The rice germplasm line CAAS-11308X315 (CX315) was sourced from the Germplasm Resources Center of CAAS and identified through systematic resistance screening against diverse Xoo strains under controlled inoculation conditions. Two susceptible rice cultivars, IR24 and JG30 used from laboratory-maintained materials as controls in comparison with the resistant line CX315. Statistical analysis of the data was performed using a two-tailed Student’s t-test. Plants were cultivated at CAAS experimental fields located in Beijing and Sanya, Hainan, under net house or open field conditions, with conventional water and fertilizer management practices.

The Xoo strains used to assess the resistance of CX315 to BB included races from China and other countries. These strains included PXO61, PXO86, PXO79, PXO71, PXO112, PXO99_A, PXO280, PXO339, and PXO341 from the Philippines; T7174, T7147, and T7133 from Japan; KXO85 and KXO576 from Korea; and HLJ72, HB17, NX42, ZHE173, GD1358, LN57, JS49-6, GX15-2, Yun17-3, LN44, and others from China. Xoo strains were cultured in NA medium (Polypeptone 5g/L, sucrose 10g/L, yeast extract 1g/L and beef extract 3g/L, Bacto agar 15g/L (for solid media)) at 28°C for three days, then resuspended in sterile distilled water and maintained OD₆₀₀ of 1.0 (measured using Eppendorf spectrometer 1000). Inoculation was performed using either the leaf-clipping method or needleless syringe infiltration, with lesion lengths measured at 14 days post-inoculation (dpi) for leaf clipping and 5 dpi for infiltration.

The susceptible rice cultivar IR24 was crossed with the resistant germplasm line CX315 to develop an F₂ mapping population through selfing of heterozygous F₁ plants. Both F₁ and F₂ populations were phenotypically screened for resistance or susceptibility following inoculation with T7174 strain. Segregation patterns in the F₂ population were analyzed using the Chi-square test (χ²)to determine the genetic basis of resistance. Polymorphic insertion-deletion (InDel) markers were developed based on the Shuhui498 reference indica genome (https://mbkbase.org/R498/) and used to identify genetic differences between the parents IR24 and CX315 ( Supplementary Table 2 ). Specific primers targeting these polymorphic regions were designed using the Shuhui498 genome sequence. Genomic DNA was extracted from F₂ individuals using the CTAB method. PCR amplification with polymorphic InDel markers was performed on these DNA samples, and PCR products were resolved by electrophoresis on 5% agarose gels to detect marker segregation. Linkage analysis was then conducted to map candidate genes associated with resistance.

Total RNA was extracted from plant samples using the Trizol reagent according to the manufacturer’s protocol (Rio et al., 2010). The extracted RNA was then reverse-transcribed into first-strand cDNA using the TIANScript-II RT Kit (Tiangen Biotech, Beijing). Quantitative real-time PCR (qRT-PCR) was performed using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme) on an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, USA). The sequences of primers used for qRT-PCR are given in Supplementary Table 5 . The 20μL reaction mixture contained 10μL of 2X SYBR Master Mix, 0.4μL each of forward and reverse primer, 2μL of cDNA template, and 7.2μL of nuclease-free water. The PCR program consisted of an initial denaturation at 95°C for 3 minutes, followed by 40 cycles of 95°C for 5 seconds and 60°C for 34 seconds. Relative gene expression levels were calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001), with the rice Actin gene serving as the internal reference.

Target sequences within the exon of the Xa4 gene were selected using the TargetDesign tool (http://skl.scau.edu.cn/targetdesign/). These sequences were ligated into sgRNA expression cassettes via overlapping PCR to generate two sgRNAs, sgRNA-U6a-T1 and sgRNA-U6b-T2 ( Supplementary Table 4 ). The sgRNA cassettes were cloned into the binary vector pYLCRISPR-Cas9-Xa4-KO using Golden Gate cloning (Ma et al., 2015). Agrobacterium-mediated transformation was performed to introduce the CRISPR construct into rice calli. Genomic DNA was extracted from transformed plant leaves using the CTAB method and used as template for PCR amplification with Xa4-KO-seq primers listed in Supplementary Table 4 . Sanger sequencing of the PCR products used for identification of knockout mutations in Xa4.

Total RNA was extracted from rice leaf tissues using the Trizol reagent, and RNA quality was assessed using a Nanodrop spectrophotometer and Bioanalyzer. RNA libraries were prepared using the TrueSeq RNA Library Prep Kit (Illumina) with fragmentation, reverse transcription, and amplification steps, followed by sequencing on an Illumina NovaSeq 6000 platform with paired-end 150 bp reads. Raw reads were processed to remove low-quality bases and adapter sequences using Trimmomatic and aligned to the rice reference genome Shuhui498 using STAR. Gene expression levels were quantified with feature Counts and analyzed for differential expression using DESeq2, considering a fold change ≥ 2 and an FDR < 0.05. Functional annotation of differentially expressed genes was performed using the NCBI Nr and GO databases, with KEGG pathway analysis to identify enriched biological processes. RNA-seq data was submitted to NCBI SRA database under project PRJNA1209632.

Initial screening of germplasm lines identified CX315 as highly resistant line to Xoo strains from the Philippines, China, Japan, and Korea. To ascertain the genetic resistance, CX315 and the susceptible cultivar JG30 were inoculated at the tillering stage with 21 representative Xoo strains. CX315 exhibited significantly robust resistance to all strains, with lesion lengths ranging from 0.1 to 6 cm, whereas JG30 showed consistent susceptibility, with lesion lengths exceeding 7 cm ( Figure 1A ). These findings highlight CX315 as a promising source of broad-spectrum resistance for bacterial blight in rice.

To determine whether CX315’s resistance is genetically controlled, we crossed CX315 (♂) and the susceptible cultivar IR24 (♀), and the resulting F₁ plants were evaluated after inoculation with the Xoo strain T7174. The F₁ plants exhibited resistance comparable to CX315, as evidenced by shorter lesion lengths ( Figure 1B ). Evaluation of F₂ population informed 1,767 plants exhibited resistance, while 555 plants were susceptible, fitting a 3:1 Mendelian segregation ratio (χ² = 1.4935, p = 0.222), thereby confirming that resistance in CX315 is controlled by a single dominant gene ( Supplementary Table 1 ). To further validate that this resistance is not only genetically inherited but also associated with active defense responses, we analyzed the expression of key pathogenesis-related (PR) genes following pathogen challenge. PR genes are molecular markers of plant immune activation, and their upregulation reflects the activation of basal and systemic defense pathways. qRT-PCR results showed significant induction of OsPR1a, OsPR1b, OsPR5, and OsPBZ1 in CX315 compared to JG30 ( Figure 1C ), indicating that CX315 confers a strong defense response upon infection. Additionally, the bacterial population of Xoo in CX315 was significantly lower than in JG30, as shown by reduced expression of the bacterial effector gene XopA ( Figure 1C ). These findings collectively support the hypothesis that CX315 harbors a genetically controlled resistance mechanism that actively suppresses pathogen proliferation through enhanced immune signaling.

To fine-map the R gene in CX315 and elucidate the genetic basis of its resistance, an F₂ mapping population was generated by selfing heterozygous F₁ plants derived from a cross between CX315 and IR24. This population was phenotyped for resistance to the Xoo strain T7174. To locate the resistance locus, 256 insertion-deletion (InDel) markers distributed across all 12 rice chromosomes were screened for polymorphism between CX315, IR24, and their F₂ progeny. Totally, 112 markers were polymorphic and used for genotyping. Linkage analysis based on recombination frequencies revealed that markers ID11–7 and M11-540, located on chromosome 11, were tightly associated with the resistance phenotype, with recombination events observed in 30 and 25 individuals, respectively ( Figure 2 ). Subsequent fine mapping narrowed the candidate interval to a 147.7 kb region between markers M11–588 and M11-602, with M11–489 showing complete co-segregation with the resistance trait. This high-resolution mapping region harbors 13 predicted open reading frames (ORFs) according to the reference genome (Shuhui498). Gene annotation within the 147.7 kb mapping interval on chromosome 11 identified a high-confidence candidate region for the novel resistance gene Xa50(t) ( Supplementary Table 3 ). While ORF9 is predicted to be Xa4 gene locus we named ORF9 to Xa4 subsequently. The presence of WAK, receptor-like kinase, and leucine-rich repeat encoding ORFs within the candidate region warrants further investigation to precisely identify the Xa50(t) gene.

Transcriptome profiling revealed significant differential gene expression (DEG) patterns in CX315 and JG30 at both 0 and 48 hpi with Xoo strain T7174, highlighting key molecular mechanisms of resistance. Functional enrichment analyses emphasized the activation of defense-related pathways in CX315, along with suppression of susceptibility-associated processes. A total of 6,221 differentially expressed genes (DEGs) were identified between CX315 and JG30 at 48 hpi, with 2,275 upregulated and 3,946 downregulated in CX315 ( Figure 3C ). The Venn diagram ( Figure 3A ) shows the distribution and overlap of DEGs across genotypes and timepoints, revealing a distinct set of CX315-specific DEGs at 48 hpi. The volcano plot ( Figure 3B ) illustrates the magnitude of differential expression, highlighting genes with strong pathogen-responsive activation or repression.

At baseline (0 hpi), 2,220 DEGs were identified between CX315 and JG30, including 798 upregulated and 1,422 downregulated genes in CX315. GO enrichment of these genes indicated that upregulated transcripts were predominantly involved in oxidative stress response and carbohydrate derivative metabolic processes, suggesting that CX315 maintains a primed defense state even prior to infection. Pathway analysis further revealed enrichment in phenylpropanoid biosynthesis and MAPK signaling—two key components of plant immune responses.

At 48 hpi, GO enrichment of CX315 DEGs demonstrated activation of defense-related processes, including “response to biotic stimulus,” “oxidoreductase activity,” and “cell wall organization,” while downregulated genes were enriched in photosynthesis-related pathways ( Figure 3E ). This shift reflects a strategic reallocation of metabolic resources from growth to defense under pathogen attack. In line with this, members of the wall-associated kinase (WAK) family showed higher expression in CX315 compared to JG30 at 48 hpi ( Figure 3D ), consistent with their involvement in Xa50(t)-mediated immune signaling.

Temporal comparison within CX315 (0 h vs. 48 hpi) identified 5,321 DEGs, of which 2,491 were upregulated and 2,830 were downregulated post-inoculation. Enriched biological processes among upregulated genes included oxidative stress and chitin metabolic responses, while downregulated transcripts were associated with photosynthetic pathways such as photosystem I and thylakoid membrane components.

Overall, these findings demonstrate that CX315 mounts a rapid and coordinated transcriptional defense in response to Xoo, involving the activation of key immune pathways and suppression of susceptibility-associated and energy-intensive metabolic processes. This dynamic transcriptional reprogramming provides insight into the molecular mechanisms by which Xa50(t) confers enhanced resistance to bacterial blight.

It was indicated that pathogen-triggered induction of Xa4 expression contributes to its disease resistance (Hu et al., 2017). To prioritize candidate genes within the 147.7 kb interval linked to Xa50(t), qRT-PCR was conducted on all 13 predicted ORFs in CX315 and the susceptible cultivar JG30 at 0- and 48-hours post-inoculation with Xoo strain T7174. Three genes ORF5, Xa4, and ORF13 were significantly upregulated in CX315 at 48 hpi, while expression of ORF5 and Xa4 remained unchanged in JG30 ( Figure 4 ). Interestingly, ORF13 displayed moderate induction in JG30, suggesting it may be partially responsive to pathogen inoculation even in the absence of functional resistance ( Figure 4 ). This pathogen-responsive expression in the resistant background suggests their potential roles in Xa50(t)-mediated defense. The expression of ORF4 could not be reliably determined, due to technical limitations in primer design.

ORF5 encode wall-associated kinase (WAK)-like proteins, which act as sensors of cell wall perturbation or pathogen-derived signals, initiating immune responses. ORF13 encodes a receptor-like kinase (RLK) with WAK-related domains, suggesting a role in downstream signal transduction or receptor complex formation. The co-induction of these kinase-domain genes points to a multilayered signaling cascade contributing to CX315 resistance. Their selective activation in response to Xoo highlights them as promising candidates underlying Xa50(t) and warrants further functional analysis.

Since the fine-mapped interval of Xa50(t) overlapped with the known Xa4 locus, comparative pathotyping was performed to determine whether Xa50(t) is allelic to Xa4 or confers resistance through an independent mechanism. For this purpose, the Xa4-containing line IRBB4 and the Xa50(t)-harboring line CX315 were inoculated with the Xoo strain T7174 at the seedling stage and further challenged with a panel of diverse Xoo strains at the tillering stage. At the seedling stage CX315 exhibited a hypersensitive-like response and significantly shorter lesions compared to IRBB4 ( Figure 5A ). At the tillering stage, the resistance responses significantly varied between CX315 and IRBB4 for several strains, indicating that the underlying mechanisms differ between Xa50(t) and Xa4 ( Figures 5B, C ).

To confirm the presence of Xa4 in the CX315 background, we amplified and sequenced its coding region, which verified that Xa4 is intact in CX315 and suitable for targeted editing. To further validate whether Xa50(t) acts independently of Xa4, we employed CRISPR/Cas9-mediated knockout of Xa4 in the CX315 background. Two guide RNAs targeting the first exon of Xa4 were designed ( Figure 6A ), and the sgRNA expression cassettes were cloned into a CRISPR/Cas9 binary vector. Two homozygous T₁ transgenic lines were obtained by Sanger sequencing confirmed indel mutations at the targeted sites, resulting in frameshift knockouts of Xa4 ( Figure 6B , Supplementary Figure S1 ). Protein alignment confirmed that both Xa4-KO lines produce truncated proteins due to premature stop codons ( Supplementary Figure S2 ). T₁ homozygous knockout plants were inoculated with Xoo strain T7174 at the maturity stage. These Xa4-KO lines retained resistance phenotypes similar to the wild-type CX315 ( Figure 6C ), demonstrating that Xa50(t)-mediated resistance remains functional in the absence of Xa4.

Together, the phenotypic divergence between IRBB4 and CX315, along with the retained resistance observed in Xa4-knockout lines, confirms that Xa50(t) operates independently and represents a functionally distinct resistance gene. These findings underscore the potential of Xa50(t) as a valuable genetic resource for breeding broad-spectrum and durable resistance in rice. However, the current mapping interval contains multiple candidate resistance genes, which prevents definitive identification of Xa50(t) at this stage. Further studies aimed at dissecting the regulatory networks and functional mechanisms associated with Xa50(t) will be essential to elucidate how this novel R gene contributes to immune specificity and complements existing resistance pathways in rice.