Functional Analysis Of Macrophage Migration Inhibitory Factor(MIF)during Symbiotic Nitrogen Fixation In Medicago Truncatula

In previous research,we found that the expression of MIF was significantly up-regulated in Medicago lupulina roots after inoculation,suggesting its involvement in symbiosis.In this study,the model legume Medicago truncatula was used as the research object to investigate whether the MIF genes play a role in root nodule symbiosis.The MIF gene family was surveyed and the spatiotemporal expression analysis,tissue and subcellular localization,m RNA interference and overexpression of the MIF were performed.The main results are as follows:The MIF genes of 11 species?8 legumes,2 non-legumes and humans?were identified in genome-wide and found that MIF is present in the 11 species and has a certain degree of conservation.There are more MIF genes in the genomes of the legumes than in the non-legumes except Cicer arietinum with three MIFs,as many as in Arabidopsis thaliana and in Oryza sativa.The MIFs can be clustered into three clades in the plants,and each clade is found in the 10 considered plants.Six MIF genes were identified in M.truncatula,which distribute in the chromosome 4 and 7.Among them,five Mt MIFs display higher synteny with the homologous genes from Glycine max,the close living relatives of M.truncatula.Additionally,one homologous gene with higher synteny was found in A.thaliana and none in O.sativa.Finally,the expression of Mt MIFs displayed three patterns: a)significantly high expression in roots and nodules;b)significantly high expression in stems and leaves;c)constitutive expression.The results showed that MIF is widely found in plants and has more functional differentiation.In addition,MIF may be involved in the symbiotic nodulation of the legumes.The spatiotemporal expression and localization analyses showed that,compared with the control,the expression level of the selected gene,MTR?4g127360,was increased significantly in the late stage of nodulation whether under exogenous nitrogen treatment or inoculation with rhizobia.MTR?4g127360 was strongly inhibited in the early stage and up-regulated in the late stage of symbiosis.Using transformants carrying the MTR?4g127360 promoter-GUS,MTR?4g127360 was showed to be expressed in the nodule primordia,young nodules,mature nodules and root stele.Furthermore,it was strongly induced in mature root nodules.Subcellular location assay showed that MTR?4g127360 protein was located in the plasma membrane and nucleus.These indicated that MTR?4g127360 was involved in the symbiotic process and may play an important role in the mature root nodules.RNA interference and overexpression analyses showed that MTR?4g127360 had no detectable effect on the infection process of rhizobia.Compared with the control,the stem and root length of the RNAi plants decreased at 28 days post-inoculation?dpi?,while no significant change on the chlorophyll content.And after overexpression,there was no noteworthy difference in plant growth and chlorophyll content.Knockdown of MTR?4g127360 resulted in the number of nodules increased,whereas the nitrogenase activity and the expression of leghemoglobin gene decreased significantly.In contrast,the gene overexpression led to a decrease in the number of nodules,while the nitrogenase activity and leghemoglobin gene expression were up-regulated significantly.Paraffin section and transmission electron microscopy analyses showed that the control nodules were still vigorous in nitrogen fixation at 28 dpi,while the RNAi nodules began to senesce,in which the area of nitrogen fixation zone decreased and there were fewer bacteroids.Besides,the degradation of symbiosomes was observed.At 45 dpi,when the control nodules were in the aging and the symbiosomes displayed noticeable shrinkage and deformation in the nodule cells,a large area of nitrogen-fixing zone was still maintained without any sign of senescence in the overexpression nodules containing the normal bacteroids.Above all,RNA interference of MTR?4g127360 caused the premature aging and poor nitrogen-fixation ability of root nodules,while its overexpression delayed the aging process in nodules and enhanced the nitrogen fixation capacity of nodules.In conclusion,MTR₄g127360 is involved in the symbiotic nodulation of M.truncatula,and played an important role in preventing nodules from premature senescence and maintaining the nitrogen fixation ability of nodules in the late stage of symbiosis.It seems that MTR?4g127360 is not related to the early infection process.