liquid medium containing rifampicin (Rif), gentamicin (GMS), and kanamycin (Kana), collected by centrifugation, and resuspended. The pTRV1 and pTRV2 bacterial suspensions were then mixed at a 1:1 (v/v). B: thirty to fifty B. napus seeds were sterilized with 75% ethanol for 1-2 min, rinsed three times with sterile water, treated with 5 mmol L-1 GA3 for 1-2 h to promote germination, rinsed three additional times, and incubated on moist filter paper in the dark for 48 h to induce germination. C: germinated seeds were immersed in freshly prepared Agrobacterium infiltration solution, vacuum-infiltrated at 15 psi for 10 min, placed on 1/2 MS solid medium and incubated in the dark for 24 h, rinsed, and then sown in greenhouse soil under a 16 h light/8 h dark photoperiod until maturity."

Fig. 2

Amino acid sequence alignment of JMJ13 from Arabidopsis thaliana and Brassica napus Multiple amino acid sequence alignment of AtJMJ13 and its homologs in Brassica napus. Sequences were aligned using the ClustalW algorithm and visualized with ESPript 3.0. In the aligned sequences, identical residues are highlighted with a black background, similar residues are shaded in gray, and gaps introduced to optimize the alignment are indicated by dashes."

Fig. 3

Amino acid sequence alignment of SNAP33, VAMP722, and CDF3 from Arabidopsis thaliana and their homologs in Brassica napus Multiple amino acid sequence alignment of AtSNAP33, AtVAMP722, AtCDF3, and their homologs in Brassica napus. Sequences were aligned using the ClustalW algorithm and visualized with ESPript 3.0. In the aligned sequences, identical residues are highlighted with a black background, similar residues are shaded in gray, and gaps introduced to optimize the alignment are indicated by dashes."

Fig. 4

Conserved domain architecture of JMJ13, SNAP33, VAMP722, and CDF3 from Arabidopsis thaliana and Brassica napus A-D: schematic diagrams of the conserved domains in JMJ13, SNAP33, VAMP722, and CDF3. The horizontal axis represents protein sequence length, and boxes of different colors represent different conserved domains."

Fig. 5

Detection of target gene expression in seedling leaves after TRV-mediated gene silencing A-D: relative expression levels of the four target genes in leaf tissues after TRV-mediated silencing, as determined by RT-qPCR. Ev: empty vector. Data were normalized using Actin as the internal reference gene and are presented as the mean ± standard deviation of three independent biological replicates. ns: no significant difference; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001."

Fig. 6

Detection of target gene expression in flower buds after TRV-mediated gene silencing A-B: relative expression levels of BnaJMJ13 and BnaSNAP33 in flower buds after TRV-mediated silencing, as determined by RT-qPCR. Ev: empty vector. Data were normalized using Actin as the internal reference gene and are presented as the mean ± standard deviation of three independent biological replicates. ns: no significant difference; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001."

Fig. 7

TRV-mediated silencing of BnaJMJ13 promotes early flowering in Brassica napus Ev: plants transformed with empty vector; ****: P < 0.0001."

Fig. 8

Pollen adhesion and germination rates in TRV-mediated BnaSNAP33-silenced lines A-B: representative images showing pollen adhesion and germination after pollen from the control plants (Ev) and VIGS-Bnasnap33 lines was simultaneously pollinated onto stigmas of control plants (Ev). The upper panels show bright-field images, and the lower panels show fluorescence images. At least six stigmas were observed for each line. Ev: empty vector. Scale bar = 200 μm. C: quantification of pollen grains adhering to stigmas after self-pollination and cross-pollination. D: quantification of pollen grain germination on stigmas after self-pollination and cross-pollination. In all crosses, the female parent was the control plant (Ev, plants transformed with the empty vector), and the male parents were Ev and VIGS-BnaSNAP33 lines. **: P < 0.01; ***: P < 0.001."

Fig. 9

Editing results of bnasnap33 mutants A: gene structure of BnaSNAP33 and positions of the target sites. B: mutations at the target sites of BnaSNAP33 in T1 plants. Blue regions indicate mutations, and red regions indicate target sites. Ref: reference sequence; i: insertion mutation; d: deletion mutation."

Fig. 10

Pollen adhesion and germination rates in bnasnap33 mutants A-C: representative images showing pollen adhesion and germination after pollen from wild-type (WT) plants and bnasnap33 mutants was pollinated onto WT stigmas. The upper panels show bright-field images, and the lower panels show fluorescence images. At least 10 stigmas were observed for each line. Scale bar = 200 μm. D: quantification of pollen grains adhering to stigmas after self-pollination and cross-pollination. E: quantification of pollen grain germination on stigmas after self-pollination and cross-pollination. In all crosses, the female parent was WT, and the male parent was either WT or a bnasnap33 mutant. ****: P < 0.0001."