Domain Swap Approach Reveals the Critical Roles of Different Domains of SYMRK in Root Nodule Symbiosis in Lotus japonicus

Abstract

Symbiosis receptor kinase (SYMRK) is a cell membrane-localized protein kinase containing extracellular malectin-like domain (MLD) and leucine-rich repeat (LRR) domains, which is critically required for both root nodule symbiosis (RNS) and arbuscular mycorrhizal symbiosis (AMS). SYMRK is widely distributed in the genomes of different plant species; however, the contribution of different domains of SYMRK and its homologs from other plant species to RNS is largely unclear. In this study, SYMRK and its homologs from three typical plant species including <i>Medicago truncatula</i> (for both RNS and AMS), <i>Oryza sativa</i> (for AMS but not RNS), and <i>Arabidopsis thaliana</i> (for neither RNS or AMS) were investigated using domain swap approach in response to rhizobia in <i>Lotus japonicus</i>. Full-length SYMRK from rice and <i>Medicago</i> but not from <i>Arabidopsis</i> could complement <i>Lotus symrk-409</i> mutant plants to contribute RNS. The chimeric protein with the extracellular domain (ED) of LjSYMRK and cytoplasmic domains (CD) of SYMRK from both <i>Medicago</i> and rice but not <i>Arabidopsis</i> could contribute to RNS in <i>Lotus</i>, suggesting that the CD of SYMRK is required for symbiotic signaling. The chimeric receptors containing the CD of LjSYMRK (SYMRK^CD) and the EDs of MtDMI2 (MtDMI2^ED), OsSYMRK (OsSYMRK^ED), AtSYMRK (AtSYMRK^ED), NFR1 (NFR1^ED), and NFR5 (NFR5^ED) could complement <i>Lotus symrk-409</i> mutant plants to develop nodules. However, MtDMI2 could partially complement <i>Lotus symrk-409</i> mutants to form both effective nodules and ineffective bumps, which is similar to the complementation results from MtDMI2^ED-LjSYMRK^CD and LjSYMRK^GDLC in <i>Lotus symrk-409</i> mutants, suggesting that ED of SYMRK has a very fine-tune regulation for RNS in <i>Lotus</i>. The deletion of either MLD or LRR on SYMRK^GDLC (a mutant version of SYMRK with GDPC motif replaced by GDLC) could contribute to RNS when overexpressed in <i>Lotus symrk-409</i> mutants, suggesting that MLD and LRR domains might work together to be involved in symbiotic signaling and the LRR domain might play a negative role in LjSYMRK^GDLC-mediated RNS. By mutagenizing the conserved amino acids on LRR domain, five serine residues were found to be required for the function of LjSYMRK^GDLC in RNS. These finding precisely refine the molecular mechanisms of SYMRK function in symbiotic signaling in <i>L. japonicus</i>.