Molecular Mechanisms Of Nodulation Signal Transduction And The Application Of CRISPR/Cas9 In Lotus Japonicus

The global nitrogen cycle plays an extremely important role in earth biochemistry.The most abundant form of nitrogen is N2,accounting for about 78%of the atmosphere by volume.Nevertheless,N2 could not be directly assimilated by animals,plants and most microorganisms.Instead,legume plants have an exceptional capability forN2 fixation due to the endosymbiotic association with nitrogen-fixing rhizobium,which generally leads to the formation of nodules,where the bacteria depends on the nitrogenase enzyme for the reduction of N2 to ammonia.When the soil is deficient in the biologically available nitrogen(ammonia,nitrate,or nitrite),the host plants release flavonoids which are perceived by rhizobia.In response,bacteria move to the rhizosphere and can also produce the Nod factors(NFs).Successful recognition of NFs by the host elicits the establishment of root nodule symbiosis(RNS).Nodule formation consists of the rhizobial infection process and the subsequent nodule organogenesis.A complex signal exchange involving nod factor receptors recognizing NF,other receptors perceiving EPS and LPS which leads to the infection thread formation.Once the rhizobia are recognized by the host,the cortical cells initiate the dedifferentiation process leading to the root nodule primordia development,which involves the subset of plant phytohormones,for example cytokinin,auxin,and ethylene.This work engineered the CRISPR/Cas9 system to make the methodology appropriate for the leguminous model plant Lotus japonicus.We also identified the cytokinin signal transduction cascade related genes and lectin domain like receptor kinase genes and their symbiotic roles based on the following methods:CRISPR/Cas9-mediated target gene knockout,phenotypic analysis of corresponding mutants,bimolecular fluorescence complementation and gene expression.In chapter 1,we discussed several aspects related to this thesis:?the nitrogen cycle on the earth,the indispensable role of nitrogen fertilizer on agricultural industry and the application prospect of symbiotic nitrogen fixation to solve nitrogen deficiency in farmland and environmental pollution caused by excessive use of chemical fertilizers;?the origin of symbiotic nitrogen fixation,current states of knowledge and prospects of symbiotic signal transduction cascade;?the molecular mechanisms of representative plant phytohormones,namely cytokinin,auxin,gibberellin,and ethylene,in the nodule organogenesis;?the principle and application of CRISPR/Cas9 system in plant genome editing.In chapter 2,we identified nine candidate genes of U6 snRNA,and chose the 0.8 kb fragment of LjU6-1 gene promoter region to drive the expression of single guide RNA(sgRNA),which was suitable for the application of CRISPR/Cas9 system in Lotus japonicus.Specifically,we observed:?successful single gene(LjSYMRK)editing via one sgRNA in stable transgenic lines and the efficiency was 35%(7 out of 20 plants);?multiple genes(LjLbl,LjLb2,LjLb3)modification via two common sgRNAs in transgenic hairy root lines and the efficiency was 28.5%;?disruption of LjLbl/2/3 genes in stable transgenic lotus plants;?nodule-specific expression of Cas9 confers similarly efficient gene modifications in transgenic hairy roots.In chapter 3,using yeast two-hybrid screening and bimolecular fluorescence complementation(BiFC)technique,we identified two LHK1-interacting proteins,Lotus histidine phosphotransfer protein LjLHPl and LjLHP3.Both LjLHP1 and LjLHP3 could interact with Lotus histidine kinase 1(LHK1)at plasma membrane.The phosphorylated LjLHP1 and LjLHP3 moved into nucleus and interacted with response regulators(RRs).RNA interference(RNAi)knockdown of LjLHP3 expression in Ljlhpl LORE1 insertion mutant led to further inhibited nodule formation in L.japonicus.These results indicated that both LjLHPl and LjLHP3 play an essential role in nodulation and might work redundantly to mediate cytokinin signal transduction.We also detected the protein interactions between two LjLHPs and LjRRs(including 7 A-type RRs and 7 B-type RRs),and proposed a straightforward signal transduction network.In chapter 4,we characterized a Lotus mutant with hyper-infection and reduced nodule numbers.This mutant was generated by insertion of retrotransposon LORE1 in a S-Domain like reception kinase gene loci.Hence,we named this gene as LjSDK(S-Domain like Kinase).The LjSDK protein contains an extracellular bulb-type lectin domain,a plant PAN/APPLE-like domain,a transmembrane domain and an intracellular serine/threonine kinase domain.LjSDK was localized at plasma membrane in Nicotiana benthamiana leaves and in transgenic hairy roots.Lj SDK could interact with nod factor receptor NFR5 in Nicotiana benthamiana leaves.These results suggest that LjSDK may participate in early process of symbiotic signal transduction.