The Role And Molecular Mechanism Of Map Kinase Cascade During Symbiosis Signaling In Lotus Japonicus

SymRK (symbiosis receptor-like kinase) is first cloned to recognition of both arbuscular mycorrhizal fungal and nitrogen-fixing rhizobial bacteria, likely active near the junction of fungal and rhizobial signaling cascades. SymRK is required for an early signal transduction pathway in the rhizobium-legume symbiosis. In our study, we used SymRK kinase domain as a bait to screen a yeast two-hybrid cDNA library prepared from early rhizobium-inoculated Lotus roots, one cDNA encoding a novel protein was designated as SIP2(SymRK-interacting protein2), which is a typical MAP kinase kinase (MAPKK). MAPK cascades are minimally composed of three kinase modules, which are involed in the regulation of development, growth, differentiation, programmed cell death, hormonal and stress response. Signaling through MAPK cascades is a fundamental and conserved process in animals, plants and yeast. MAPKK is in the middle part of MAPK cascades, needed to be activatied by upstream receptors MAPKKKs through phosphorylation, and phosphorylate MAPKs as downstream targets. Extracellular signal progressively amplified and transmitted to the cell nucleus through MAPK signaling cascades, led to changes in the transcriptone. This work studied the role and mechanism of MAPK signaling cascade during the rhizobium-legume symbiosis signaling pathway in Lotus japonicus. The main results are as follows:1. SymRK is specific interact with MAPKK. Using the yeast two-hybrid system, MAPKK (SIP2) interacted with the protein kinase domain of SymRK, but didn't interact with protein kinase domain of NFR1or NFR5. Other MAPKKs, such as MAPKK2and MAPKK10from L. japonicus didn't interact with SymRK neither. In addition, the interaction is conserved in medicago. NORK from alfalfa is the ortholog of SymRK interact with SIMKK, which is the ortholog of SIP2from alfalfa, the cross interactions among them. We further verified the interactions between SIP2constructs and SymRK-PK using an in vitro protein-protein pull-down assay. The technique of BiFC (bimolecular fluorescence complementation) confirms this interaction in Nicotiana benthamiana epidermis leaves. These results indicate that the interaction between SymRK and MAPKK is specific and conserved in the legume, the two proteins may have a common mechanism in the leguminous plants. 2. SIP2is a functional protein kinase. SIP2is a MAPKK, did it has autophosphorylate or transphosphorylate a protein substrate activity? Purified GST-SIP2and His-SIP2proteins were subjected to in vitro kinase assays using casein as a substrate, after autoradiography, we detected that SIP2was able to autophosphorylate itself and phosphorylate casein. SIP2can use MPK6as a substrate for phosphorylation, so SIP2is a typical MAPKK. To test if SymRK could be phosphorylated by SIP2or SIP2could be phosphorylated by SymRK. The kinase activity of SymRK and SIP2had to be abolished, we created kinase negative forms by replacing the absolutely conserved K residue with R in the ATP-binding site. The results showed that SIP2could not phosphorylate SymRK-PK-KR and SymRK failed to phosphorylate the SIP2-KR, these suggesting that SymRK is neither a potential phosphorylation target nor a kinase source of S1P2.3. SymRK is an inhibitor of SIP2kinase. To learn more about the interaction mechanism between SymRK and SIP2, we added an increasing amount of either kinase-active (SymRK-PK) or kinase-negative SymRK (SymRK-PK-KR) as an effector to the kinase-active SIP2assays in the presence of MPK6-KR as a substrate. The results showed that the kinase activity of SIP2decreased as the increase of SymRK in a dose-dependent manner. When added an increasing amount of NFR1-PK or bovine serum albumin (BSA) to the SIP2kinase assays, we did not observe any inhibitory effect on the SIP2kinase activity, suggesting that SymRK is a specific inhibitory effect on the kinase activity of SIP2towards the MPK6substrate.4. Expression of SIP2gene and subcellular localization of SIP2protein. Lotus SymRK is constitutively expressed in early symbiosis signaling pathway, we asked if the SIP2expression patterns similar with SymRK. In this study, young roots, stem, leaves and nodules were harvested at different time points after inoculation with M. loti MAFF303099, the expression levels of SIP2were measured using real-time PCR. Our results showed that SIP2was expressed in all tissues tested, including roots, stem, leaves and nodules, did not observe significant changes in SIP2mRNA level, SIP2is constitutively expressed. Through Agrobacterium rhizogenes LBA1334-mediated transformation, GFP:SIP2expressed in Lotus hairy roots, the fluorescence was observed under a confocal laser scanning microscope, GFP::SIP2localized to the plasma membrane and cytoplasm, GFP alone was used as a control, which was distributed in the cytoplasm and nucleus. The plasmid GFP::SIP2was delivered to the onion epidermal cells via particle bombardment. After plasmolysis, the fluorescence signal was observed in the cytoplasm and plasma membrane, while the GFP control fluorescence throughout the cytoplasm. We conclude that the SIP2protein is localized to the plasma membrane and cytoplasm.5. Lotus SIP2regulate of Rhizobium infection and nodule primordia formation. To identify the SIP2biological functions in the symbiotic process, two RNAi constructs were used to generate L. japonicus hairy roots via A. rhizogenes LBA1334infection, the hairy roots were inoculated with M. loti MAFF303099to induce nodule formation and their nodulation phenotypes were analyzed4weeks post inoculation. The suppression of SIP2expression was confirmed using real-time PCR amplification of a region in the SIP2mRNA. The average nodule number per root of SIP2-RNAi were significantly lower than that of the control hairy roots, IT formation and nodule initiation appeared to be impaired by the knockdown expression of SIP2in RNAi hairy roots. The expression levels of SIP2and three marker genes for infection thread and nodule primordial formation were down-regulated drastically, while the expression of two other MAPKKs genes were not altered. These observations demonstrate an essential role of SIP2in the early symbiosis signaling and nodule organogenesis.6. SymRK-SIP2interaction is not required for arbuscular mycorrhizal colonization. We infected the SIP2-RNAi hairy roots with Glomus intraradices, a common arbuscular mycorrhizal fungus. There was no observable difference in the efficiency of hyphal and arbuscular colonization between SIP2-RNAi hairy roots and the control hairy roots expressing the empty vector. Therefore, we conclude that SymRK-SIP2interaction is specific for the root response to Rhizobium infection and is not required for arbuscular mycorrhizal colonization.