The Molecular Mechanism Of MtWRKY76 In Response To Abiotic Stress And Nodule Pathway In Medicago Truncatula

WRKY transcription factora have formed the complex mechanisms to regulate their own growth,against biotic and abiotic stress.Legumes symbiotic interacting with Rhizobia convert nitrogen mol-ecules in the air into ammonia available for plant,and this process is affected by the genes both from the host and Rhizobia.Based on the transcriptional dynamics in Medicago truncatula under salt stress,with deep data mining,we chose a transcription factor gene,MtWRKY76,for further experimental validation.MtWRKY76,858 bp and 285 aa,belongs to WRKYII sub-group.MtWRKY76 was located in nuclei by sub-cellular localization and without transactivation in yeast.MtWRKY76 was mainly expressed in leaves,roots and nodules,which was induced by ABA,salt and drought.Homologous expression of MtWRKY76 in Medicago truncatula R108,the plant of T3 generation was used for study.During 100 mM NaCl stress,the root length of wild type was 1.36 cm while 2.36 cm and 2.27 cm for transgenic L2 and L4 respectively,and the plant dry weight of wild type was 4.15 mg while 5.52 mg and 5.30 mg for transgenic L2 and L4 respectively.When deprive of water for 42 d the survival rate for wild-type was only 26.7%,88.9%and 73.3%for L2 and L4 respectively,meanwhile,the relative electrolyte leakage of transgenic lines was lower than wild-type.In conclu-sion,the transgenic plants displayed salt and drought resistance.The expression of MtMYB61,MtMYB3,MtCBF4 and MtCAS31 increased in transgenic lines relative to wild type.MtWRKY76 was used as a bait protein to screen the cDNA library of M.truncatula in yeast two-hybrid system.MtASR interacted with MtWRKY76 in yeast and in planta.Our study indicated that MtASR inter-acting with MtWRKY76 might act as part of a transcription-regulating complex involving in abiotic stress signaling pathway based on prevous studies.MtWRKY76 was induced by S.meliloti 1021 and reached the top at 4 d interestingly.When inoculat-ed with S.meliloti 1021 for 30 d,the number of nodules in transgenic lines was significantly higher than that of wild-types.The number for wild-type was about 7,furthermore L2 and L4 increased 1.79 and 1.65 times respectively.Moreover,the expression of MtEFD,MtCRE1,MtNIN,MtNSP1 and MtNSP2 increased in transgenic lines relative to wild type,instead MtCDK3 which was negatively regulated nodule number was down-regulated.MtWRKY76 could bind to W-box cis-element in the promoter of MtNSP1 gene through EMSA.DGE analysis implicated that the genes about abiotoc and nodule processes were increased,simarly to our studies.We first reports that MtWRKY76 participates in the crosstalk of abiotic stress and nod-ule pathway in M.truncatula.Therefore,this paper confers a simple relation model of MtWRKY76.