Legume Genomics and Genetics 2025, Vol.16, No.2, 81-90 http://cropscipublisher.com/index.php/lgg 84 bridge but also cooperates with many signal proteins and transcription factors to integrate the changes in the external environment with the plant's own developmental signals. There are also some other kinases, such as GmSK2-8, which are quite "busy". They link the stress response of plants with nodule signals by phosphorylating transcription factors like GmNSP1 (He et al., 2020; Wang et al., 2021). Interestingly, these kinases do not act alone. They often interact with plant hormone signals (such as strigolactone, brassinolactone), which makes the regulation of the tumor formation process more flexible and detailed (Ahmad et al., 2019). 3.3 Transcription factors regulating downstream symbiotic gene expression (e.g., NIN, ERN1) Ultimately, root nodules still rely on gene expression to complete their specific construction, and it is a group of transcription factors that truly "direct" the expression of these genes. NIN (nodule initiation factor) is a typical representative. Its activity can drive the expression of downstream genes such as ENOD40 and CLE peptide, thereby affecting the formation process and quantity control of root nodules (Zheng et al., 2023). But NIN is not the only "dispatcher". There are also ERN1, NSP1, NSP2, WRKY17 and some members of the ERF family, which are respectively involved in the extension of infected filaments, the structural development of root nodules, and even the transmission of hormone signals (Zhao et al., 2025). Of course, their "superiors" are often those kinases mentioned earlier, and environmental factors also interfere from time to time, making the entire regulatory process both precise and full of variations (Zhang et al., 2023). 4 Functional Characterization of Signaling Genes in Soybean-Rhizobium Interaction 4.1 Gene knockout and overexpression studies in soybean Not all genes are "reticent"; some, once their expression is enhanced, will bring about significant changes. Genes like GmPHT1-4, GsIMaT2 and GmD27c, once overexpressed in soybeans, can be observed to have an increase in the number of root nodules and more vigorous root growth. This indicates that they play a promoting role in nodular signaling or related metabolic pathways (Rehman et al., 2022a; Zhu et al., 2023). Conversely, for those genes that originally had restrictive effects, such as GmD53a and GmSK2-8, when they were knocked down or silenced, the number of root nodules increased instead, and the sensitivity of plants to stress decreased (Rehman et al., 2022b). Therefore, through this "addition and subtraction" operation, it can basically be determined that these signal genes are indeed directly involved in the regulation of tumor formation efficiency and the process of signal transmission. 4.2 RNA interference and CRISPR-Cas technologies for functional validation If one only relies on expression changes to guess the function of genes, it will always be inaccurate. Thus, RNA interference (RNAi) and CRISPR-Cas have become more reliable "proven tools". For instance, after knocking down GmD53a or GmSK2-8 by RNAi, or removing miR169c with CRISPR-Cas9, the results showed that the number of root nodules, nitrogenase activity, and the expression of some symbiotic related genes were significantly affected (Xu et al., 2020). This targeted approach not only verified the actual role of these genes in tumor formation but also enabled researchers to more clearly observe their positions and functions within the entire regulatory network. 4.3 Use of transcriptomic and proteomic profiling to identify candidate genes Of course, not all functions need to be tested one by one. Sometimes, researchers tend to use high-throughput methods for screening. Rna-level analysis methods such as transcriptome sequencing can quickly identify genes that are upregulated during root tumor formation, such as GmNIN2b, GmENOD40, GmNFR1, GmNFR5, GmDRR1, etc. (Shi et al., 2020). But merely looking at transcription is not enough; proteomics is also crucial - after all, it is the proteins that do the work in the end. These analyses revealed some changes in protein abundance and post-translational modifications, thereby further supporting the importance of these candidate genes in root tumor development and information exchange between soybeans and rhizobia. 5 Cross-Talk with Other Signaling Pathways in Soybean-Rhizobium Interaction 5.1 Interaction between symbiotic signaling and plant hormone pathways Nodulation cannot be accomplished merely by rhizobia signals; the intervention of plant hormones often alters the entire process. For instance, brassinolide (BR) is a "disruptor", which can interfere with the signals of
RkJQdWJsaXNoZXIy MjQ4ODYzNA==