A newly evolved chimeric lysin motif receptor‐like kinase in Medicago truncatula spp. tricycla R108 extends its Rhizobia symbiotic partnership

Abstract

Rhizobial lipochitooligosaccharidic Nod factors (NFs), specified by nod genes, are the primary determinants of host specificity in the legume-Rhizobia symbiosis.We examined the nodulation ability of Medicago truncatula cv Jemalong A17 and M. truncatula ssp. tricycla R108 with the Sinorhizobium meliloti nodF/nodL mutant, which produces modified NFs. We then applied genetic and functional approaches to study the genetic basis and mechanism of nodulation of R108 by this mutant.We show that the nodF/nodL mutant can nodulate R108 but not A17. Using genomics and reverse genetics, we identified a newly evolved, chimeric LysM receptor-like kinase gene in R108, LYK2bis, which is responsible for the phenotype and can allow A17 to gain nodulation with the nodF/nodL mutant. We found that LYK2bis is involved in nodulation by mutants producing nonO-acetylated NFs and interacts with the key receptor protein NFP. Many, but not all, natural S. meliloti and S. medicae strains tested require LYK2bis for efficient nodulation of R108.Our findings reveal that a newly evolved gene in R108, LYK2bis, extends nodulation specificity to mutants producing nonO-acetylated NFs and is important for nodulation by many natural Sinorhizobia. Evolution of this gene may present an adaptive advantage to allow nodulation by a greater variety of strains.

Medicago truncatulaSOBIR1 controls specificity in the Rhizobium-legume symbiosis

“…WT strains of Sinorhizobium meliloti 2011 and Sinorhizobium medicae WSM419 carrying lacZ reporter genes were used. For S. meliloti 2011, the previously constructed strain S. meliloti 2011 (pXLGD4) was used (Luu et al 2022). For S. medicae WSM419, the plasmid pLS310-1 was constructed by subcloning the BamH I- EcoR I fragment containing the full length lacZ coding sequence from pQF-lacZ (Kaczmarczyk et al, 2013) into the similarly cut pCM62 plasmid (Marx and Lidstrom, 2001).…”

“…A sister line from the same transformation with no detected modification in MtSOBIR1, without Cas9 was used as control, or 2HA. For in vitro tests, germinated seedlings were transferred to tubes or Petri dishes containing gelified Fahreus medium, 0.2 mM NH4NO3 (Luu et al, 2022). Alternatively, germinated seedlings were transferred into pots (8x8x8 cm), filled with sterilized attapulgite clay granules (Oil Dri, UK).…”

“…Assays were performed on inoculated plants at 21 dpi as described (Luu et al 2022). Briefly, 1 ml of acetylene was injected into a test tube containing one single inoculated plant and closed with a septum.…”

“…Among them are plant LysM-RLKs, and a notable difference recently reported between A17 and R108 is the presence of a LysM-RLK, MtLYK2bis, specifically in R108. This receptor confers on R108 the ability to nodulate with certain rhizobial strains, including a S. meliloti 2011 mutant producing modified Nod factors, and S. medicae WSM419 (Luu et al 2022). In addition, MtLYK2bis is an interactor of MtNFP.…”

“…MtLYK3 is part of a cluster of closelyrelated LysM-RLKs (Limpens et al 2003;Luu et al 2023) and other genes in this cluster may also interact with NFP and play roles in nodulation. For example, in the M. truncatula genotype R108, MtLYK2bis was recently shown to be another MtNFP partner, and involved in extending nodulation ability to certain natural strains of rhizobia as well as to a Sinorhizobium meliloti 2011 rhizobial mutant producing modified Nod factors (Luu et al 2022). Another interesting aspect of MtNFP is its involvement in plant immunity.…”