Functional Research Of MtENOD20 During Symbiotic Nitrogen Fixation In Medicago Truncatula

Our previous analyses revealed that the early nodulin gene MtENOD20?MTR₄g130800?belongs to the Phytocyanins gene family.In this study,tissue specific expression,subcellular localization,RNA interference?RNAi?and gene overexpression were conducted to further explore the function of MtENOD20 in symbiosis with the model plant Medicago truncatula.The results are summarized as follows:1.Expression pattern analysis of MtENOD20 geneThe qPCR was performed to analyze the spatio-temporal expression situation of two types of alternative splicing,namely,MtENOD20-1 and MtENOD20-2 in the symbiotic nodulation process of M.truncatula.The results showed that the MtENOD20-1 and MtENOD20-2 were strongly induced in the inoculated roots compared with the un-inoculated roots,but they have different expression patterns.In addition,MtENDO20-1was almost not expressed in roots,stems and leaves of both uninoculated and inoculated plants,but was highly expressed in nodule.Compared with MtENDO20-1,MtENDO20-2was also expressed in roots,stems and leaves of inoculated plants,but it had a lower expression than that in nodule.In general,it was speculated that the two alternatives splicing of MtENOD20 may perform different functions in nodule development because of their different expression patterns.2.Tissue specific expression of MtENOD20The fusion expression vector(pro^MtENOD20::GUS)was constructed and transferred into the roots of the M.truncatula by Agrobacterium rhizogenes-mediated transformation.Then the transformed roots were taken for the GUS staining at different stages.The results showed that MtENOD20 was mainly expressed in cortex cells?ie,the initial part of the root nodule primordium?,the root hair cells containing the infection threads and infection zone of nodule.3.Subcellular localization of MtENOD20Subcellular localization vector was transferred into tobacco leaves infected by the A.tumefaciens and roots of M.truncatula transformed by A.rhizogenes to localize the MtENOD20,respectively.In the tobacco leave cells,the green fluorescence signals mainly appeared on the plasma membrane,endoplasmic reticulum and nucleus.For the nodule cells,MtENOD20 mainly locate on the symbiosome.To sum up,these results suggest that MtENOD20 may target in symbiosome through the secretory pathway mediated by endoplasmic reticulum.4.Analyses of MtENOD20 RNAi and overexpressionBy comparison,the plant height,root length,the number of nodules,nitrogenase activity and leghemoglobin all decreased in MtENOD20 RNAi plants.Abviously,the nodules of RNAi roots were white,small and ineffective.The statistical results of infection events showed that the number of the root hair curlings,the extension infection threads,cortical infection threads and nodule primordia reduced significantly compared with the control at 7 days after inoculation?dpi?.The nodule paraffin section and transmission electron microscopy?TEM?results showed that the bacteroids were not differentiated in RNAi nodules.Compared with the wild type,the bacteroids were type I or II without nitrogen fixation activity in RNAi nodules.Expression analyses of symbiotic related genes showed that infection related genes,MtNSP1,MtERN1,MtERN2 and MtERN3,were no changes in RNAi roots.However,the expression levels of MtNIN,MtENOD11 and MtENOD40 significantly decreased.The genes related to bacteroid differentiation,RSD,DNF2,NCR001 and NCR035,significantly reduced in the RNAi roots.The results indicated that MtENOD20 could be involved in the infection process and differentiation of bacteroids.Overexpression analysis of MtENOD20 showed that the plant height,root length,fresh weight,dry weight and chlorophyll content increased,and the number of nodules,nitrogenase activity and leghemoglobin increased as well.Compared with the control,the number of root hair curls,epidermal infection threads,cortical infection threads and nodule primordia increased at 7 dpi in MtENOD20 overexpression plant.The nodule paraffin section and TEM analyses showed that the bacteroids were normal and closely arranged in the infected cells of MtENOD20 overexpression nodules at 45 dpi,while the symbiosome fusion occurred in the control nodules,indicating that the nodule cells began to senesce.