Functional Analysis Of MtWOX13 In Medicago Nodule Development

Legumes and rhizobia can establish a mutually beneficial symbiosis relationship.Nodule provides a suitable living environment for rhizobia and carbohydrates necessary for their growth.In turn,rhizobia convert molecular nitrogen into ammonium nitrogen for plants to absorb and utilize.The nodule organ is equivalent to a miniature nitrogen fertilizer factory,which provides a steady stream of nitrogen nutrients for the growth of plants.Nodule is the epiphytic organ of the plant,and nodule meristems develop from root cortex cells.Two types of nodules,indeterminate and determinate nodules,form in legume plants.Indeterminate nodules,which have a sustained nodule meristem,first mitotically activate inner cortical and pericycle cells,whereas nodule meristems originate from the third cortical layer,or the inner cortical layers in Medicago truncatula.Determinate nodules initiate from the outermost one or two layers of cortical cells and do not maintain a nodule meristem form on Lotus japonicus and Glycine max.However,the molecular mechanism underlying the development of legume root nodules is still unclear.Plant-specific WOX(WUSCHEL-related homeobox)transcription factor family plays an important role in the maintenance of plant roots and stem apical meristems,the formation of lateral organs and floral organs,and the development of embryos.Especially,it has an important regulatory effect on areas where cell proliferation and differentiation.In this study,we found that transcription factors Mt WOX13 and Mt WOX13-like1 were significantly induced by nodulation factor treatment.We cloned the gene codi ng for the GUS reporter under the control of the Mt WOX13 and Mt WOX13-like1 upstream regulatory sequences.We found that Mt WOX13 and Mt WOX13-like1 were highly expressed in the nodule primordia.We also found that Mt WOX13 and Mt WOX13-like1 were highly expressed during lateral root formation.In mature LR no staining were detected within the root tip.This is consistent with the expression pattern of At WOX13 in root development.The data imply that Mt WOX13 and Mt WOX13-like1 are involved in nodule organogenesis.Further research found that the number of infections and pink nodules with nitrogen fixation ability in Mtwox13-like1 single mutant were significantly reduced,although the total number of nodules did not change significantly,indicating that Mt WOX13-Like1 plays important role in regulating the infection of rhizobia.Furthermore,the dominant negtive of Mt WOX13 and Mt WOX13-like1 can significantly reduce the number of nodules and the number of infection threads on the hairy roots of transgenic plants,which proves that Mt WOX13 and Mt WOX13-like1 participate in the development of nodules and the formation of infection threads in nodule symbiosis.In addition,we found through real-time reverse transcription PCR analysis that the loss of function of Mt WOX13 and Mt WOX13-like1 and the mutation of Mtwox13-like1 down-regulate the expression of symbiosis-related marker genes.In particular,the expression of RIP gene,which controls Rhizobium infection,decreased most significantly.It suggests that Mt WOX13-like1 may participate in the formation of infection threads by directly activating RIP.