<scp> <i>OsTB1</i> </scp> Coordinately Enhances Rice Culm Strength and Blast Resistance by Promoting Lignin Biosynthesis

Maintaining stable and high-yielding rice production is crucial for food security. Among the major challenges in rice cultivation, lodging remains a persistent agronomic problem that significantly compromises harvesting efficiency, reduces yield, and diminishes grain quality. Generally, rice varieties with stronger culms exhibit better lodging resistance. Rice culm strength is determined by its morphology and cell wall composition (Li et al. 2015). Although multiple quantitative trait loci (QTLs) controlling culm morphology have been successfully cloned (Jiao et al. 2010; Tu et al. 2022), the molecular mechanism underlying cell wall components remains largely unexplored. Meanwhile, rice blast disease, caused by Magnaporthe oryzae, represents another critical yield-limiting factor (Dean et al. 2012). Recent studies have revealed that enhanced lignin deposition in cell walls and increased sclerenchyma thickness constitute important physical barriers against fungal penetration (Li et al. 2020; Liu et al. 2025). Previously, we identified qSCSA3-1, a QTL associated with culm cross-sectional area that is allelic to SCM3/OsTB1 (Cui et al. 2020). In the present study, we demonstrate that OsTB1 plays a dual role in enhancing both culm strength and blast resistance through direct transcriptional regulation of lignin biosynthesis genes. Using CRISPR/Cas9 genome editing technology, we created two independent OsTB1 knockout mutants (yostb1-1 and yostb1-2) in the Yangchannuo1 (YCN1) background, which carries a natural 5ʹ-UTR TGTG insertion associated with elevated OsTB1 expression (OsTB1YCN1 allele, Figures S1 and S2). Parallel experiments were conducted in the japonica rice cultivar Nipponbare (NIP), which carries a low-expression OsTB1NIP haplotype, yielding two additional knockout lines (nostb1-1 and nostb1-2, Figures S2 and S3). All the mutants in the two backgrounds displayed significant reductions in culm diameter, culm wall thickness, and breaking resistance, compared to the corresponding wild-type plants (Figure 1a–d). Field observations during late developmental stages revealed that NIP-derived mutants (nostb1-1 and nostb1-2) developed notably fragile culms that were highly susceptible to lodging (Figure S4). Intriguingly, the nostb1-1 and nostb1-2 mutant culms exhibited significantly reduced mechanical strength compared to diameter-matched wild-type controls. This suggests that OsTB1 influences culm strength not only through morphology but also through compositional changes. We performed transcriptome analysis of culm tissues from wild-type YCN1 and the yostb1-1 mutant and identified 2459 differentially expressed genes (DEGs) (Figure S5a). Gene Ontology (GO) analysis of these DEGs revealed significant enrichment in multiple metabolic processes (Figure S5b). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment in phenylpropanoid biosynthesis (Figure 1e), a key metabolic pathway generating lignin, flavonoids, lignans, and phenylpropanoid esters. The function of lignin in cell wall reinforcement and vascular development is well-reported (Dong and Lin 2021). Given the demonstrated role of OsTB1 in culm strength enhancement, its downstream targets are predicted to modulate lignin accumulation. Both histochemical staining and quantitative assays showed substantially decreased lignin accumulation in ostb1 mutants (Figure 1f,g). Collectively, these data establish that OsTB1 functions as a positive regulator of lignin biosynthesis in rice culms. To elucidate how OsTB1 regulates this metabolic cascade, we analysed the ~2 kb promoter regions of potential genes encoding key enzymes in this pathway, including OsPALs, Os4CLs, OsCCRs, OsCADs, and COMT. Among the analysed gene promoters, at least 24 contained the reported OsTB1 binding motif “TGGGCC” (Figure 1h; Figure S6a). Yeast one-hybrid (Y1H) assays revealed direct interactions between OsTB1 and the promoters of eight core lignin biosynthetic genes (Os4CL2, Os4CL3, Os4CL4, OsCCR19, OsCCR29, OsCAD1, OsCAD4, and OsCAD5) (Figure S6b). Electrophoretic mobility shift assays (EMSAs) with GST-OsTB1 fusion protein demonstrated sequence-specific binding to biotin-labelled promoter fragments containing the “TGGGCC” motif (Figure 1i). Dual-luciferase reporter assays confirmed OsTB1's capacity to directly activate transcription (Figure 1j). qRT-PCR analysis revealed that the expression levels of these lignin biosynthetic genes were significantly downregulated in ostb1 mutants (Figure 1k). Taken together, these findings demonstrate that OsTB1 serves as a master transcriptional regulator of the lignin biosynthetic pathway, orchestrating lignin deposition through simultaneous binding and transactivation of multiple core lignin synthesis gene promoters. Phloroglucinol-HCl staining revealed reduced lignin deposition in ostb1 mutant leaf sclerenchyma cells (Figure 1l), with biochemical assays confirming significantly lower total lignin content (Figure 1m). Given the role of lignin in reinforcing cell walls against pathogen invasion, we systematically assessed the response of ostb1 mutants to M. oryzae infection using both punch and spray inoculation methods. Compared to wild-type plants, ostb1 mutants exhibited increased susceptibility to blast fungus under both methods (Figure 1n,o). Relative fungal growth in inoculated ostb1 mutant leaves was also significantly higher than wild-type controls (Figure 1n,o). In addition, OsTB1 expression was induced by M. oryzae infection (Figure S7). Collectively, these findings indicate that OsTB1 promotes lignin accumulation to simultaneously enhance lodging resistance and blast resistance in rice. To assess the agronomic potential of OsTB1 alleles in japonica rice improvement, we employed a targeted breeding strategy by crossing the YCN1 with the elite japonica cultivar Wuxiangnuo109 (WXN109, carrying the OsTB1NIP allele). Through backcrossing and marker-assisted selection, we developed a pair of near-isogenic lines (NILs) in the WXN109 genetic background: WXN109-OsTB1NIP and WXN109-OsTB1YCN1 (Figure 1p; Figure S8). Compared with WXN109-OsTB1NIP, WXN109-OsTB1YCN1 exhibited significant improvements in both culm diameter and lignin content (Figure 1q,r). Notably, the breaking resistance of WXN109-OsTB1YCN1 culms was enhanced by 11.87% (Figure 1s), indicating superior mechanical strength properties. WXN109-OsTB1YCN1 showed a reduction in lesion area and decreased relative fungal growth compared to WXN109-OsTB1NIP, demonstrating enhanced blast resistance conferred by the OsTB1YCN1 allele (Figure 1t; Figure S9). Investigation of yield components displayed that the introduction of the OsTB1YCN1 allele reduced the panicle number per plant but significantly generated more grains per panicle, with no effect on grain weight, resulting in no overall yield penalty (Figure S10). No significant difference in rice appearance, viscosity index, and taste value was found (Figure S11). These results demonstrate that introducing the OsTB1YCN1 allele can effectively enhance culm strength and blast resistance without compromising yield and quality. In summary, our findings elucidate the dual role of OsTB1 in simultaneously enhancing culm strength and blast resistance through direct regulation of lignin biosynthesis genes (Figure 1u). These comprehensive results establish OsTB1 as a premier molecular target for breeding programs focused on developing high-yielding rice varieties with enhanced lodging and disease resistance. Y.Z., G.L., S.W., and A.Y. supervised the project. W.T., Y.W., T.S., Z.Z., X.W., S.M., and Q.W. performed most of the experiments. J.H., J.M., J.Z., and Z.G. assisted in the experimental procedures. Z.Y. and G.Y. contributed to data analysis. W.T. and Y.Z. wrote the manuscript. T.Z. helped revise the manuscript. All authors read and approved the final manuscript. This work was supported by grants from the Biological Breeding-Major Projects in National Science and Technology (2023ZD040680105), Basic Research Program of Jiangsu (BK20230013), the Project of Zhongshan Biological Breeding Laboratory (ZSBBL-KY2023-01), the China Postdoctoral Science Foundation (2023M742964), and the Jiangsu Funding Program for Excellent Postdoctoral Talent (2024ZB827). The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data S1. Figures S1–S11. Table S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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