Functions And Regulation Mechanisms Of Dihydroflavonol-4-reductase-like Protein LjDFL1 During Rhizobial Infection

The establishment of nitrogen-fixing symbiosis between rhizobia and legume plants starts with the communication of signaling molecules.In almost all symbiotic interactions between rhizobia and leguminous plants,host flavonoid-induced synthesis of Nod factors in rhizobia is required to initiate symbiotic response in plants.Legume flavonoids released into the rhizosphere are recognized by compatible rhizobia through constitutively expressed NodD proteins,which induce the transcriptional of Nod factor genes.Nod factors are synthesized by the induced nod gene products,and are recognized by compatible legume NF receptors,such as LjNFR1 and LjNFR5 from Lotus japonicus.Then the symbiosis signaling pathway is activated and leads to formation of infection threads and organogenesis.Although LjNFR5 has been identified for 15 years,there are few reports on the regulation mechanisms of rhizobial infection and nodulation associated with LjNFR5.In this study,a yeast two-hybrid analysis revealed that a dihydroflavonol-4-reductase-like protein(LjDFL1)interacts with LjNFR5,and plays a positive role in regulation of rhizobial infection.The specific research results are as follows:1.Using the atypical kinase domain of LjNFR5(LjNFR5-KD)as bait to screen a L.japonicus root cDNA library,we identified a dihydroflavonol-4-reductase-like protein(LjDFL1).The interaction between LjDFL1 and LjNFR5 was verified in vitro and in planta,using the yeast two-hybrid system,vitro protein pull-down,coimmunoprecipitation assays and bimolecular fluorescence complementation(BiFC)assays.The interaction between NFR5 and DFL1 homologs is conserved in legumes.2.The results of quantitative reverse transcriptional PCR(qRT-PCR)indicated that LjDFL1 transcripts are in all tested tissues,with the highest level of expression in root tissues.Promoter-GUS analysis of LjDFL1pro:GUS revealed that LjDFL1 was highly expressed in root tips,especially in the root hairs near the root tip,as well as in the epidermal cells of the elongation and maturation zones of root tip.These results showed LjDFL1 was highly expressed in the regions of roots,which are highly sensitive to rhizobial infection.3.qRT-PCR analysis revealed that LjDFL1 expression was significantly downregulated in roots inoculated with rhizobia compared with uninoculated roots,and declined in roots treated with Nod factors.In the L.japonicus mutants nfr5,symrk,ccamk,nsp2 and nin,which failed to establish symbiosis,LjDFL1 expression did not significantly decreased after rhizobial inoculation.LjDFL1 was expressed at much higher levels in roots inoculated with the Mesorhizobium loti MAFF303099 nodC or nodB mutant than in roots inoculated with wild-type M.loti.These results indicate that the activation of the NF signaling pathway plays a role of the negative feedback in the expression of LjDFL1.4.We used CRISPR/Cas9 technology and stable transformation of L.japonicus to obtain LjDFL1 knock-out mutants,ljdfl1-1 and ljdfl1-2.The numbers of ITs per plant and per centimeter of root were significantly lower in ljdfl1-1 and ljdfl1-2 than in the wild-type MG20 plants at 5 days postinoculation(dpi)with M.loti strain MAFF303099 constitutively expressing LacZ.The expression of NIN,NPL,NF-YA1 and EPR3 was decreased in the ljdfl1 mutant roots compared to the wild-type roots with qRT-PCR.The results implied that LjDFL1 gene mutation leads to the decrease of the numbers of ITs during infection.5.We obtained two stable transgenic L.japonicus line overexpressing LjDFL1 under the LjUbq1 promoter were generated using Agrobacterium tumefaciens-mediated transformation.Transcripts of LjDFL1 were 29-fold higher in LjDFL1-OX-2 and 38-fold higher in LjDFL1-OX-8 than in control roots.The numbers of ITs per plant and per centimeter of root are significantly increased in LjDFL1-OX-2 and LjDFL1-OX-8 compared to the control at 5 dpi with M.loti strain MAFF303099.Gene expression analysis revealed that NIN,NPL and NF-YA1 expression increased significantly in LjDFL1 overexpression roots compared to control roots.These results suggested that over expressing LjDFL1 could benefit the formation of ITs in L.japonicus.