Plant and bacterial symbiotic mutants define three transcriptionally distinct stages in the development of the Medicago truncatula/Sinorhizobium meliloti symbiosis

Abstract

In the Medicago truncatula/Sinorhizobium meliloti symbiosis, the plant undergoes a series of developmental changes simultaneously, creating a root nodule and allowing bacterial entry and differentiation. Our studies of plant genes reveal novel transcriptional regulation during the establishment of the symbiosis and identify molecular markers that distinguish classes of plant and bacterial symbiotic mutants. We have identified three symbiotically regulated plant genes encoding a beta,1-3 endoglucanase (MtBGLU1), a lectin (MtLEC4), and a cysteine-containing protein (MtN31). MtBGLU1 is down-regulated in the plant 24 h after exposure to the bacterial signal, Nod factor. The non-nodulating plant mutant dmi1 is defective in the ability to down-regulate MtBGLU1. MtLEC4 and MtN31 are induced 1 and 2 weeks after bacterial inoculation, respectively. We examined the regulation of these two genes and three previously identified genes (MtCAM1, ENOD2, and MtLB1) in plant symbiotic mutants and wild-type plants inoculated with bacterial symbiotic mutants. Plant (bit1, rit1, and Mtsym1) and bacterial (exoA and exoH) mutants with defects in the initial stages of invasion are unable to induce MtLEC4, MtN31, MtCAM1, ENOD2, and MtLB1. Bacterial mutants (fixJ and nifD) and a subset of plant mutants (dnf2, dnf3, dnf4, dnf6, and dnf7) defective for nitrogen fixation induce the above genes. The bacA bacterial mutant, which senesces upon deposition into plant cells, and two plant mutants with defects in nitrogen fixation (dnf1 and dnf5) induce MtLEC4 and ENOD2 but not MtN31, MtCAM1, or MtLB1. These data suggest the presence of at least three transcriptionally distinct developmental stages during invasion of M. truncatula by S. meliloti.

Figure 1.

Identification of symbiotically regulated M. truncatula genes. a, Clone 108 (MtBGLU1) is suppressed early in the symbiosis. A representative northern blot is shown of RNA from roots exposed to buffer, wild-type S. meliloti (Rm1021), or the SL44 bacterial mutant which is unable to make Nod factor. Plants were harvested 6, 24, or 48 h after inoculation. Experiments were repeated at least four times. Blots were serially probed for clone 108 (top), RIP1 (middle), or Actin (lower). b, Representative northern blots showing induction of TC77066 (MtLEC4), TC86036 (MtN31), MtCAM1, ENOD2, and MtLB1 during nodulation. Plants were harvested 0, 4, 7, 14, or 28 d after inoculation with wild-type S. meliloti. Experiments were repeated three times. The upper blot was serially probed with TC77066 (top), TC86036 (upper middle), MsENOD2 (lower middle), or Actin (lower) probes. The lower blot was serially probed with MtCAM1 (top), MtLB1 (middle), or Actin (lower) probes.

Figure 2.

Nod factor is sufficient to down-regulate MtBGLU1. Shown is a representative northern blot of RNA from plants exposed to 100 pm Nod factor or to buffer for 24 or 48 h. Experiments were repeated five times. The same blot was serially probed with an MtBGLU1 (top), RIP1 (middle), or Actin (lower) probe.

Figure 3.

dmi1 is defective in the ability to suppress MtBGLU1. Shown is a representative northern blot of RNA from wild-type or dmi1-2 plants exposed to S. meliloti (Rm1021) for 48 h. Experiments were repeated at least three times. The blot was serially hybridized to an MtBGLU1 (top), RIP1 (middle), or Actin (lower) probe.

Figure 4.

Model of infection and fixation defects. Bacterial (blue lettering) and plant mutants (black lettering) define distinct developmental blocks in the symbiosis. Bacterial invasion of plants (blue) can be arrested at one of three points: before inner cortical cell penetration (upper), after release into plant cells (upper middle), and after differentiation into bacteroids (lower middle). These phases can be transcriptionally separated using the expression patterns of MtLEC4, ENOD2, MtN31, MtCAM1, and MtLB1.

Figure 5.

MtLEC4, MtN31, MtCAM1, ENOD2, and MtLB1 exhibit altered expression in response to bacterial mutants. a, Representative northern blots of RNA from M. truncatula plants exposed to bacterial mutants: exoA (Rm7031), exoH (DW223), bacA (VO2119), fixJ (VO2683), and nifD (VO2746) or to wild-type bacteria (Rm1021) for 4 weeks. Experiments were repeated at least three times. The upper blot was serially hybridized to an MtLEC4, MtN31, or actin probe. The lower blot was serially hybridized to an MtCAM1, MsENOD2, Actin, or MtLB1 probe. b, Representative photographs of M. truncatula root sections inoculated with each bacterial mutant. All photographs are at the same magnification. Scale bar = 0.5 mm.

Figure 6.

MtLEC4, MtN31, MtCAM1, ENOD2, and MtLB1 exhibit altered expression in plant mutants. Shown are representative northern blots of RNA from M. truncatula plant mutants exposed to wild-type bacteria (Rm1021) for 4 weeks. Experiments were repeated at least three times. The upper blot was serially hybridized to an MtLEC4, MtN31, MsENOD2, or Actin probe. The lower blot was serially hybridized to an MtCAM1, MtLB1 or Actin probe.