Role Of Rboh Gene Family During The Symbiotic Nitrogen Fixation In Lotus Japonicus

A hallmark of legume plants is their ability to establish a mutualistic symbiosis with rhizobia,leading to the formation of a new organ,the root nodule,whose primary function is nitrogen fixation.In the root nodules,endosymbiotic rhizobia obtain photosynthates from the host legume and in turn fix atmospheric nitrogen into ammonia to provide the host legume with nitrogen nutrition.The symbiotic interaction involves a complicated developmental processes governed by genes from both bacteria and plants.Reactive oxygen species(ROS),as important signaling molecules,are involved in regulation of plant growth and development and plant response to biotic and abiotic stresses.The involvement of ROS in the legume-rhizobial symbiosis has also been highlighted.The NADPH oxidases(respiratory burst oxidase homologs;RBOHs)are the main enzymatic sources of ROS generation.In this work,we studied the function of Rbohgenes in symbiosis in Lotus japonicus.Based on their transcriptions,three Rbohgenes,RbohA(Lj5g3v1497840),RbohB(Lj6g3v1549190)and RbohC(Lj5g3v1497820)were found to be primarily expressed in nodules.Their function involved in symbiosis were further studied in Lotus japonicus.1.Subcellular localizations of GFP-LjRbohA,GFP-LjRbohB and GFP-LjRbohC fusion proteins in N.benthamiana leaf cells.The results showed that LjRbohA,LjRbohB and LjRbohC proteins were localized at the plasma membrane.2.The relative expression of each LjRbohgene was evaluated using qRT-PCR in L.japonicus roots.The transcript of LjRbohA accumulated abundantly in roots and nodules.The transcript of LjRbohB and LjRbohC also significantly increased after inoculation with rhizobia.3.Transgenic roots with LjRobh A_Pro:GUS and LjRobh B_Pro:GUS generated using hairy roots transformation were used to analyze gene expression pattern inoculated with rhizobia.The activities of LjRbohA and LjRbohB promoters were detected at vascular tissue,nodule,and the lateral root primordia.The spatiotemporal expression of the LjRbohA and LjRbohB promoters suggest that these two genes might play a role in nodule organogenesis.4.The function of L.japonicus Rbohin symbiosis was studied using the knock-out mutant plants generated using CRISPR-Cas9.Multiple numbers of g RNAs targeting a single Rbohgene were designed.The numbers of nodules were reduced in LjRbohB,LjRbohA-LjRbohC and LjRbohA-LjRbohB-LjRbohC knockout transgenic roots compared with control transgenic roots.Our data suggests that these three Rbohs might have an overlapping function in mediating nitrogen fixation symbiosis in L.japonicus.The evidence collected to date indicates that ROS may act as signaling molecules that regulate nodulation.