Rice Transcription Factor BHLH25 Confers Resistance to Multiple Diseases by Sensing H2O2
Academic Background
When plants face pathogen invasion, they activate a series of complex defense mechanisms. Among these, reactive oxygen species (ROS) play a crucial role in plant immune responses. Hydrogen peroxide (H₂O₂), as a major component of ROS, is considered a key signaling molecule in plant immunity. However, how H₂O₂ is sensed within plant cells and converted into defense signals, particularly how transcription factors directly perceive H₂O₂ and regulate gene expression, remains an unresolved mystery.
Previous studies have shown that H₂O₂ can modulate protein function by oxidizing cysteine and methionine residues in proteins. Nevertheless, the mechanism by which transcription factors directly sense H₂O₂ and regulate plant immune responses remains unclear. This study aims to reveal how the rice transcription factor BHLH25 senses H₂O₂ to confer resistance to multiple diseases and to explore the universality of this mechanism in the plant kingdom.
Source of the Paper
The research was conducted by Haicheng Liao, Yu Fang, Junjie Yin, and other scientists from Sichuan Agricultural University, Technical University in Zvolen (Slovakia), Teikyo University (Japan), and several other institutions. The paper was published online on January 14, 2025, in the journal Cell Research, titled “Rice transcription factor BHLH25 confers resistance to multiple diseases by sensing H₂O₂”.
Research Process and Results
1. H₂O₂ Promotes Plant Immunity through OsLAC7/28/29-Mediated Lignin Biosynthesis
The study began by treating rice roots with exogenous H₂O₂, which significantly enhanced rice resistance to the blast fungus (Magnaporthe oryzae). Through RNA sequencing analysis, the researchers identified 1,596 genes upregulated by H₂O₂, with genes related to cell wall biosynthesis being particularly prominent. Further analysis revealed that the expression of three lignin biosynthesis genes (OsLAC7, OsLAC28, and OsLAC29) was significantly upregulated after H₂O₂ treatment.
Using knockout (KO) and overexpression (OE) experiments, the researchers confirmed the important role of OsLAC7/28/29 in lignin biosynthesis and disease resistance. Triple knockout plants (OsLAC7/28/29-KO) exhibited significantly reduced lignin content and disease resistance, while overexpression plants showed higher lignin content and stronger disease resistance. Additionally, H₂O₂-induced disease resistance was significantly weakened in OsLAC7/28/29-KO plants, indicating that H₂O₂ enhances plant immunity through OsLAC7/28/29-mediated lignin biosynthesis.
2. OsLAC7/28/29 is Targeted and Repressed by miR397b
The researchers further discovered that the expression of OsLAC7/28/29 is regulated by miR397b. Through yeast one-hybrid screening and DNA affinity purification experiments, they identified the transcription factor BHLH25 as directly binding to the miR397b promoter and repressing its expression. Overexpression of BHLH25 significantly reduced miR397b expression, thereby increasing OsLAC7/28/29 expression. Conversely, knockout of BHLH25 led to increased miR397b expression and decreased OsLAC7/28/29 expression.
3. BHLH25 Regulates Lignin Biosynthesis and Disease Resistance via miR397b
Overexpression of BHLH25 significantly enhanced lignin biosynthesis and disease resistance in rice, while knockout of BHLH25 resulted in reduced lignin content and weakened disease resistance. Additionally, knockout of miR397b significantly increased lignin content and disease resistance, whereas overexpression of miR397b had the opposite effect. These results indicate that BHLH25 promotes lignin biosynthesis by repressing miR397b expression, thereby enhancing plant disease resistance.
4. BHLH25 Enhances Disease Resistance through CPS2-Mediated Phytoalexin Biosynthesis
In addition to regulating lignin biosynthesis, BHLH25 also enhances disease resistance by promoting the expression of the phytoalexin biosynthesis gene CPS2. BHLH25 directly binds to the CPS2 promoter and activates its expression. Overexpression of BHLH25 significantly increased the content of phytoalexin C, while knockout of BHLH25 led to a significant reduction in phytoalexin C content. Furthermore, overexpression of CPS2 significantly enhanced rice disease resistance, whereas knockout of CPS2 had the opposite effect.
5. BHLH25 Regulates Defense Responses through Oxidation State Changes at M256
The researchers found that BHLH25 is oxidized by H₂O₂ during pathogen invasion, particularly at the methionine 256 (M256) site. Oxidized BHLH25 preferentially binds to the miR397b promoter, thereby repressing miR397b expression and promoting lignin biosynthesis. As lignin biosynthesis progresses, H₂O₂ is consumed, and the oxidation state of BHLH25 gradually recovers, allowing it to promote CPS2 expression and enhance phytoalexin biosynthesis.
Through gene mutation experiments, the researchers discovered that replacing M256 with valine (V) caused BHLH25 to lose its ability to bind to the miR397b and CPS2 promoters, rendering it unable to regulate lignin and phytoalexin biosynthesis, and significantly reducing disease resistance. This indicates that M256 is a key site for BHLH25 to sense H₂O₂ and regulate defense responses.
6. Broad Conservation of BHLH25 in the Plant Kingdom
The researchers further analyzed BHLH25 homologous genes in 110 plant genomes and found that M256-like methionine residues are highly conserved in these homologs. Additionally, the BHLH25 homolog in Arabidopsis thaliana (AtBHLH25) exhibited a similar H₂O₂-sensing mechanism, suggesting that this mechanism is widely present in the plant kingdom.
Conclusions and Significance
This study reveals a novel mechanism by which the rice transcription factor BHLH25 senses H₂O₂ to regulate multiple disease resistance in plants. Through oxidation state changes at its M256 site, BHLH25 separately regulates lignin and phytoalexin biosynthesis, thereby enhancing both physical barriers and chemical defenses in plants. This mechanism not only helps plants effectively resist pathogen invasion but also prevents the negative effects of excessive accumulation of H₂O₂, lignin, and phytoalexins on plant growth.
Moreover, the broad conservation of BHLH25 and its homologs in the plant kingdom suggests that this mechanism may be universally present in various plants, providing new insights for developing broad-spectrum disease-resistant crops.
Research Highlights
- Discovery of a Novel Mechanism: The first revelation of how the transcription factor BHLH25 senses H₂O₂ to regulate multiple disease resistance in plants.
- Dual Defense Pathways: BHLH25, through oxidation state changes, separately regulates lignin and phytoalexin biosynthesis, forming a dual defense system.
- Identification of a Key Site: The oxidation of the M256 site is crucial for BHLH25 to sense H₂O₂ and regulate defense responses.
- Broad Conservation: The widespread conservation of BHLH25 and its homologs in the plant kingdom indicates that this mechanism may be universally present.
Additional Valuable Information
The study also provides detailed experimental methods and data analysis procedures, including RNA sequencing, yeast one-hybrid assays, DNA affinity purification, chromatin immunoprecipitation (ChIP-qPCR), and more, offering valuable technical references for researchers in related fields. Additionally, the research team developed a specific antibody recognizing oxidized M256, providing a powerful tool for further studies on the oxidation state of BHLH25.
Through this research, we not only gained a deeper understanding of the molecular mechanisms of plant immune responses but also provided new theoretical foundations and technical support for the future development of disease-resistant crops.