Legume Genomics and Genetics 2025, Vol.16, No.2, 81-90 http://cropscipublisher.com/index.php/lgg 83 receptors will increase rapidly after rhizobia inoculation, and without them, the subsequent formation of root nodules is basically difficult to carry out. So, although they are "sentries at the door", their status is by no means low. Figure 1 GmROP9 is involved in root nodule symbiosis (Adopted from Gao et al., 2021) Image caption: (A) Representative hairy roots expressing empty vector (EV), GmROP9-RNAi-2-1, or GmROP9-RNAi-2-2 construct at 21 days post-inoculation (dpi) with rhizobia. Scale bar represents 1 cm. (B) Transcript levels of GmROP genes in EV and GmROP9-RNAi-2 transgenic roots. Data are normalized to GmELF1b and are mean ± SD from three replicates. Statistically significant differences were detected by Student’s t test; ns indicates not statistically significant, ∗p < 0.05, and ∗∗p < 0.01. (C) Nodule numbers per plant transformed with EV and two independent GmROP9-RNAi-2 constructs (GmROP9-RNAi-2-1 and GmROP9-RNAi-2-2) at 21 dpi. Three biological replicates were performed, and at least 20 independent transgenic hairy roots per construct were analyzed and used for statistical analysis. Boxes show the first quartile, median, and third quartile; whiskers show minimum and maximum values; and dots show data points. Statistically significant differences were detected by Student’s t test; ∗∗p < 0.01. (D–G) Transcript levels of GmNIN (D), GmENOD40 (E), GmNSP1 (F), and GmERN1 (G) in EV and GmROP9-RNAi-2 transgenic roots. Data are normalized to GmELF1band are mean ± SD from three replicates. Statistically significant differences were detected by Student’s t test; ∗∗p < 0.01. (H) Representative hairy root expressing EV, GmRO9-KO, or GmROP6 GmROP9-KO construct at 21 dpi. Scale bars represent 5 mm. (I) Nodule numbers per independent roots transformed with EV, GmROP9-KO, or GmROP6 GmROP9-KO construct at 21 dpi. Three replicates were performed, and at least 10 independent transgenic roots per construct were analyzed and used for statistical analysis. Boxes show the first quartile, median, and third quartile; whiskers show minimum and maximum values; and dots show data points. Statistically significant differences were detected by Student’s t test; ∗∗p < 0.01. (J) Transcript levels of GmNIN, GmENOD40, GmNSP1, and GmERN1 in EV, GmROP9-KO, or GmROP6 GmROP9-KO transgenic roots. Data are normalized to GmELF1band are mean ± SD from three replicates. Statistically significant differences were detected by Student’s t test; ∗∗p < 0.01 (Adopted from Gao et al., 2021) 3.2 Calcium/calmodulin-dependent kinases and nodulation signaling pathways Once the Nod factor is perceived, the next step is to "translate" the signal into a language that the cells can understand. Here, the calcium signal comes into play. Calcium/calmodulin-dependent kinases (CCaMKs), such as GmDMI3, are key tools responsible for reading calcium oscillations in the cell nucleus. It not only serves as a
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