A Lotus Japonicus Rboh Is Involved In Symbiotic Nodule Functioning

Fixed nitrogen is one of the limiting factors required for plant growth.The root nodule symbiosis(RNS)between legume plants and rhizobia is the most efficient and productive source of fixed nitrogen,and has critical importance in agriculture.But this endosymbiosis is specific for host plant and is restricted to legume plants and occasionally some nonlegume plants.Its complex symbiotic interaction relations,the plants and bacteria of multiple genes involved in transcription regulation.The release of flavonoid by legume plant,which can induce the rhizobium to product nod factors,is the first step to initiate nodulation.The formation of nitrogen-fixing nodules requires the coordinated development of rhizobial infection and nodule organogenisis,which can be separated genetically.During the past decade,a lot of genes have been cloned from various symbiotic mutants of the two model legumes:Lotus japonicus and Medicago truncatula.A model of early symbiotic signaling cascade has been built based on these studies.Indeed,accumulating experimental evidence shows that ROS are key players infundamental processes,such as cellular growth,stomatal closure and plant defence against pathogens,the involvement of ROS in the legume—rhizobia symbiotic interaction has also been highlighted.Plants contain a large amount of explosive oxidase(respiratory burst oxidase homologs;RBOHs).And the main of ros is produced by RBOHs.In this paper,based on the LjRbohA gene.Here,we got some results as follows:1.Transient expression of a green fluorescent protein(GFP)-LjRbohA fusion gene in tobacco protoplasts clearly showed that LjRbohA proteinis localized to the plasma membrane of tobacco cells.2.Real-time PCR approach showes that LjRbohA transcripts are highly expressed in the nodulation of L.japonicus after inoculation with M.loti.3.To investigate the spatial pattern of LjRbohA expression at the cellular level,the LjRbohA gene promoter is used to drive the expression of the GUS reporter in transgenic hairy roots.Histochemical staining showes that LjRbohAPro::GUS was highly expressed in nodule primordial(E,F)and appeared to be restricted to the nitrogen-fixing zone of the functional nodule.These observations are consistent with the results of real-time PCR and confirm that the expression of LjRbohA gene is indeed dependent on rhizobial infection.These results indicate that LjRbohA involves in the process of rhizobial infection and nodule development in L.japonicus.4.LjRbohA LORE1 inserted mutant show defects about nodulation.The number of nodule primordia were collected 6 days after Mloti inoculation and there was significant difference between LjRbohA mutant and wild type.The size of nodule primordia were analysed 6 days after M.loti inoculation and there was significant difference between LjRbohA mutant and wild type.5.The ros in wt was much more abundant than mutant 30032291 by histochemical staining.Violet particles was elevated as time goes on.6.BiFC was used to assess the interaction between LjRac2 and LjRbohA in tobacco leaves.LjRac2 interacted with LjRbohA and its constitutively active mutant at dots in plasma membrane.