LIN, a Medicago truncatula gene required for nodule differentiation and persistence of rhizobial infections

Abstract

Ethyl methanesulfonate mutagenesis of the model legume Medicago truncatula has previously identified several genes required for early steps in nodulation. Here, we describe a new mutant that is defective in intermediate steps of nodule differentiation. The lin (lumpy infections) mutant is characterized by a 4-fold reduction in the number of infections, all of which arrest in the root epidermis, and by nodule primordia that initiate normally but fail to mature. Genetic analyses indicate that the symbiotic phenotype is conferred by a single gene that maps to the lower arm of linkage group 1. Transcriptional markers for early Nod factor responses (RIP1 and ENOD40) are induced in lin, as is another early nodulin, ENOD20, a gene expressed during the differentiation of nodule primordia. By contrast, other markers correlated with primordium differentiation (CCS52A), infection progression (MtN6), or nodule morphogenesis (ENOD2 and ENOD8) show reduced or no induction in homozygous lin individuals. Taken together, these results suggest that LIN functions in maintenance of rhizobial infections and differentiation of nodules from nodule primordia.

Figure 1.

Symbiotic phenotypes of wild-type and lin roots. Plants were inoculated with the wild-type Rhizobium strain ABS7M containing the chimeric hemA∷lacZ gene. A, Distribution of nodules (arrow) in the nodulation zone of a wild-type root (left) and primordia (arrowhead) in the corresponding zone of a lin root (right) at 18 dpi. B to G, Plant roots were harvested at different time points after inoculation and processed for β-galactosidase activity. B and C, Segments of lin root showing the recurrent occurrence of primordia (arrowheads in B) and infections (arrows in C) at 18 dpi. D and F, Wild-type infections. A root harvested at 1 dpi (in D) shows an infection thread (arrow) penetrating through the root hair and into the cortical cell layer, while F depicts a successful infection (arrow) ramifying within a developing primordium (np) at 3 dpi. E and G, Infections in the lin mutant. E, Infection (arrow) arrested within the root hair at 1 dpi. G, Arrested infection (arrow) that fails to penetrate the nodule primordium (np) at 3 dpi. Bars = 50 μm.

Figure 2.

Cytological characterization of infection arrest and nodule primordia of lin. Sinorhizobium-infected lin roots were fixed and stained for β-galactosidase activity. Semithin sections of infected roots were stained with methylene blue plus azure II. A, A longitudinal section through a lin root at 21 dpi showing a nodule primordium (np) with an infection (arrowhead) arrested in the root hair. B, A cross-section of a lin root at 21 dpi shows two arrested infections (arrowheads), one opposite a xylem pole and one opposite a phloem pole. A developing vascular strand (double arrowhead) is found within the nodule primordium (np) adjacent to a xylem pole. Bars = 50 μm.

Figure 3.

Genetic mapping of lin to LG1 of M. truncatula. Localization of the LIN locus on LG1 of the M. truncatula genetic map. Markers DK369R, TUP, DSI, and SCP from the genetic map of LG1 (Choi et al., 2004) were found to be linked to the LIN locus. The recombination frequency between the two flanking markers DSI and SCP is 6.4 based on the lin mapping population.

Figure 4.

Northern-blot analysis of expression of early nodulins in nodulating roots of lin and wild-type plants. Total RNA from the main root zone (see “Materials and Methods”) of plants before and after inoculation was electrophoresed, blotted, and probed sequentially with cDNA probes. The rDNA probe is a loading control.

Figure 5.

Relative transcript abundance of marker genes for primordium development in wild-type and lin roots. A to C, Relative transcript abundance of ENOD20 (A), MtN6 (B), and CCS52A (C) in root samples of A17 and lin over a time course (x axes) with and without inoculation with rhizobia. Lined bars represent uninoculated root tissue of A17; black hatched bars represent inoculated root samples of A17; spotted bars represent uninfected samples of lin; and gray hatched bars represent infected root samples of lin. Transcript abundance (y axes) for all samples was estimated relative to A17 uninoculated roots on day 6. Each point represents the mean of three or four replicates with error bars representing the sd. Note that the y axis is in logarithmic scale and the scale is different for each gene. Uninoculated samples are indicated in terms of their physiological age in days and infected samples are indicated in dpi; for each time point, uninoculated and inoculated samples were harvested at the same age.