Generation of Brassica napus with enhanced Sclerotinia sclerotiorum resistance through CRISPR /Cas9‐mediated inhibition of the PROTEOLYSIS6 N‐degron pathway

Sclerotinia sclerotiorum is a devastating necrotrophic fungal pathogen with an exceptionally broad host range, infecting over 700 plant species. Brassicaceae species are particularly affected by this pathogen, including economically important species such as Brassica napus and various vegetable plants like Brassica rapa L. ssp. pekinensis (Liu et al., 2025). This pathogen causes significant reductions in both yield and quality, and to date, no fully resistant germplasm has been identified. Consequently, the identification and genetic manipulation of disease-resistant genes represent a pivotal strategy for enhancing host resistance against S. sclerotiorum. Accumulating evidence indicates that biotic stresses, including those by necrotrophic pathogens such as Botrytis cinerea (Valeri et al., 2021) and in the clubroot agent Plasmodiophora brassicae (Gravot et al., 2016) and bacterium Agrobacterium tumifaciens (Kerpen et al., 2019), induce hypoxic stress in plant tissues. Intriguingly, studies in Arabidopsis thaliana demonstrate that hypoxia may compromise plant immunity by suppressing PAMP-triggered immunity (Mooney et al., 2024), highlighting the potential role of oxygen (O2) deprivation as a common aspect during different biotic stresses. In this study, we applied multiple approaches to demonstrate that infection by S. sclerotiorum induces hypoxic stress in host plants. Building on this finding, we used CRISPR/Cas9-mediated knockout of BnaPROTEOLYSIS (PRT)6 to disrupt the low-oxygen-associated N-degron pathway, thereby conferring strong resistance to S. sclerotiorum in B. napus. To investigate whether S. sclerotiorum infection induces hypoxic stress in host plants, a comparative RNA-Seq analysis was conducted on B. napus. Plants were subjected to either 3-h submergence, which has been used as a well-established hypoxia treatment, or 12-h S. sclerotiorum inoculation. Transcriptomic analysis revealed that 34 out of 43 (79%) hypoxia-responsive marker genes (Mustroph et al., 2009) exhibited nearly identical upregulation patterns under both conditions, strongly suggesting that S. sclerotiorum infection triggers a strong hypoxia response (Fig. 1a; Supporting Information Fig. S1). This finding was further substantiated using a planar O2 sensor system, which comprises an O2-sensitive optical foil integrated with a high-definition detection microscope unit for detailed spatial measurement of O2 tension (Figs 1b, S2). The measured ratio shows a negative correlation with the relative O2 concentration. Real-time monitoring over a 500-s period demonstrated that O2 concentrations in infected tissues were consistently and significantly lower than in adjacent uninfected regions, establishing a clear negative correlation between pathogen infection and local O2 availability (Fig. 1c; Video S1). To accurately quantify O2 concentration at the cellular level, we utilized high-resolution microsensor technology to measure O2 concentrations in S. sclerotiorum infected and uninfected abaxial lamina (Figs 1d, S2). The O2 tension in uninfected leaf tissues was c. 20 kPa, whereas in infected tissues it decreased to c. 5 kPa (Fig. 1e). These measurements confirmed a statistically significant reduction in O2 availability in pathogen-infected leaves compared to uninfected controls. The PLANT CYSTEINE OXIDASE (PCO) N-degron pathway is the only known mechanism mediating cellular sensing and response to hypoxia in plants (Gibbs et al., 2011; Licausi et al., 2011). Central to this pathway are the Group VII Ethylene Response Factor (ERFVII) transcription factors (in A. thaliana RAP2.12, RAP2.2, RAP2.3, HRE1, and HRE2), which function as physiological substrates for oxygen-dependent N-degron-mediated proteasomal degradation catalyzed by candidate E3 ubiquitin ligase PROTEOLYSIS 6 (PRT6) (Zubrycka et al., 2023) (Fig. 1f). Transduction of oxygen sensing is dependent on Cys-2 of ERFVIIs, in the presence of oxygen this residue is oxidized resulting in protein degradation, whereas in hypoxia the protein is stable (Manrique Gil et al., 2025). This residue is part of a larger amino-terminal conserved region in ERFVIIs (Nakano et al., 2006). We identified the conserved amino-terminal residues of BnaERFVIIs (Fig. 1g–i) and generated GUS reporter proteins beginning with the ERFVII sequences MCGGAI/VI (Fig. 1j) to investigate the influence of S. sclerotiorum inoculation on stabilization through these sequences. Whereas little GUS activity was observed in mock leaves with the wild-type sequences, substitution of Cys-2 with Ala (a residue that does not act as a destabilizing residue in the N-degron pathway) resulted in constitutive activity of GUS regardless of infection. Following infection with S. sclerotiorum, GUS reporter activity using both wild-type sequences greatly increased (Fig. 1k). These results suggest that hypoxic conditions induced by S. sclerotiorum infection promote protein stabilization through Cys-2, thereby providing a mechanistic explanation for how reduced oxygen availability enhances protein stability and function. To further investigate the functional role of this pathway in plant resistance to S. sclerotiorum, we performed S. sclerotiorum inoculation assays using A. thaliana mutants defective in either all five ERFVII transcription factors (pentuple mutant erfVII (Abbas et al., 2015)) or the N-recognin PRT6 (prt6-1) (Fig. 1l). Quantitative analysis revealed that leaves of erfVII mutants developed significantly larger lesion areas compared to wild-type plants following S. sclerotiorum inoculation (Fig. 1m), indicating that ERFVII transcription factors play an important role in defense against this pathogen. By contrast, the prt6-1 mutant (in which substrates are stable) exhibited enhanced resistance, with lesion areas reduced by 16.7% relative to controls. These data demonstrate that the PRT6 N-degron pathway negatively regulates plant defense against S. sclerotiorum, likely through its control of ERFVII protein stability. Our findings suggest that targeted genetic disruption of PRT6 function represents a promising strategy for engineering S. sclerotiorum resistance in plants. Brassica napus is not only a crop of major global economic importance but is also widely regarded as a model system for studying polyploidy within the Brassicaceae family, and specifically in the genus Brassica. In B. napus, we identified six PRT6 homoeologs through genome-wide analysis, with four homoeologs (BnaA02.PRT6 (A), BnaC02.PRT6 (B), BnaA10.PRT6 (C), and BnaC09.PRT6 (D)) exhibiting relatively high expression levels across multiple tissues (Fig. S3). Using CRISPR/Cas9 genome editing, we designed four specific guide RNAs targeting conserved functional domains in these homoeologs (Fig. 2a,b). Among the 13 positive transgenic T0 plants, five (38.5%) exhibited successful genome editing events. These included two single mutants (#2 and #6), one double mutant (#30), one triple mutant (#29), and one quadruple mutant (#23) (Fig. 2c). All mutations were heterozygous in the T0 generation. Following two generations of segregation and genotyping, we isolated plants carrying stable homozygous and multiplex edited alleles, including single mutant bnac02.prt6 (bb), double mutant bnac02.prt6 bnac09.prt6 (bbdd), triple mutant bnac02.prt6 bnaa10.prt6 bnac09.prt6 (aabbdd), and quadruple mutant bnaa02.prt6 bnac02.prt6 bnaa10.prt6 bnac09.prt6 (aabbccdd) genotypes. These were verified via PCR amplification followed by Sanger sequencing using allele-specific primers (Fig. S4). Previously, it was shown that in the Arabidopsis prt6 mutant, hypoxia response genes are constitutively upregulated (Gibbs et al., 2011). Here, we also found strong upregulation of hypoxia key response genes (including ADH1 and PDC2) in the bnaprt6 (aabbccdd) mutant compared to wild-type plants (Fig. S5). Pathogenicity assays using S. sclerotiorum isolates revealed a striking gene dosage effect, with disease resistance strongly correlating with the number of disrupted PRT6 copies (Fig. 2d,e). Quantitative analysis showed that while single mutants (bb) exhibited only a modest, non-significant reduction of 1.4% in lesion area, higher-order mutants demonstrated progressively greater resistance. Double mutants (bbdd) showed 11.7% reduction, triple mutants (bbccdd) 27.3%, and quadruple mutants (aabbccdd) 29.1% compared to wild-type controls (Fig. 2e). Comprehensive evaluation of key agronomic traits in field-grown plants revealed that these PRT6 null lines exhibited no significant alterations in plant height, branch number, main inflorescence silique number, or thousand-seed weight compared to wild-type plants (Fig. 2f). In addition, no significant impact on rapeseed quality was observed (Table S1). These findings suggest that targeted disruption of BnaPRT6 genes can enhance S. sclerotiorum resistance without compromising essential agronomic performance, a crucial consideration for crop improvement strategies. To facilitate molecular marker-assisted selection, allele-specific markers were developed to accurately distinguish among wild-type, heterozygous, and homozygous mutant genotypes of four BnaPRT6 homoeologs (Figs 2g, S6). Future studies should evaluate the performance of these mutants under field conditions, with particular emphasis on resistance of the stem and other key organs to S. sclerotiorum. In summary, our study demonstrates that S. sclerotiorum infection induces hypoxic conditions in plant tissues. Furthermore, the A. thaliana prt6 mutant exhibits enhanced resistance to this fungal pathogen, as has been shown for other biotic stresses (Vicente et al., 2019; Valeri et al., 2021), indicating an important role for the PRT6 N-degron pathway in resistance to this fungus. Based on these findings, we successfully generated Sclerotinia-resistant B. napus by disrupting the N-degron pathway through targeted knockout of BnaPRT6 using CRISPR/Cas9 technology (Fig. 2h). The evolutionary conservation of the PRT6 N-degron pathway across higher plants, combined with our demonstration of its role in S. sclerotiorum resistance, highlights its potential as a broad-spectrum target against S. sclerotiorum. Notably, PRT6 orthologs show high sequence conservation across the Brassicaceae family (Table S2), suggesting our findings will be translatable to other economically important species vulnerable to S. sclerotiorum. Beyond the findings presented in this study, removal of PRT6 function has previously been shown to confer tolerance to abiotic stresses such as drought, salinity, and waterlogging in Arabidopsis and barley (Mendiondo et al., 2016; Vicente et al., 2019). This raises the possibility that the genetic resources generated here may underpin broad-spectrum tolerance to both biotic and abiotic environmental challenges, an avenue that warrants further investigation. The Columbia-0 (Col-0) ecotype of Arabidopsis thaliana, originally obtained from the Nottingham Arabidopsis Stock Centre (NASC), was used as the wild-type background for this study. Arabidopsis mutant seeds erfVII and prt6-1 utilized in this study were previously developed (Gibbs et al., 2011; Weits et al., 2014; Abbas et al., 2015). Brassica napus cultivar J9712 was used as explant for oilseed rape hypocotyl transformation, that is originally provided by Prof. Yongming Zhou (Huazhong Agricultural University, Wuhan, Hubei, China). BnaPRT6 genome sequence, gene coding sequence (CDS), and protein sequence were acquired from an online database (https://yanglab.hzau.edu.cn/BnIR). Four sequence-specific single guide RNAs (sgRNAs) were designed using the online tool CRISPR-P 2.0 (http://cbi.hzau.edu.cn/CRISPR2/). These sgRNAs were incorporated into a CRISPR/Cas9 construct using the pRGE32 multiplex genome targeting vector, generously provided by Prof. Kabin Xie (Huazhong Agricultural University, Wuhan, Hubei, China). The genome-edited plants were obtained through Agrobacterium-mediated transformation. Positive transformants harboring T-DNA insertions were initially screened by PCR amplification. Homozygous BnaPRT6 knockout mutants were subsequently confirmed using PCR followed by Sanger sequencing. Allele-specific molecular markers were designed according to the precise nucleotide insertion sites in each homoeologous gene (Lin et al., 2024). After obtaining the mutant lines, specific primers were designed to detect potential off-target mutations in the non-targeted PRT6 homeologs (BnaA03.PRT6 and BnaC03.PRT6) (Table S3). Arabidopsis seeds were germinated on vertical plates containing an agar medium, which consisted of 1/2 Murashige & Skoog basal salt mixture (Duchefa, the Netherland), 0.8% plant agar, and 1% sucrose (Sigma-Aldrich, Darmstadt, Germany). The seeds were stratified at 4°C in the dark for 48 h before being transferred to a growth environment at 22°C with a 16 h : 8 h light : dark photoperiod. After 7 d, the seedlings were transplanted into pots with soil for continued growth. Similarly, B. napus plants were cultivated in a climate-controlled chamber under the same light conditions (16-h light/8-h dark photoperiod) at 22°C, with a maintained humidity level of 65%. To assess susceptibility to Sclerotinia sclerotiorum (Lib.) de Bary, wild-type J9712 and mutant plants were cultivated in the field for c. 5 months before leaf collection. The protein sequences of ERFVII transcription factors (RAP2.12, RAP2.2, RAP2.3, HRE1, and HRE2) in B. napus Zhongshuang11 (ZS11) were retrieved from the online database BnIR (https://yanglab.hzau.edu.cn/BnIR). The first 16 N-terminal amino acids of each ERFVII protein were extracted for conservation analysis. A sequence logo was generated using WebLogo (http://weblogo.berkeley.edu/), and a pie chart was prepared with Microsoft Excel. A phylogenetic tree was constructed based on the amino acid sequences of six BnaPRT6 homeologs obtained from the BnTIR database (http://yanglab.hzau.edu.cn/BnTIR). The tree was generated in MEGA11 using the neighbor-joining method with 1000 bootstrap replicates. Expression patterns of the six BnaPRT6 homeologs across different plant tissues and organs were also retrieved from the BnTIR database. Information on PRT6 orthologs in plant species of the Brassicaceae family was found in the Brassicaceae Database (BRAD) (http://brassicadb.cn). The coding sequences of MCGGAII-GUS, MCGGAVI-GUS, and MAGGAVI-GUS were cloned into the binary vector pMDC83 and subsequently introduced into the B. napus line J9712 via Agrobacterium-mediated transformation. Putative transgenic plants containing the T-DNA insertions were first identified by PCR screening. Homozygous T2 lines, derived from these primary transformants, were then used for GUS staining analysis. Detached leaves from transgenic plants were inoculated with a 7-mm-diameter S. sclerotiorum-colonized agar plug. Leaves were collected at 16 h post-inoculation, allowing sufficient hyphal colonization while retaining structural integrity for subsequent analyses. As a control, mock inoculation used a sterile agar plug of identical size. A 10-mm-diameter leaf disc was then excised from the inoculation site for histochemical GUS staining. Histochemical GUS staining was conducted according to Jefferson et al. (1987). Briefly, leaf tissues were incubated overnight at 37°C in staining solution containing 0.5 mg ml−1 X-gluc, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, 0.1% Triton X-100, and 10 mM sodium phosphate buffer (pH = 7.2). Subsequently, samples were destained in 75% (v/v) ethanol and imaged under a stereomicroscope (Leica EZ4W, Bensheim, Germany) equipped with a digital camera. Brassica napus seedlings grown on Whatman paper were harvested 6 d post-germination for total RNA extraction. cDNA was synthesized using the HiScript® II first Strand cDNA Synthesis Kit (Vazyme, Nanjing, China), which incorporates a gDNA removal step to preclude genomic DNA contamination. Quantitative real-time PCR (qRT-PCR) was then performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA) with PowerUp™ SYBR™ Green Master Mix (Applied Biosystems Inc.), in strict accordance with the manufacturer's protocols. The genes BnaActin7 (BnaC02G0037200ZS) and BnaUBC10 (BnaA10G0098100ZS) were employed as internal reference controls for normalization. Relative gene expression levels were calculated using the 2−∆∆CT method (Livak & Schmittgen, 2002). RNA-Seq analysis was conducted as previously described with minor modifications (Lin et al., 2024). Briefly, leaf samples from wild-type rapeseed plants (J9712) were either collected after 3 h of complete submergence in darkness, or after 12 h of inoculation with S. sclerotiorum, with three independent biological replicates for each condition. High-quality mRNA was extracted using the RNeasy Plant Mini Kit (Qiagen). RNA libraries were prepared and sequenced on an Illumina by China), of data in were initially for quality using The were then to the reference genome using with the B. napus genome as the genes were identified with a of and a of and of and was with The hypoxia-responsive genes in this study were based on previously under hypoxic conditions (Mustroph et al., We identified 43 genes in B. napus with identical to the hypoxia genes previously in Arabidopsis (Table S4). The S. sclerotiorum was on agar and USA) at in the was prepared by a agar plug with a of 7 from the of a S. sclerotiorum The leaves were collected for inoculation with S. sclerotiorum from the leaves were with 7 in agar as a The leaves of the B. napus plants were inoculated with S. sclerotiorum for The susceptibility of Arabidopsis leaves to S. sclerotiorum was using a inoculation Briefly, agar collected from the of S. sclerotiorum were the leaves and maintained for 20 The leaf inoculation method is a used for evaluation of S. sclerotiorum response in rapeseed and other et al., et al., et al., The lesion by S. sclerotiorum infection was at different with (Lin et al., 2024). 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Fig. for O2 level measurement with planar O2 sensor and in Brassica napus. Fig. analysis and expression of BnaPRT6 homeologs in Brassica napus. Fig. of mutations by PCR followed by Sanger sequencing. Fig. The analysis of genes revealed expression between the bnaprt6 quadruple mutant (aabbccdd) and the J9712 plants. Fig. The of allele-specific molecular markers in Brassica napus of and protein acid between different mutants and the wild-type cultivar J9712 in Brassica napus. Information on PRT6 orthologs in plant species of the Brassicaceae used in this study. The of marker Video Relative O2 levels in and Sclerotinia sclerotiorum infected areas with planar O2 is not for the or of Supporting Information by the than should be to the Central The is not for the or of by the than should be to the for the The with to in and in