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

Rhizobial lipochitooligosaccharidic Nod factors, specified by nod genes, are the primary determinants of host specificity in the legume-Rhizobia symbiosis. A Sinorhizobium meliloti nodF/nodL mutant produces Nod factors that differ from wild-type ones in lacking an O-acetate, and with a different acyl chain on the terminal non-reducing sugar. This mutant is defective in nodulation with various Medicago hosts. We examined the nodulation ability of M. truncatula cv Jemalong A17 and M. truncatula ssp. tricycla R108 with the nodF/nodL mutant. 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 gene in R108, LYK2bis, which is responsible for the phenotype. Transformation with LYK2bis allows A17 to gain nodulation with the nodF/nodL mutant. We found that LYK2bis is involved in specific NF signalling and interacts with the key receptor protein NFP. Our findings reveal that a newly-evolved gene in R108, LYK2bis, extends nodulation specificity to strains producing non-O-acetylated NFs. Interaction between LYK2bis and NFP provides a means of integrating the nodulation signalling pathways.

Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti – Medicago laciniata symbiosis

Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation specific, nodulating only with a restricted range of Sinorhizobium meliloti, e.g., strain 102L4, but not with most strains that nodulate Medicago sativa (alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of M. laciniata. Nucleotide sequencing revealed that this fragment contained nodABCIJ genes whose overall arrangement was similar to those found in strains RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only subclones of the 11-kb DNA fragment containing a functional nodC gene from strain 102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The practical implications of these findings are discussed in the context of the development of a specific M. sativa – S. meliloti combination that excludes competition for nodulation by bacterial competitors resident in soil.Key words: Sinorhizobium meliloti, Medicago laciniata, nodulation specificity,nod gene, nucleotide sequence.

Comparison of partial 23S rDNA sequences fromRhizobiumspecies

A hypervariable region of Rhizobium 23S rDNA was amplified by polymerase chain reaction and phylogenetic relationships of several strains were determined by comparing nucleotide sequences of the amplified product. Variation in the 23S rDNA nucleotide sequences was consistent with phylogenetic relationships determined by host nodulation specificity and (or) 16S rDNA sequence analysis. Six strains representing three Rhizobium species (R. leguminosarum bv. trifolii, R. meliloti, and R. etli), and two strains each of Bradyrhizobium and Agrobacterium were clustered into five rDNA groups. Unique features identified by secondary structure analysis of the 23S rRNA sequenced region were consistent with the hypothesis that 23S rDNA could be used to design species- or strain-specific Rhizobium probes.Key words: Rhizobium, rDNA, strain identification, phylogeny.

Nodulation and Nodulin Gene Expression in an Interspecific Hybrid Between Glycine max and Glycine tomentella

When the genome of a wild species, Glycine tomentella, is combined with that of the cultivated soybean, Glycine max in a hybrid, nodulation specificity is altered. Two strains of Rhizobium fredii that nodulate the G. rnax parent fail to nodulate the hybrid, indicating the presence of a restriction gene, while three Bradyrhizobium japonicum strains that nodulate G. max are highly effective on the hybrid. Some strains of (Brady)rhizobium, originally isolated from wild Australian Glycine species, nodulate only one of the two parental species. One strain ineffective on G. max is effective on the hybrid. Two other strains, while effective on one parent, appear more effective on the hybrid. In three fully examined cases in which effective nodules are produced on the hybrid, the late nodulins leg-hemoglobin, glutamine synthetase, and xanthine dehydrogenase are expressed from both genomes. Thus, although a rhizobial strain may nodulate only one or the other of the parental types, if it successfully nodulates the hybrid, the nodulation process provides signals or internal conditions that lead to expression of late nodulins from both genomes.