Abstract
Summary
2024年10月7日， Molecular Plant在线发表了杜克大学董欣年教授实验室题为Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade的研究文章，报道水杨酸信号转导枢纽中新组分的发现和相关信号转导途径的调控机制。
https://doi.org/10.1016/j.molp.2024.08.008
Background:
In plant immunity research, salicylic acid (SA) is a key signaling molecule that plays a central role in systemic acquired resistance in plants. SA reprograms about 20% of the transcriptome by activating the NPR1 protein, triggering a cascade of immune responses. Although the importance of NPR1 in plant immunity is widely recognized, its specific molecular mechanism is still not fully understood.
Outline of Research
To better understand how NPR1 regulates immune responses, the authors used TurboID and green fluorescent protein-labeled cleavage release technology (greenCUT&RUN) to study NPR1's signaling network and transcriptional cascade. To explore how SA-bound NPR1 bridges with the TGA complex and initiates transcriptional reprogramming, the researchers constructed Arabidopsis transgenic plants expressing the NPR1-33HA-TurboID (NPR1-TbID) fusion protein and performed label-free quantification using LC-MS/MS mass spectrometry. After 4 h of SA treatment, 234 NPR1 neighbor proteins were identified (Figure 1), which play a key role in regulating gene expression and SA-induced resistance, and most of the proteins are also present in the neighbor proteome of GBPL3, a key protein in the SA synthesis pathway, in addition to certain transcription factors.
Fig.1 Some transcriptional regulation and chromatin remodeling proteins in NPR1 neighbor proteins are also present in the GBPL3 neighbor proteome.
Through analysis, the researchers found that NPR1-TbID captured almost all known NPR1-interacting proteins, including NPR3, NPR4, NIMIN1, TGA5, and WRKY18, among others. This validates the specificity of the TurboID method and demonstrates that the identified NPR1 neighbor proteins are predominantly concentrated in the nucleus and may contain components of NPR1 enhancers, rather than SA-induced NPR1 condensates in the cytoplasm. In addition to the known NPR1-interacting proteins, many new neighbor proteins have been identified. Based on gene ontology analysis, these proteins are rich in functions associated with histone modification, chromatin remodeling, transcriptional machinery, and RNA splicing complexes, suggesting that these nuclear functional modules are involved in SA-mediated transcriptome reprogramming. These findings support NPR1's role as a central signaling hub, which mediates a wide range of resistance responses in plants by remodeling chromatin and regulating gene expression. In order to further validate the newly discovered NPR1 neighbor proteins, the researchers paid special attention to two histone proteins: (1) chromatin remodeling proteins, represented by BRM; (2) Histone demethylated proteins, represented by LDL3. Experiments have shown that the interaction between BRM and LDL3 and NPR1 is significantly increased under SA induction. In addition, the knockout of BRM and LDL3 genes partially weakened plant resistance to pathogens, which could be restored through complementary experiments, suggesting that they play a key role in SA-induced defense.
Figure 2.NPR1 targets the TF gene promoter by binding to TGA TFs.
NPR1 is known to interact with a variety of transcription factors, including TGAs and WRKYs. In this study, the researchers paid special attention to WRKY54 and WRKY70 because although WRKY70 has been shown to be associated with NPR1, its single mutant has a relatively small transcriptional effect. QuantSeq analysis showed that the gene expression of the WRKY54/70 double mutant was significantly reduced under SA induction, and the transcriptional response of the mutant to SA was abnormal compared with that of the wild type. These results suggest that WRKY54 and WRKY70 play an important positive regulatory role in SA-mediated transcriptional reprogramming.
Figure 3.WRKY54/70 are the major TFs downstream of NPR1-TGA and positively regulate SA-mediated gene expression and immune resistance.
In order to further explore the transcription target of NPR1, the researchers used greenCUT&RUN technology to analyze NPR1 under SA induction. The results showed that NPR1 was mainly bound to the promoter region of the target gene and enriched in the TGA binding site (Fig. 2). Although NPR1 and WRKY70 share a subset of target genes, they bind at different loci, suggesting that NPR1 regulates the transcriptional output of SA-responsive genes by binding to TGA transcription factors, which WRKY70 is involved as a downstream transcription factor (Figure 3).
Figure 4.The formation of biomolecular condensates stabilizes the binding of NPR1 to TGA TF and enhances its transcriptional activity.
Finally, the investigators experimentally verified that the formation of NPR1 condensates is essential for chromatin binding and transcriptional activity (Figure 4). Studies have shown that the condensate formed by NPR1 induced by SA can significantly enhance its interaction with TGA transcription factors, thereby improving transcriptional activity. In contrast, mutant RDR3, despite accumulating in the nucleus, has significantly reduced chromatin binding capacity and transcriptional activity due to its inability to form condensates. This result supports the important role of NPR1 condensates in SA signaling. In summary, the researchers systematically identified the neighbor proteins and target genes of NPR1, an important component of the SA signaling pathway, through TurboID label-free quantitative analysis and greenCUT&RUN method. The results showed that NPR1 was associated with protein complexes involved in chromatin remodeling, histone modification and RNA splicing. In addition, the direct target genes of NPR1 are mainly concentrated in the promoter region and achieve their transcriptional activation function by binding to TGA transcription factors. NPR1 has also been found to enhance its chromatin binding and transcriptional activity through the formation of intranuclear condensates. These findings reveal the mechanism by which NPR1 coordinates gene expression through molecular complexes induced by SA, and further deepen our understanding of plant immune regulatory networks.
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