Identification of a dominant gene in Medicago truncatula that restricts nodulation by Sinorhizobium meliloti strain Rm41

Abstract

Background: Leguminous plants are able to form a root nodule symbiosis with nitrogen-fixing soil bacteria called rhizobia. This symbiotic association shows a high level of specificity. Beyond the specificity for the legume family, individual legume species/genotypes can only interact with certain restricted group of bacterial species or strains. Specificity in this system is regulated by complex signal exchange between the two symbiotic partners and thus multiple genetic mechanisms could be involved in the recognition process. Knowledge of the molecular mechanisms controlling symbiotic specificity could enable genetic improvement of legume nitrogen fixation, and may also reveal the possible mechanisms that restrict root nodule symbiosis in non-legumes.

Results: We screened a core collection of Medicago truncatula genotypes with several strains of Sinorhizobium meliloti and identified a naturally occurring dominant gene that restricts nodulation by S. meliloti Rm41. We named this gene as Mt-NS1 (for M.truncatulanodulation specificity 1). We have mapped the Mt-NS1 locus within a small genomic region on M. truncatula chromosome 8. The data reported here will facilitate positional cloning of the Mt-NS1 gene.

Conclusions: Evolution of symbiosis specificity involves both rhizobial and host genes. From the bacterial side, specificity determinants include Nod factors, surface polysaccharides, and secreted proteins. However, we know relatively less from the host side. We recently demonstrated that a component of this specificity in soybeans is defined by plant NBS-LRR resistance (R) genes that recognize effector proteins delivered by the type III secretion system (T3SS) of the rhizobial symbionts. However, the lack of a T3SS in many sequenced S. meliloti strains raises the question of how the specificity is regulated in the Medicago-Sinorhizobium system beyond Nod-factor perception. Thus, cloning and characterization of Mt-NS1 will add a new dimension to our knowledge about the genetic control of nodulation specificity in the legume-rhizobial symbiosis.

Figure 1

Nodulation and growth phenotypes of M. truncatula plants after inoculation with S. meliloti strain Rm41. A, F83005.5 and A20 lines after inoculation by S. meliloti strain Rm41. A20 (right) grew normally under nitrogen-free conditions, whereas F83005.5 (left) can hardly survive under the same conditions. B, Nodulation phenotypes of the same plants in panel A. A20 roots formed nitrogen-fixing root nodules (right), while F83005.5 cannot nodulate with Rm41. C and D, a closer look at the nodulation phenotypes of A20 and F83005.5, showing fully developed functional nodules on the roots of A20 (D) and the nodule primordial (arrows) formed on the roots of F83005.5 (C).

Figure 2

Fluorescence microscopy analyses of infection process of compatible and incompatible interactions between M. truncatula plants and Rm41. All images are composite images of GFP-expressing Rm41 cells (green) and root cells (red). A, Typical infection thread formed by compatible interaction between A20 and Rm41. The infection thread extends from the colonized, curled root hair to the base of the root hair cell. B, In the incompatible interaction between F83005.5 and Rm41, the bacteria can normally colonize the curled root hairs but typical infection threads cannot be detected. Occasionally, we can detect aborted, aberrant infection threads present on the F83005.5 roots. C, The nodule primordium on the A20 roots contained bacteria, while the nodule primordia on the F83005.5 roots (D) contained no bacteria despite frequent presence of bacterial colonies on the epidermal surface of the nodule primordia.

Figure 3

Genetic mapping of Mt-NS1. A, Growth phenotypes of A20 (Nod+, ns1/ns1), F83005.5 (Nod-, NS1/NS1), and a subpopulation of F2 plants three weeks post inoculation with S. meliloti Rm41. Nodulated plants are green and healthy, whereas the non-nodulated plants showed nitrogen starvation symptoms with yellowish leaves under the nitrogen-free condition. B, Fine mapping of Mt-NS1. The Mt-NS1 locus was delimited to a genomic region between markers SNP103 and SNP127. Numbers indicate the number of recombination breakpoints separating the marker from Mt-NS1 based on genotyping ~3,900 F2 plants. C, Annotation of the 50-kb genomic DNA of Jemalong A17 (Nod + Fix-, ns1/ns1) that covers the candidate gene region identifies 7 putative genes (Medtr8g28070-Metr8g28115). D, Identification of an insertion/deletion polymorphism between the two copies of the receptor-like kinases (Metr8g28110 and Metr8g28115) among the genomes of Jemalong A17, A20, and F83005.5.