A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis

See allHide authors and affiliations

Science  05 Jan 2007:
Vol. 315, Issue 5808, pp. 101-104
DOI: 10.1126/science.1132514


In legumes, Nod-factor signaling by rhizobia initiates the development of the nitrogen-fixing nodule symbiosis, but the direct cell division stimulus that brings about nodule primordia inception in the root cortex remains obscure. We showed that Lotus japonicus plants homozygous for a mutation in the HYPERINFECTED 1 (HIT1) locus exhibit abundant infection-thread formation but fail to initiate timely cortical cell divisions in response to rhizobial signaling. We demonstrated that the corresponding gene encodes a cytokinin receptor that is required for the activation of the nodule inception regulator Nin and nodule organogenesis.

The development of nitrogen-fixing nodules on roots of leguminous plants commences with a molecular dialogue between the host plant and a compatible strain of rhizobia, leading to the synthesis of bacterially encoded lipochito-oligosaccharide signaling molecules, the Nod factors (NFs). Plant plasma membrane–derived structures called infection threads (ITs), which originate within root hairs of the host root in a NF-dependent manner, act as conduits for rhizobia to enter the root tissues and to progress toward the root cortex where a nodule primordium (NP) has been initiated. The release of bacteria from ITs into the cytosol of a subset of NP cells and subsequent cellular specialization of both symbionts lead to the formation of fully functional nitrogen-fixing organs, the root nodules (1).

The initiation of cell divisions for NP organogenesis is presumed to arise from the relay of a signal from the epidermally perceived NFs to the root cortex. Several genes in the NF-dependent signaling pathway have been identified, including putative NF receptors (24) and a number of downstream elements (512). Deleterious mutations in any of these genes abolish bacterial entry into the root and NP development, indicating a crucial role for NF signaling in both processes.

The identification of plants that spontaneously form nodules (1315), together with the observations that ectopic application of cytokinins (16) or auxin transport inhibitors (17) to the root surface lead to the development of nodule-like structures, demonstrates that the machinery required for NP development is intrinsic to the plant. Consequently, the NF pathway is presumed to trigger nodule organogenesis by regulating the endogenous plant mechanism; however, the nature of the mechanism involved remains unclear.

We performed a screen for genetic suppressors of the Lotus japonicus har1-1 hypernodulation phenotype that identified three allelic suppressor lines characterized by a low-nodulation phenotype and an excessive formation of ITs. The corresponding locus was named HYPERINFECTED 1 (HIT1) (18). Further detailed phenotypic analysis, performed in both double (hit1 har1-1) and single (hit1) mutant backgrounds, showed that the three suppressor lines had indistinguishable mutant phenotypes, with the bacterial root invasion by way of ITs intact and the timely onset of associated cortical cell divisions for NP organogenesis aborted. The hit1-1 har1-1 and hit1-1 mutants were chosen as reference lines.

When analyzed 10 days after inoculation (dai) with a Mesorhizobium loti strain carrying a constitutive hemA::lacZ reporter gene fusion, the most noticeable feature of hit1-1 har1-1 roots was hyperinfection (Fig. 1A and fig. S1A). The large number of ITs that formed in the hit1-1 har1-1 mutant roots originated within curled root hairs, but their progression toward the root cortex was blocked at the interface between the epidermis and the cortex (Fig. 1B). Infrequent ITs that escaped this early blockage and managed to penetrate within the hit1-1 har1-1 root cortex looped frequently, suggesting that they were misguided (Fig. 1C). In spite of abundant infection events at the root epidermis, the root cortex of the hit1-1 har1-1 mutant failed to initiate NP (Fig. 1C). By 14 dai, many ITs overcame the initial blockage and progressed deeper into the mutant root (Fig. 1D). Cortical cell divisions were initiated coincident with the accumulation of ITs within the root cortex, but NP did not develop (Fig. 1, D and E).

Fig. 1.

Root segments stained for β-galactosidase (LacZ) activity 10 dai [(A) to (C)] and 14 dai [(D) and (E)] with M. loti. (A) A large number of ITs gave a blue appearance to the hit1-1 har1-1 root. (B) An IT (blue) traversed a root hair but became blocked at the interface between the epidermis and cortex. (C) Misguided ITs looping within the root cortex. The subepidermal infection would normally be associated with subtended cell divisions (28). VB, vascular bundle. (D and E) Negative images of (D) a longitudinal section through hit1-1 har1-1 root showing a large number of ITs (red) within the root cortex and (E) a cross-section of the hit1-1 har1-1 root showing accumulation of ITs around the entire midcortex perimeter of the section plane.

hit1-1 displayed the same mutant phenotypic features as hit1-1 har1-1, although the overall number of symbiotic events observed was reduced, likely reflecting the presence of the functional HAR1 autoregulatory receptor kinase (19). In hit1-1, an initial lack of NP formation in response to rhizobial infection (fig. S1B) was accompanied by the early onset of hyperinfection (fig. S1A) with a large number of ITs located within the root cortex (fig. S1C). Most root cortical cells associated with ITs in hit1-1 and hit1-1 har1-1 remained small and uncolonized; M. loti was confined to ITs (fig. S1D). Occasionally, a local release of bacteria from clustered ITs resulted in enlarged and often flattened nodules (fig. S2, A and B), giving rise to the low-nodulation phenotype (fig. S2C). Intermittently, we also observed the development of nodules similar to those in the wild-type roots in both hit1-1 and hit1-1har1-1 (fig. S2B).

We further investigated a role for the HIT1 locus in NP organogenesis by studying Early Nodulin 40 (ENOD40) and Nin expression, two markers for NP initiation (12, 20). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that steady-state levels of the corresponding transcripts in the inoculated roots were strongly reduced in the hit1-1 har1-1 compared with those of the har1-1 parental line, although this difference was not statistically significant between inoculated hit1-1 and wild-type roots (fig. S3, A and B). We concluded that the presence of a functional HIT1 locus is required for normal ENOD40 and Nin expression during the organogenesis of NPs, at least in the har1-1 hit1-1 background.

Additional validation of HIT1 function in nodule organogenesis was provided by the analysis of the L. japonicus root hairless (Ljrhl1-1) and hit1-1 double mutant. In response to inoculation with M. loti, Ljrhl1-1 initially develops a large number of uncolonized NPs, a consequence of the absence of root hairs and associated ITs (21). We reasoned that if HIT1 mediates nodule primordia organogenesis, the presence of a mutant hit1-1 allele should prevent or notably attenuate NP formation in Ljrhl1-1. The analysis of the Ljrhl1-1 hit1-1 mutant showed that the development of NP was almost entirely aborted, providing strong evidence for the indispensable role of HIT1 in nodule organogenesis (Fig. 2). The hit1-1 phenotype resembled the infection-thread “symbiosis” proposed to have been a precursor to nodulation (22). Could HIT1 be the missing evolutionary link?

Fig. 2.

Numbers of nodules and nodule primordia (10 dai) on wild-type roots and hit1-1, Ljrhl1-1, and Ljrhl1-1 hit1-1 mutants. Error bars show means ± 95% CI (n =20).

To begin addressing this question, we set out to map-base clone the HIT1 locus. While this work was in progress, Jens Stougaard's group at Aarhus University (Denmark) cloned the L. japonicus Spontaneous nodule formation 2 locus (13), renamed as Lotus histidine kinase 1 (Lhk1) (23), which is localized to the same genetic interval on chromosome IV as HIT1. Given the opposing nodulation phenotypes of snf2 and hit1-1, we tested the hypothesis that hit1 mutants represent loss-of-function alleles of Lhk1.

Lhk1 specific primers were used to amplify the analogous genomic region from the three hit1 lines. In all three lines, mutations were found that were predicted to result in premature stop codons (Fig. 3A). This finding, along with the ability of a wild-type Lhk1 gene to complement the hit1-1 har1-1 and hit1-1 phenotypes in transgenic L. japonicus hairy root experiments (fig. S4), confirmed the identity of the underlying gene as Lhk1.

Fig. 3.

(A) The exon (box)/intron (line) structure of the Lhk1 gene with positions of the molecular lesions for each of the hit1 mutant alleles indicated. The corresponding protein domains are indicated: transmembrane domains (TM) (black); CHASE domain (white hatched); histidine kinase (HK) domain (gray hatched); receiver (REC) domain (gray). hit1-1 carries a nucleotide substitution from G1695 to A that leads to alternatively spliced products, as shown by RT-PCR in (B). hit1-2 contained two consecutive transversions (G2407 and A2408 to TT) followed by a single base (C2409) deletion. This frame shift results in a premature stop codon 45 base pairs (bp) downstream. hit1-3 has a transition from C4922 to T resulting in a premature stop codon before the REC domain. (C) Expression of Lhk1 in various L. japonicus tissues as assayed by RT-PCR. Ubiquitin (Ubi) was used as the RNA loading control. (D) Lhk1 cDNA confers cytokinin responsiveness to yeast cells. TM182 cells were transformed with either p415CYC carrying the Lhk1 cDNA, the hit1-2 cDNA or the vector alone. Transformants were plated on a minimal medium with or without galactose (gal), or on a galactose-free medium supplemented with the indicated hormone. NAA, 1-naphthaleneacetic acid

Because the hit1-1 allele carried a G1695-to-A nucleotide substitution in the splice donor site of intron four, we used oligonucleotide primers flanking this site to amplify the corresponding cDNA. Seven aberrant hit1-1 splice variants were identified (Fig. 3B). In addition, a polymorphic species was found among the PCR products. A search for the corresponding L. japonicus genomic sequence identified a previously unrecognized gene, here named Lhk2 (Lotus histidine kinase 2), of which the predicted product showed 85% identity at the amino acid level with LHK1. Whereas Lhk1 transcripts were present in roots, nodules, and shoots (Fig. 3C), the Lhk2 mRNA was detectable only in roots (fig. S5).

Analysis of the full-length Lhk1 cDNA revealed a 2979–base pair open reading frame encoding a predicted protein of 993 amino acids (Fig. 3A). The LHK1 protein had 64% identity with the Arabidopsis cytokinin histidine kinase receptor AHK4, and 49 and 45% identity with AHK2 and AHK3, respectively. Similar to LHK1, LHK2 was more closely related to AHK4 than to other Arabidopsis cytokinin receptors (fig. S6).

When expressed in the sln1Δ yeast strain carrying a lethal mutation in SLN1 histidine kinase (24), Lhk1 rescued the growth of the yeast strain in a cytokinin-dependent manner, demonstrating that LHK1 is a cytokinin receptor (Fig. 3D). In agreement with this notion, roots of hit1-1 mutants exhibited strong insensitivity to exogenously applied cytokinin (Fig. 4A). A similar cytokinin-insensitive root phenotype was observed in all three hit1-1 har1-1 double-mutant lines (fig. S7).

Fig. 4.

(A) hit1-1 roots are insensitive to exogenously applied cytokinin. (B) qRT-PCR showing significant attenuation (*P < 0.05) of Nin mRNA in hit1-1 versus wild-type roots upon exogenous application of 50 nM BA. BA-stimulated accumulation of Nin transcripts in hit1 roots was significantly higher (P < 0.05) in comparison to water treatments. Error bars show means ± 95% CI. (C) The proposed role of LHK1 in NF-induced nodule organogenesis. Perception of NF by a presumed NFR1 and NFR5 receptor complex stimulates local cytokinin biosynthesis in, or redistribution of cytokinin to, the root epidermis and cortex (29). This is perceived by LHK1, which activates Nin expression in the root cortex leading to initiation of nodule organogenesis.

Because the accumulation of Nin and ENOD40 transcripts was significantly attenuated in the hit1-1 har1-1 mutant, we next tested whether exogenous application of cytokinin regulates expression of these genes in the wild-type roots. ENOD40 has been shown to be induced by external application of cytokinin to the roots in several legume species (25), and this was also the case in L. japonicus, although the overall induction of ENOD40 was rather modest (fig. S8A). In contrast, 50 nM benzyl adenine (BA) increased the steady-state level of Nin transcript by a factor of 20 (fig. S8A). This induction required de novo protein biosynthesis (fig. S8B). In hit1-1 roots, BA-stimulated accumulation of Nin transcripts was significantly diminished in comparison with wild-type roots, indicating that the high level of Nin expression requires the functional Lhk1 (Fig. 4B).

NF signaling regulates Nin expression (3, 15), which is required for the formation of ITs in the root epidermis and initiation of nodule primordia organogenesis in the root cortex (12). Our data indicate that although necessary for Nin expression and nodule organogenesis, Lhk1 is not required for IT formation (Fig. 4C). The reported partitioning of Nin expression between the root epidermis and cortex could provide a plausible explanation for this apparent conundrum (3, 15, 26). Nin expression, supporting IT formation, may be regulated by an Lhk1-independent mechanism in the root epidermis, possibly involving another cytokinin receptor. Diminished nodule organogenesis in hit1-1 har1-1 and hit1-1 likely restricts local and/or systemic feedback mechanisms that limit root susceptibility to Rhizobium infection, resulting in hyperinfection (Fig. 4C).

The LHK1 homologs, such as LHK2 and LHK3, are likely to function as cytokinin receptors, which may explain a leaky (formation of some nodules) symbiotic phenotype and lack of more general developmental abnormalities in mutants carrying hit1 alleles. The snf2 mutant described in the accompanying manuscript (23) and strongly reduced nodulation in Medicago truncatula plants carrying a MtCREI silencing construct (27) further demonstrate that cytokinin sensing is required to stimulate nodule development. Together, these results specify that the regulators of cytokinin biosynthesis and/or action are crucial downstream targets of NF perception (Fig. 4C) and that recruitment of a cytokinin receptor could have been an essential event during the evolution of nitrogen-fixing nodule symbiosis.

Supporting Online Material

Materials and Methods

Figs. S1 to S8

References and Notes

References and Notes

View Abstract

Stay Connected to Science

Navigate This Article