Legume Genomics and Genetics 2025, Vol.16, No.2, 81-90 http://cropscipublisher.com/index.php/lgg 85 tumor-forming factors and exert an inhibitory effect. The transcriptional regulatory factor GmBES1-1 will bind to GmNSP1 and GmNSP2, thereby rendering them "ineffective" and ultimately inhibiting the tumor formation process. On the contrary, some hormones are "helpful", such as strigolactone (SL) and karigin (KAR), which indirectly affect multiple signals including auxin, jasmonic acid and abscisic acid by regulating genes like GmMAX2a, GmD14s and GmKAI. This signal series connection makes nodules more likely to occur (Rehman et al., 2018). It is worth mentioning that the phosphorus transporter GmPHT1-4 has also been found to be present in multiple hormone signaling pathways and may even be a key node for integration effect signaling. 5.2 Overlap between defense and symbiotic gene regulatory networks The process of symbiosis is actually not completely "peaceful", and the boundary between it and the defense responses of plants is often blurred. RIN4, a familiar immune regulatory factor, is rapidly phosphorylated shortly after rhizobia inoculation. Studies have found that it is not only involved in defense but also promotes nodule formation (Toth et al., 2025). However, rhizobia do not just sit and wait to die. The type III effector molecules they release, such as NopL, NopT, NopP, NopAA, and NopD, will "communicate" with proteins like GmREM1a, GmNFR5, and GmPBS1 in the host, thereby influencing the plant's existing defense mechanisms. It also rewrites the tumor formation process (Li et al., 2023; Ma et al., 2024). From the transcriptome perspective, these changes are not just individual phenomena. Many genes regulated by these effector factors occur simultaneously in pathogen responses and hormone signals, indicating that defense and symbiosis are not completely separate paths (Wang et al., 2023). 5.3 Role of environmental cues in modulating signaling Internal signals alone are far from enough; sometimes the influence of the external environment is more direct. Take nitrogen as an example. Under high nitrogen conditions, miR169c will be activated, thereby inhibiting the expression of GmNFYA-C. The result is a reduction in the number of root nodules - this phenomenon has occurred in various experiments. However, when GmNFYA-C is forcibly overexpressed, even if there is more nitrogen, it can still retain some of its tumorigenesis ability. As for salt stress, it blocks the subsequent steps of tumor formation by activating the kinase GmSK2-8, which limits the function of GmNSP1. Another factor that is often overlooked is light, not the light directly shining on the roots, but the light signals perceived by the above-ground parts. Mobile factors such as GmSTF3/4 and GmFTs from the stem are transmitted to the roots to participate in regulation, bringing the "opinion of the sun" to the symbiotic mechanism. That is to say, environmental signals are not just observing from the periphery; they are actually involved and can even determine the extent to which tumor formation progresses. 6 Case Study 6.1 Functional role of GmNARK as a negative regulator of nodulation Root nodules cannot grow as many as you want. Plants also have their own "braking mechanism". GmNARK is precisely such a receptor kinase that plays a limiting role. It has a leucine repeat sequence and belongs to the receptor-like kinase family. Whenever rhizobia infect, plant roots produce some CLE signaling peptides, such as GmRIC1 and GmRIC2, which are transported to the above-ground parts and recognized by GmNARK. After recognition, the above-ground part will "reply" to generate some inhibitory signal, which is sent back to the root to prevent the further formation of new root nodules. This mechanism, simply put, is a feedback system to prevent the number of root nodules from getting out of control (Figure 2) (Gresshoff et al., 2025). It can help plants keep the number of root nodules within the range permitted by resources and the environment, avoiding the overall growth being hindered by too many root nodules. 6.2 Insights from mutant analysis and phenotypic characterization What would happen if GmNARK broke down? Researchers discovered the answer through several mutants. Loss-of-function mutants like nts1007, SS2-2, and Sakukei 4 exhibit a state of "rampant root nodules". Even in environments that usually inhibit nodule formation, such as high nitrate nitrogen or phosphorus deficiency, these mutants still grow a large number of root nodules (Isidra-Arellano et al., 2020). Further analysis revealed that their expression patterns on CLE peptides and downstream related signals were also different from those of normal
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