Invasion of Lotus japonicus root hairless 1 by Mesorhizobium loti involves the nodulation factor-dependent induction of root hairs

Abstract

In many legumes, including Lotus japonicus and Medicago truncatula, susceptible root hairs are the primary sites for the initial signal perception and physical contact between the host plant and the compatible nitrogen-fixing bacteria that leads to the initiation of root invasion and nodule organogenesis. However, diverse mechanisms of nodulation have been described in a variety of legume species that do not rely on root hairs. To clarify the significance of root hairs during the L. japonicus-Mesorhizobium loti symbiosis, we have isolated and performed a detailed analysis of four independent L. japonicus root hair developmental mutants. We show that although important for the efficient colonization of roots, the presence of wild-type root hairs is not required for the initiation of nodule primordia (NP) organogenesis and the colonization of the nodule structures. In the genetic background of the L. japonicus root hairless 1 mutant, the nodulation factor-dependent formation of NP provides the structural basis for alternative modes of invasion by M. loti. Surprisingly, one mode of root colonization involves nodulation factor-dependent induction of NP-associated cortical root hairs and epidermal root hairs, which, in turn, support bacterial invasion. In addition, entry of M. loti through cracks at the cortical surface of the NP is described. These novel mechanisms of nodule colonization by M. loti explain the fully functional, albeit significantly delayed, nodulation phenotype of the L. japonicus ROOT HAIRLESS mutant.

Figure 1.

Zones of root hair development in L. japonicus. An image of a portion of a L. japonicus root is shown and different zones of root hair development are indicated. The transition from the root hair emergence zone to the root hair elongation zone has been assigned arbitrarily and is indicated by a dotted line.

Figure 2.

Features of root hairs in wild-type L. japonicus. A, A confocal fluorescent image of a propidium iodide stained epidermal cell that has initiated root hair growth from the center of the periclinal cell wall. B, A scanning electron micrograph of a portion of wild-type L. japonicus root within the root hair mature zone. C, A scanning confocal image of an infection thread originating from a microcolony at the curled root hair tip and proceeding down into the base of the epidermal cell. M. loti bacteria are tagged with GFP (green fluorescence) and the root tissue has been counterstained with propidium iodide (red fluorescence).

Figure 3.

Root hair phenotypes of L. japonicus mutant lines. A, The har1-1 parental line and double mutants Ljrhl1 har1, Ljprh1 har1, Ljsrh1 har1, and Ljvrh1 har1 are shown. B, Scanning electron micrographs of double mutants Ljrhl1 har1, Ljprh1 har1, and Ljsrh1 har1. Root segments were photographed at approximately 0.3 to 0.5 cm from the root tip.

Figure 4.

Positions of root hair loci LjSRH1, LjVRH1, LjPRH1, and LjRHL1 on L. japonicus chromosomes 3, 5, and 6. Flanking simple sequence repeat markers are given. For all markers shown, the observed ratios of the genotypic classes deviated significantly from the ratios expected if independent segregation from the mutant locus was assumed (P < 0.001; chi-square tests). Thus, all markers shown are linked to their respective root hair loci. The number of recombinants as a fraction of the total number of mutants tested is given for each marker in brackets.

Figure 5.

Symbiotic phenotypes of wild-type (Gifu) and mutant plants (har1-1, and double mutants Ljrhl1 har1, Ljprh1 har1, Ljsrh1 har1, and Ljvrh1 har1. A, Roots of plants 10 dai with M. loti strain NZP2235 carrying a hemA::LacZ reporter gene construct. Roots of all plants shown were cleared and stained for β-galactosidase activity. B, A longitudinal section (25 μm) of the double mutant Ljrhl1 har1 10 dai. NP and emerging lateral root primordium can be easily distinguished. C, Numbers of nodules and NP on the roots of wild type, har1-1, and root hair double mutants (Ljrhl1 har1, Ljprh1 har1, Ljsrh1 har1, and Ljvrh1 har1) and single mutant (Ljrhl1-1) 21 dai with M. loti strain NZP2235 (hemA::LacZ). Roots were cleared and stained for β-galactosidase activity before counting. Mean values ± 95% confidence interval are given for each genotype (n = 10 to 15).

Figure 6.

Representative infection events 21 dai on double mutants Ljprh1-1 har1-1, Ljsrh1 har1-1, and Ljvrh1-1 har1-1. Roots were cleared and stained for β-galactosidase activity to detect the infecting M. loti strain NZP2235 (hemA::LacZ). A, An abnormally broad IT formed within a mutant root hair of Ljprh1-1 har1-1. The IT has ramified within the nodule cortex (B) IT traversing an uncurled root hair on Ljsrh1 har1-1. C, A Ljvrh1-1 har1-1 root hair exhibiting a curled root hair tip encircling a microcolony from which an infection thread descends.

Figure 7.

Infection phenotypes of Ljrhl1-1 (42 dai). A, A longitudinal section of a NP stained for β-galactosidase activity. B, A light micrograph of a semithin section of an NP. Note the absence of infected cells. Activated cortical cells with centrally located enlarged nuclei and many starch granules are visible. C, A longitudinal section of an infected nodule stained for β-galactosidase activity showing deep-blue color in the nodule central zone and an IT extending from the nodule apex. D, A light micrograph of a semithin section of an infected nodule. Infected cells (IC) are clearly discernible; NC, nodule cortex; VB, vascular bundle. E, Roots of a 4-month-old M. loti infected Ljrhl1-1 plant showing an abundance of nodules.

Figure 8.

Root hair-independent invasion of NP in the Ljrhl1 mutant by M. loti (hemA::LacZ). A, A longitudinal thick section of an NP showing a bacterial patch on the surface visualized by β-galactosidase staining (21 dai). B, A light micrograph of a toluidine blue stained thin section of an NP showing a surface bacterial patch (asterisk) which progresses via an intercellular route through 3 layers of cortical cells creating an infection pocket (21 dai). C, A transverse section (30 μm) of an infected NP with a surface patch of bacteria leading to the infection pocket and the IT that has ramified within the NP cortex (42 dai). D, Close-up of C. The bacterial patch is continuous with an infection pocket (arrow) that narrows down into an IT.

Figure 9.

Root hair phenotypes of the Ljrhl1-1 mutant after inoculation with M. loti. A, Three long nodule-associated (cortical) hairs emerging from the apex of a NP. B, A non-nodule-associated (epidermal) root hair with presumed NF induced branching and deformations. C, A differential interference contrast image of an uncolonized NP showing two cortical root hairs, one fully emerged and the second one just emerging (asterisk). D, IT traversing a root (cortical) hair that has emerged from the site of a fissure in the root epidermis at the apex of an enlarged NP.

Figure 10.

Numbers of nodule-associated (gray bars) and non-nodule-associated (white bars) root hairs for individual plants (1–10) that were either uninoculated or were inoculated with the M. loti strains as shown. A to C, Single mutant Ljrhl1-1. D to F, Double mutant Ljrhl1-1 har1-1.