A Molecular Mechanism For The Synergistic Effect Of MtDMI2 And MtPUB2 Regulating Nodulation Homeostasis In Medicago Truncatula

The mechanism underlying the modulation of nodulation homeostasis in legumes is a critical biological topic for a long time.Previous studies show an autoregulation of nodulation(AON)mechanism that regulates the nodulation from shoot to root.People also speculate a negative feedback that controls the nodulation locally;however,the mechanism that maintains the balance of nodulation remains largely unknown.Does not make infection 2(MtDMI2,also known as symbiosis receptor kinase,SymRK,in Lotus japonicus)is the key leucine-rich repeat(LRR)receptor-like kinase(LRR-RLK)in the noted MtDMI2-MtDMIl-MtDMI3 nodulation signaling pathway,so it is vital to search for the regulated proteins on MtDMI2.As one critical receptor-like kinase in nodulation signaling pathway,MtDMI2/LjSymRK is able to trigger irreversible reactions once passes the signal to the downstream proteins.Some studies have found the interacting proteins of MtDMI2,such as 3-hydroxy-3-methylglutaryl CoA reductase 1(MtHMGRI)and plant U-box 1(MtPUB1).Although these studies revealed the interacting proteins of MtDMI2/LjSymRK and associated functions,up to date,it is highly uncertain that how the LRR-RLK is strictly regulated during common symbiosis signaling pathway.In this study,the novel E3 ubiquitin ligase,MtPUB2,and the LRR-type receptor-like kinase,MtDMI2,form a negative feedback module to regulate nodulation homeostasis that is mediated by MtDMI2-induced phosphorylation and MtPUB2-induced ubiquitination and resulting degradation of MtDMI2.For the first time,we discovered that MtPUI32 utilizes MtDMI2 as a direct substrate for ubiquitination.Moreover,we found that MtDMI2 phosphorylates MtPUB2 through the Ser421 site.More importantly,this ubiquitination depends on the phosphorylation both in vitro and in vivo.Mtpub2 mutants were not available in the fast-neutron or Tntl insertion collection(Noble Foundation).Therefore,to functionally characterize MtPUB2y we performed Agrobacterium rhizogenes-mediated RNAi hairy roots and stable Agrobacterium tumefaciens-mediated RNAi lines.Once the RNAi efficiency reached 0.95,severe reactions such as the delayed growth,sterility,and eventual death may occur.We found that MtPUB2-RNAi roots exhibited fewer infection threads(ITs),and compared to the wild-type plants,fewer nodules,and shorter root length.Meanwhile,we also found that MtPUB2 may also be involved in the plant growth,flowering time and defense signalling pathways.Next,to directly substantiate the aforementioned hypothesis,we performed the quantitative western blotting assays(with two biological repeats,using actin protein as the loading control)in vivo and analysed the data using the numerical solutions in detail.The dmi2-1(TR25)and A17 plants(wild type)were inoculated with S.meliloti 1021(and mock)and sampled at different time slots[0 h(hours),5 h,12 h,24 h,3 DAI,5 DAI,7 DAI,14 DAI,21 DAI,and 28 DAI(days after inoculation)].The results showed that increases in MtPUB2 were accompanied by upregulation of MtDMI2.Vice versa,with downregulation of MtDMI2,MtPUB2 decreased.We then adopted the numerical solutions of Lotka-Volterra equations(LV equations)to describe the protein levels of MtDMI2 and MtPUB2 during the nodulation from 24 h to 21 DAI.The results demonstrated the interaction between MtDMI2 and MtPUB2 formed a negative feedback loop of the prey(MtDMI2)-predator(MtPUB2)manner.Taken together,we proposed a working model that M.truncatula perceives nod factors(NFs)signals via nod factor perception(MtNFP)and lysin motif-containing receptor-like kinase 3(MtLYK3).Then the signals are transferred to MtDMI2.MtDMI2 activates MtPUB2 by trans-phosphorylation through Ser421.Finally,the activated MtPUB2 mediates the degradation of MtDMI2 through ubiquitination to maintain the homeostasis of nodulation.Once this homeostasis is broken,abnormalities in ITs and nodulation will appear.Similar to the functions of negative feedback mechanisms revealed in cancer studies,such as the tumor protein 53(p53)_murine double minute 2(MDM2)working mechanisms.P53 acts as a tumor suppressor and functions in many biological pathways.MDM2 is an E3 ubiquitin ligase,which can promote the degradation of p53 via ubiquitination.This p53-MDM2 negative feedback needs to be sustained in a balanced state in healthy cells.Negative feedback loops have been found in cancer research,but such a loop is novel in legume nodulation.In conclusion,it is a breakthrough to propose a dynamic model involving an E3 ubiquitin ligase and a protein receptor-like kinase in plants.