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). 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