In this experiment, a susceptible variety 9311 and a Guangxi common wild rice accession DY19 carrying the BLS resistance gene bls2 were used as materials. The bacterial strain was the Guangxi Xoc strain gx01 with high pathogenicity, which was provided by professor He Yongqiang of the State Key Laboratory for Conservation and Utilization of Subtropical Agricultural Biological Resources. The experiment was carried out in the scientific research base of the College of Agriculture, Guangxi University, adopting the method of single-plant transplanting.

Guangxi common wild rice material DY19 carrying bls2 resistance gene was used as the donor parent, and susceptible indica rice varieties 9311 was used as the recipient parent. The progeny population from the cross were inoculated by pricking with Guangxi prevailing Xoc strain gx01 at the tillering stage. Then, the high-resistant individuals were screened and backcrossed with 9311, combined with SL03 marker-assisted multi-generation selection (Shi et al., 2019). In BC₄F₂ populations, resistance inoculation assays and molecular marker detection were conducted. The plants were resistant to BLS and exhibited the donor parent’s homozygous genotype at molecular marker SL03 were selected for self pollination. Similarly, the plants were susceptible to BLS and carried the recipient parent’s homozygous genotype at SL03 were also selected for self pollination. Subsequently, the BLS resistant and susceptible NILs were obtained in BC₄F₃ populations. Finally, the genetic background of the near-isogenic lines was detected by using polymorphic primers evenly distributed on 12 chromosomes of rice. In order to fine map the introgression fragment of the resistant near-isogenic lines, simple sequence repeat (SSR) and insertion–deletion (InDel) markers were designed using the Gramene website (http://www.gramene.org/) and the software Primer 5. The molecular markers analysis follows the method of Shi et al. (2019), with products analyzed by polyacrylamide denaturing gel electrophoresis and visualized by silver staining.

The methods of bacterial inoculation and lesions identification referenced from Tang et al. (2022). Three rice plants were inoculated with strain gx01, while three rice plants were inoculated with sterile water and used as blank control. Five leaves with the best growth from each plant were selected for inoculation. Among them, 3 leaves were inoculated 3 times per leaf, with a pinhole spacing of 1 cm for the transcription sequencing experiment; the remaining 2 leaves were inoculated once per leaf to measure the length of the lesions. Leaves used in the transcriptome experiment were harvested at 12 hpi, 24 hpi and 48 hpi. Three biological replicates of leaf samples, each containing a pool of three individual plants, were collected for each time points. The lesions were investigated 15 days after inoculation. According to the identification criteria of the International Rice Research Institute (IRRI), the resistance level was identified according to the lesion length ( Table 1 ).

The total RNA of the leaves was extracted using the TIANGEN RNA prep Pure polysaccharide polyphenol plant total RNA extraction kit (DP441; TianGen; China). The RNA concentration was measured by Thermo Qubit 3.0 fluorescence quantitative analyzer (Thermo Fisher Scientific, USA). The RIN value of RNA was assessed by Agilent 4200 Tape Station Bioanalyzer (Agilent Technologies, USA) to evaluate its integrity. The purity of RNA was detected by Thermo Scientific NanoDrop spectrophotometer ND8000 (Thermo Fisher Scientific, USA). Qualification criteria: total RNA greater than 1μg, RIN value greater than 7, OD260/OD280 between 1.8-2.1, OD230/OD260 between 0.4-0.5.

The RNA libraries were prepared using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (NR604; Vazyme Biotech Co., Ltd; China) following the manufacturer’s instructions. After library construction, fragment size and concentration were assessed using an Agilent 4200 Tape Station Bioanalyzer (Agilent Technologies, USA) and ABI Quant Studio 6 Flex Real-Time PCR (Thermo Fisher Scientific, USA) for quality control. Qualification criteria: library fragment size between 300–500 bp, molar concentration greater than 10 nM. Once the libraries passed quality control, sequencing was performed using the Illumina NovaSeq 6000 sequencing platform PE150 (Illumina, USA). The sequencing data output for each sample was not less than 6 G.

Clean reads were obtained by removing reads containing adapters and low-quality reads from the raw data. The software hisat2 was used to compare the clean reads with the rice reference genome Oryza sativa Japonica Group. Mapped reads are spliced and assembled by using the software StringTie, and functional annotation was carried out through databases such as GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genome), and NR (NCBI non-redundant protein sequences). The R language package edgeR was used for gene differential expression analysis, and the DEGs screening threshold was FDR (false discovery rate)<0.05, log₂FC (fold change)>1 or<-1. The expression of the same gene between the experimental group and the control group at each time point of different inoculation treatments was analyzed. After the genes with no expression difference between the experimental group and the control group were removed, the genes with different expressions between the two treatments and the control group were merged at each time point. DEGs among different inoculation treatments in this gene set were analyzed, and Venn map and volcano map were drawn. GO function annotation and KEGG enrichment analysis of DEGs were carried out by using the Shengxin analysis platform set up by Anhui Microanaly Genetech Co., Ltd. (Hefei, China). The software KOBAS is used to count the abundance of DEGs in the KEGG pathway and analyze the genes and fluxes related to plant disease resistance.

The genes involved in key pathways from RNA-seq were randomly selected and verified by qRT-PCR. Specific primers of candidate genes were designed by NCBI Primer BLAST ( Table 2 ). The reverse transcription experiment was performed using VAHTS HiScript III RT Super Mix for qPCR (R323; Vazyme Biotech Co., Ltd; China). The rice gene actin was used as an internal control reference gene, and three technical replicates were set for each gene in each sample. ROCHE Light Cycler 480 SYBR Green I Master (Roche Diagnostics, Germany) reagent was used to prepare the 10 μl PCR reaction system according to the instructions. Gene expression was detected on ABI Quant Studio 6 Flex Real-Time PCR. The relative expression of the selected genes was calculated according to the 2 − ΔΔCT method.

The BLS resistant and susceptible NILs were constructed by one hybridization, four backcrosses, and two selfings. All plants in the resistant line were uniformly resistant to BLS and having DY19 genotype at molecular marker SL03, while all plants in the susceptible line were consistently susceptible to BLS and exhibiting 9311 genotype at SL03. A total of 116 markers with polymorphism between the donor and recipient parents were used to detect the genotype of the resistant NIL, and the graphical genotype was drawn ( Figure 1 ). As shown in Figure 1 , the genetic background of the resistant NIL was restored to the genotype of the recipient parent 9311 except for the segment containing SL03 on chromosome 2. BLS resistance gene bls2 should be located on this introgression segment delimited by SL01 and SL05 (spanning∼5.4 M). In addition, we found that the resistant and susceptible near-isogenic lines share the same genetic background, except for this 5.4 M region between SL01 and SL05 on chromosome 2.

Rice resistant and susceptible NILs (abbreviated as R and S) were inoculated with Xoc strain gx01 (experimental group) and sterile water (control group), respectively. Identification of lesions showed that in the control group of both materials, there were no signs of disease. In the experimental group of R, the average length of lesions was 4.78 mm, showing high resistance to BLS. In the experimental group of S, the average length of lesions was 33.35 mm, showing high susceptibility to BLS ( Figure 2 ).

Three biological replicates were designed for each rice materials (R vs S), each treatment (Xoc-inoculated vs sterile water-inoculated), and each time points (12 hpi, 24 hpi,48 hpi). There were 36 RNA samples sequenced on the Illumina platform. The sequencing results meet the analysis requirements: each sample has more than 6G of data, and the quality Q20 of sequencing data is more than 97%. Among the clean reads obtained through data filtering, the number of reads uniquely located in the reference genome was higher than 85%. These unique reads located in the reference genome were used for subsequent gene expression analysis ( Supplementary Table S1 ).

After removing genes with no expression difference between the experimental group and the control group at each time point of each material, and excluding genes unrelated to BLS resistance response, the DEGs at different time points between R and S were statistically analyzed to reveal the expression pattern of resistance-related genes. A total of 639 DEGs ( Supplementary Table S2 ) were identified between R and S at three time points. Namely, 218 DEGs (118 up and 100 down-regulated), 170 DEGs (96 up and 74 down-regulated) and 329 DEGs (185 up and 144 down-regulated) were identified at 12 hpi, 24 hpi and 48 hpi, respectively in R vs S. Notably, the number of DEGs was highest at 48 hpi. Furthermore, venn diagrams reveal the number of overlapping DEGs at different time points ( Figure 3 ).

DEGs were annotated and those related to plant resistance were screened. Resistance-related genes were divided into five categories ( Supplementary Table S3 ): 16 transcription factor related genes (MYB, MYC2, WRKY, ERF, bHLH, etc.), 14 protein kinase-related genes (WAK, CRK, LecRK), 87 resistance protein-related genes (NBS-LRR, LRR, sugar transporter, disease resistance, pathogen-related, calmodulin-like, ubiquitin family, Wax synthesis, sulphate transporter, cytochrome P450, PP2C, glucosyltransferase, cellulose synthase, terpene synthase, flavonol synthase, etc.), 9 hormone-related genes (GAs, IAA, JA, ET, BR) and 4 other genes (CoA). Among these genes related to plant resistance, eight genes were located in the 5.4 M range between SL01 and SL05, and the differential expression was significant ( Table 3 ), which were Os02g0540700 (OsP3IP1), Os02g0569400 (CYP76M8), Os02g0569900 (CYP76M7), Os02g0570700 (CYP71Z7), Os02g0571900 (CYP76M6), Os02g0597300, Os02g0599700 (OsPP2C19) and Os02g0621300 (OsCER1) respectively. CYP76M8 is a multifunctional/promiscuous hydroxylase, with CYP76M5 and CYP76M7 seeming to provide only redundant activity, while CYP76M6 seems to provide both redundant and novel activity, relative to CYP76M8 (Wang et al., 2012). CYP71Z7 is a C2 oxidase, and Ent-isokaurene C2-hydroxylase performs the initial step in the conversion of ent-isokaurene to the antibacterial oryzalides in rice (Wu et al., 2011). In addition, two identified Xoc-TALE target genes showed differential expression: the sulfate transporter gene Os01g0719300 (OsSULTR3; 6) was up-regulated at 24 hpi and down-regulated at 48 hpi; flavanone 3-hydroxylase gene Os04g0581000 (OsF3H04g; OsSAH2) was down-regulated at 12 hpi. Both were known as susceptibility genes for BLS and their downregulated expression may contribute to enhanced disease resistance.

Among the 218 DEGs identified at 12 hpi, GO annotated 167 genes, including 89 up-regulated genes and 78 down-regulated genes. The second level of GO was enriched to 18 biological process items, 11 cell component items and 9 molecular functions ( Figure 4A ). Significant analysis (P< 0.01) was performed for all enriched levels ( Figure 4B ). Among 6 biological process items, the significance of “GO: 0071554 cell wall organization or biogenesis” and “GO: 0035303 regulation of dephosphorylation” were more prominent; among 3 cell component items, the significance of “GO: 0005576 extracellular region” and “GO: 0005618 cell wall” were more prominent; among 11 molecular functional items, the significance of “GO: 0004864 protein phosphatase inhibitor activity” and “GO: 0019840 isoprenoid binding” and “GO: 0033293 monocarboxylic acid binding” were more prominent. Above analysis indicated that compared with S, R exhibited significant changes in cell wall and extracellular components with active dephosphorylation reactions at 12 hpi. Among the 170 DEGs identified at 24 hpi, GO annotated 133 genes, including 78 up-regulated genes and 55 down-regulated genes. The second level of GO was enriched to 17 biological process items, 11 cell component items and 7 molecular functions ( Figure 5A ). The significance of all enriched levels was analyzed (P< 0.01) ( Figure 5B ). Among 15 biological process items, the significance of “GO: 0019748 secondary metabolic process”, “GO: 0016101 diterpenoid metabolic process” and “GO: 0051501 diterpenoid phytoalexin metabolic process” were more prominent; among the 17 molecular functional items, the significance of “GO: 0046872 metal ion binding” and “GO: 0036202 ent-cassa-12,15-diene-11-hydroxylase activity” were more prominent. Above analysis indicated that compared with S, R had more active secondary metabolic process at 24 hpi, especially the diterpenoid phytoalexin metabolic process, which was closely related to disease resistance. Among the 329 DEGs identified at 48 hpi, GO annotated 246 genes, including 134 up-regulated genes and 112 down-regulated genes. The second level of GO was enriched to 17 biological process items, 13 cellular component items and 9 molecular function items ( Figure 6A ). Significant analysis (P< 0.01) was performed for all enriched levels ( Figure 6B ), including 10 biological process items, 2 cellular component items, and 14 molecular function items. Significant GO terms are described as follows: “GO:0044550 secondary metabolite biosynthetic process”, “GO:0009250 glucan biosynthetic process”, “GO:0009832 plant-type cell wall biogenesis”, “GO:0009834 plant-type secondary cell wall biogenesis”, etc. Above analysis indicated that compared with S, R had more active synthesis of the secondary metabolite, glucan, cellulose, cell wall and secondary cell wall at 48 hpi.

According to the time point, the identified DEGs were subjected to KEGG metabolic pathway enrichment analysis. KEGG pathways were mainly distributed in five categories: organismal systems, environmental information processing, cellular processes, metabolism and genetic information processing ( Supplementary Table S4 ).

Among the 218 DEGs identified at 12 hpi, 68 DEGs were involved in 36 KEGG pathways, including 42 up-regulated genes and 26 down-regulated genes. KEGG enrichment analysis of “ko00592 α-linolenic acid metabolism”, “ko04712 circadian rhythm-plant”, “ko04016 MAPK signaling pathway–plant”, “ko04626 plant-pathogen interaction”, “ko00591 linolenic acid metabolism”, “ko00940 phenylpropanoid biosynthesis”, “ko00908 zeatin biosynthesis” and “ko00350 tyrosine metabolism” were significant (P<0.05) ( Supplementary Table S4 ). Among them, “ko04712 circadian rhythm-plant” was mainly related to the sampling time and 12 hpi intervals. The specific gene information involved in the significantly enriched KEGG pathways is shown in Supplementary Table S5 .

Among the 170 DEGs identified at 24 hpi, 46 DEGs were involved in 28 KEGG pathways, including 30 up-regulated genes and 16 down-regulated genes. KEGG enrichment analysis of “ko00904 diterpenoid biosynthesis”, “ko00591 linolenic acid metabolism”, “ko04626 plant-pathogen interaction” and “ko00592 α-linolenic acid metabolism” were significant (P<0.05) ( Supplementary Table S4 ). By analyzing the specific gene information involved in the related KEGG pathway ( Supplementary Table S6 ), it was found that except for the downregulation of calcium-dependent protein kinase (CPK) related gene Os05g0585601 and calmodulin (CALM) related gene Os01g0955100 in “ko04626 plant-pathogen interaction” pathway, the other genes were up-regulated. In summary, the biosynthesis of diterpenoids (secondary metabolites associated with resistance) was active in R at 24 hpi, which was consistent with the results of GO enrichment analysis.

Among the 329 DEGs identified at 48 hpi, 103 DEGs were involved in 59 KEGG pathways, including 53 up-regulated genes and 50 down-regulated genes. KEGG enrichment analysis of “ko00902 monoterpene biosynthesis”, “ko00270 cysteine and methionine metabolism”, “ko00592 α-linolenic acid metabolism”, “ko00780 biotin metabolism”, “ko00591 linolenic acid metabolism”, “ko00940 phenylpropanoid biosynthesis” and “ko00950 isoquinoline alkaloid biosynthesis” were significant (P<0.05) ( Supplementary Table S4 ). And the specific gene information involved in the related KEGG pathways is shown in Supplementary Table S7 .

The key KEGG pathways reported to be related to plant disease resistance were analyzed and the relevant DEGs information is listed in Supplementary Table S8 .

According to the results of RNA-seq analysis, 9 genes (P< 0.01) were randomly selected for qRT-PCR verification. They were the NBS-LRR resistance protein-related gene Os02g0597300; a protein phosphatase 2C-related gene Os02g0599700; a expressed protein gene Os02g0598400; a proline synthetase-related gene Os01g0950866; peroxidase-related genes Os01g0172701 and Os07g0677500, which were involved in lignin synthesis through “phenylalanine synthesis” pathway; hydroperoxide lyase related gene Os02g0110101 and lipoxygenase related gene Os12g0559200 in “α-linolenic acid metabolic” pathway; transcription factor MYC2 related gene Os10g0575100, which was involved in defense response mediated by jasmonic acid in “plant hormone signal transduction” and “plant MAPK signal pathway”. The expression trend of qRT-PCR results was consistent with RNA-seq sequencing results, which indicates that RNA-seq sequencing results are reliable ( Figure 8 ).

Xoc is a pathogenic variant of the Xanthomonas family in the genus Xanthomonas. It uses its flagella to move and invades rice mainly through leaf wounds or stomata. The RLK Flagellin-sensitive 2 (FLS2) of Arabidopsis thaliana can recognize the flagellin fragment flg22 (Gómez-Gómez and Boller, 2000). the flg22 peptide directly binds to FLS2 and triggers the recruitment of the related receptor kinase BAK1 (Chinchilla et al., 2007). It is indicated that flg22 can induce the formation of a complex between FLS2 and BAK1 to initiate PTI reaction (Sun et al., 2013). In our study, six DEGs were identified that were related to the PTI pathway. When the highly pathogenic strain gx01 infected rice, compared with S, R up-regulates the protein kinase FLS2-related genes to enhance their ability to recognize and bind bacterial flagellin flg22 at 12 hpi. Phosphorylated BAK1 dissociates from the complex and activates downstream multiple immune responses, such as promoting ROS burst (Kadota et al., 2014). The activation of FLS2 downstream signaling triggers the influx of calcium ions from the extracellular to the intracellular: on the one hand, it activates the anion channels on the plasma membrane, promoting the exudation of alkaline substances for antimicrobial activity; on the other hand, it activates CPKs to regulate ROS burst. The MAPK cascade pathway is also involved in FLS2-mediated signal transduction (Bethke et al., 2012). We found that in the “plant-pathogen interaction” and “MAPK signaling” pathway, MEKK1-related gene was enriched at 12 hpi, Ca^{2 +} mediated CPKs-related genes and CALMs-related genes were enriched at 24 hpi and 48 hpi ( Figures 7C, D ). These DEGs may involve in PTI immune signaling pathways to provide BLS resistance.

To overcome virulence factors, plants have evolved disease resistance (R) genes to recognize effectors from pathogens and initiate ETI. RPM1 belongs to the NBS-LRR class of R gene proteins that acts as a signalling adaptor for the pathogen avirulence gene (avrRpm1) product, leading to the oxidative burst and hypersensitive cell death (Grant et al., 2000). In our study, Three DEGs were enriched in ETI pathway: RPM1-related gene Os11g0229333 was down-regulated at 12 hpi. The heat shock protein HSP90B gene Os06g0716750 was up-regulated at 12 hpi and 24 hpi, which may cause HR to against the pathogen invasion. Pti6 Os07g0227600 was up-regulated at 48 hpi, which functioned as transcription factors for regulating ETI process. Taken together, bls2 was proposed to regulate both PTI- and ETI-related genes, initiating a series of defense responses. Compared to S, most genes involved in PTI were up-regulated at 12 hpi but down-regulated at 24 and 48 hpi in R; whereas most ETI-related genes showed down-regulation at 12 hpi but up-regulation at 24 and 48 hpi in R. It suggests that PTI primarily occurs at 12 hpi while ETI mainly functions at 24–48 hpi in R.

In our study, two TALE target genes exhibited differential expression between R and S. For Xoc, very few TALE virulence target genes have been reported (Bi et al., 2024). The sulfate transporter gene Os01g0719300(OsSULTR3; 6) targeted by Tal2g is a major susceptibility gene for BLS (Cernadas et al., 2014). Editing EBE in the OsSULTR3;6 promoter region enhanced rice resistance to BLS (Xu et al., 2021). In this study, it was up-regulated at 24 hpi and down-regulated at 48 hpi, indicating a gradual decrease of its expression level in R from 24 to 48 hpi, thereby enhancing resistance to BLS. The flavanone 3-hydroxylase gene OsF3H04g is another BLS susceptibility gene targeted by Tal2c. Overexpressing OsF3H04g caused higher susceptibility and less SA production compared to wild-type plants (Wu et al., 2021). We found that OsF3H04g was down-regulated at 12 hpi. It was consistent with the above report.

α-linolenic acid metabolites are involved in the synthesis of the plant hormone jasmonic acid. Jasmonic acid not only induces physiological changes in plants to form defensive structures but also induces the expression of downstream defensive genes in signaling pathways, thereby enhancing plant resistance to pathogens. In our study, the genes enriched in the “α-linolenic acid metabolic” pathway were up-regulated at 24 hpi and 48 hpi. indicating that the synthesis of jasmonate and traumatic may be gradually increased from 24 hpi to 48 hpi. In the “plant hormone signal transduction” pathway, the transcription factor MYC2-related gene Os10g0575100 was down-regulated at 12 hpi and 48 hpi. MYC2 is an important regulator in the jasmonic acid signaling pathway. AtMYC2 can reduce the defense of Arabidopsis thaliana against bacterial pathogens (Song et al., 2017). Giri et al. (2017) generated multiple transgenic rice lines for over-expression and RNAi-mediated suppression of OsMYC2, then found that OsMYC2 is a negative regulator of rice Xoo resistance. In this study, Os10g0575100 was consistently highly down-regulated, indicating that the inhibition of the jasmonic acid pathway was broken and the genes involved in jasmonic acid response were activated. Moreover, PR1-related genes Os01g0382400 and Os07g0129200 were up-regulated at 24 hpi, which were involved in the salicylic acid-mediated defense response. The above results suggested that R activated jasmonic acid and salicylic acid-dependent signal transduction pathways to trigger defense responses against Xoc invasion at 24 hpi.

Almost all of the identified natural phytoalexins in rice belong to diterpenoids (Schmelz et al., 2014). Among them, the P450s involved in plant terpenoid biosynthesis are mainly distributed in the three families of CYP71, CYP85 and CYP72, among which the CYP71 family is involved in most plant secondary metabolism (Hamberger and Bak, 2013). The infection of M.oryzae can strongly induce the accumulation of these diterpenoid antitoxins (Hasegawa et al., 2010). In our study, at 24 hpi, the diterpene plant antitoxin metabolic reaction was more active, and a large number of up-regulated genes were enriched in the diterpene biosynthesis pathway. These genes are mainly involved in diterpene skeleton synthesis and sitosterol synthesis pathways, indicating that R resist Xoc infection through large-scale synthesis and accumulation of diterpene plant antitoxins. It is worth noting that in the diterpenoid biosynthetic pathway, a large number of differentially enriched genes are mainly concentrated on chromosome 2 and chromosome 4, which is highly consistent with the previous studies that rice carries two such gene clusters for the production of antimicrobial diterpenoid phytoalexins (Shimura et al., 2007; Wang et al., 2012).

Combined with background detection of R, eight DEGs, Cytochrome P450 gene Os02g0569400 (CYP76M8), Os02g0569900 (CYP76M7), Os02g0570700 (CYP71Z7) and Os02g0571900 (CYP76M6), P3-inducible U-box type E3 ubiquitin ligase gene Os02g0540700 (OsP3IP1), Protein phosphatase 2C-related gene Os02g0599700 (OsPP2C19), NBS-LRR gene Os02g0597300 and Wax synthesis gene Os02g0621300 (OsCER1) were located in the 5.4 M range of SL01 and SL05. P450s are the largest family of enzymes in plant metabolism. P450s in the same family or subfamily can catalyze the continuous steps of the same pathway and can also catalyze similar reactions of different substrates. CYP71Z18 overexpression confers elevated blast resistance in transgenic rice (Shen et al., 2019). Cyt02 encodes cytochrome P450 monooxygenase, increasing rice (Oryza sativa L.) resistance to sheath blight (Zheng et al., 2025). Knockdown of CYP76M7 and CYP76M8 simultaneously inhibited elicitor-induced phytocassanes production (Wang et al., 2012). Deletion of diterpenoid biosynthetic genes CYP76M7 and CYP76M8 induces cell death and enhances bacterial blight resistance in 9311 (Jiang et al., 2022). In our study, CYP76M8, CYP76M7 and CYP76M6 were up-regulated at 24 hpi and CYP71Z7 was down-regulated at 12 hpi and up-regulated at 24hpi, indicating that at 24 hpi, large-scale synthesis and accumulation of diterpenoid phytoalexins were occurring in R cells to resist Xoc infection. OsP3IP1 has E3 ligase activity, interacts with OsNRPD1a and OsNRPD1b to mediate their ubiquitination, and degrades them through the ubiquitin-proteasome system, thereby negatively regulating rice resistance to rice grassy stunt virus (RGSV) (Zhang et al., 2020). In our study, OsP3IP1 down-regulated at 24 hpi. It could be inferred that the gene may negatively regulate rice resistance to BLS. Protein phosphatase 2C (PP2C), a group of Ser/Thr-specific phosphatases dephosphorylating target proteins, have implications in plant immunity (Bhaskara et al., 2019). OsPP2C41, which plays positive roles in rice blast resistance and chitin-triggered immune responses (Wang et al., 2024). In our study, OsPP2C19 was up-regulated at 12 hpi and 48 hpi, suggesting that the gene affect resistance to BLS. The NBS-LRR domain has become an important component of plant defense against microbial invasion during the long-term co-evolution between plants and pathogens. The cloned BLS-resistance genes Xo1 and Rxo1 both contain the NBS-LRR domain (Read et al., 2020; Zhao et al., 2005). In our study, NBS-LRR gene Os02g0597300 was up-regulated at 48 hpi, which is likely to be a potential component of bls2 and requires further research. The outermost surfaces of plants are covered with an epicuticular wax layer that provides a primary waterproof barrier and protection against different environmental stresses. OsCER1 is a key gene in wax biosynthesis and plays an indispensable role in cold tolerance during the booting stage of rice (Hou et al., 2024). In our study, OsCER1 was up-regulated at 48hpi and secondary cell wall synthesis and cellulose synthesis in R were more active at 48 hpi. We speculate that these eight genes may be related to the resistance to BLS.