Ta-siRNA Pathway Regulates Symbiotic Nodule Formation in Lotus Japonicus
|Course||Crop Genetics and Breeding|
|Keywords||Lotus japonicus nodulation mutant nodulation autoregulation ta-siRNA|
Rhizobia are capable of infecting the roots of leguminous plants, leading to the formation nitrogen-fixing nodules. While nitrogen-fixing nodule can provide nitrogen for host plants, too many nodules become undesirable. The number of nodules is tightly regulated by environmental factors and autoregulation of nodulation.In this study, we analyzed phenotypes and molecular mechanisms of Lotus japonicus rel3 symbiotic mutant with reduced nodules. In symbiotic and non-symbiotic conditions, the length of the main root is shorter and the number of lateral roots is less in rel3 than in wild type. Moreover, the rel3 mutant developed approximately half number of nodules found in the wild type. And the ratio of nodulation zone length to the main root length in the rel3 plants was less than that in the wild type. However, there is no difference in nitrogenase activity between rel3 and wild type. The phenotypic analysis indicated that rel3 showed fewer nodules due to narrow nodulation zone. We determined the infection frequency using-NZP2235/HemA::LacZ that constitutively expresses the (3-galactosidase reporter gene. After inoculation, infection thread formation was repressed in rel3 compared to that in the wild type. This suggest that the decreased infection frequency in rel3 produce narrow nodulation zone in rel3, leading to the reduced number of nodules formed in rel3.In order to investigate molecular mechanisms of rel3 with reduced nodules, we first analyzed the sensitivity of rel3 to negative regulators of the nodulation including nitrogen and ethylene. The results show that the mutation does not affect the negative regulation of nodule development by nitrate and ethylene. In addition, the grafting experiments of rel3 and wild type indicated that the phenotype of reduced nodule number in rel3 mutant is in control of shoot-derived signals.By Agrobacterium rhizogenes-mediated root transformation, we analyzed REL3 promoter-GUS activity in inoculated roots and nodules. The GUS activity showed that REL3 gene specifically expressed in root vascular bundle and nodule vascular strands, particularly in cells that were derived from the pericycle. And REL3 also expressed in vascular bundle of developing nodules. This indicated that REL3 plays an important role for nodule initiation.REL3 mutations blocked the synthesis of TAS3-siRNA in plants, resulting in the upregulated of its target genes, ARF3 and ARF4, auxin response factors. This prompt us to detect whether the reduced nodulation number of rel3 is correspond with Auxin. Treated with auxin inhibitors NPA and TIBA, rel3 showed nor agravitropic than wild type, suggesting that the rel3 mutant is sensitive to auxin inhibitors. This implies the polar auxin transport is altered in rel3 root. The study on alternation of polar auxin transport in rel3 is ongoing.Based on current data, we speculate that the alteration of polar auxin transport in rel3 roots make change of auxin level in root cortical cells, leading to narrow the range of auxin level that required for nodule initiation in root cortical cells, and thus fewer nodules are formed in rel3 roots. This study provides the evidence that ta-siRNA pathway regulated the auxin signaling pathway, directly involved in nodule development.