KM, GG and MP conceived and designed the experiments, performed the experiments, and analyzed the data. KM and MP wrote the paper.
¤ Current address: Quintiles, Budapest, Hungary
The authors have declared that no competing interests exist.
Nitrogen-fixing root nodule symbioses (RNS) occur in two major forms—Actinorhiza and legume-rhizobium symbiosis—which differ in bacterial partner, intracellular infection pattern, and morphogenesis. The phylogenetic restriction of nodulation to eurosid angiosperms indicates a common and recent evolutionary invention, but the molecular steps involved are still obscure. In legumes, at least seven genes—including the symbiosis receptor-kinase gene
As an adaptation to nutrient limitations in terrestrial ecosystems, most plants form Arbuscular Mycorrhiza (AM), which is a symbiotic relationship between phosphate-delivering fungi and plant roots that dates back to the earliest land plants. More recently, a small group including the legumes and close relatives has evolved the ability to accommodate nitrogen-fixing bacteria intracellularly. The resulting symbiosis is manifested by the formation of specialized root organs, the nodules, and comes in two forms: the interaction of legumes with rhizobia, and the more widespread Actinorhiza symbiosis of mostly woody plants with
Root nodule symbioses with nitrogen-fixing bacteria provide many plants with a source of nitrogen. This study uncovers evidence that changes in the gene
Nitrogen limits plant growth in many terrestrial ecosystems. Evolutionary adaptations to this constraint include symbiotic associations with bacteria that are capable of converting atmospheric nitrogen into ammonium. Extracellular associations of plants with diverse groups of nitrogen-fixing bacteria are phylogenetically widespread, but only a small group evolved the ability to accommodate bacteria endosymbiotically inside cell wall boundaries. Bacterial symbionts are confined within tubular structures called infection threads, which are surrounded by a host-derived membrane that is continuous with the plasma membrane, and bound by plant cell wall–like material [
All putative
The molecular adaptations underlying the evolution of plant-bacterial endosymbioses are still a mystery, despite a substantial biotechnological interest in understanding the genetic differences between nodulating and non-nodulating plants. While the molecular communication between legumes and rhizobia has been studied in some detail, important clues are expected from the genetic analysis of the yet underexplored Actinorhiza.
Bacterial signalling molecules and corresponding plant receptors involved in RNS are known only for the legume–rhizobium interaction.
Phenotypic analysis of legume mutants has revealed a genetic link between RNS and Arbuscular Mycorrhiza (AM), which is a phosphate-scavenging association between plant roots and fungi belonging to the phylum Glomeromycota [
The link of plant–fungal and plant–bacterial endosymbioses in legumes, which involves at least seven genes [
To gain insight into the evolution of nitrogen-fixing root nodulation, we analysed common symbiosis genes across angiosperm lineages with different symbiotic abilities. Many, including the calcium/calmodulin kinase gene
Genetic evidence indicates that SYMRK acts near a point of molecular convergence of AM and legume-rhizobium signalling [
The homogenous occurrence of “full-length”
To investigate
Co-transformed roots express
(A and B) Nodulated wild-type root (left), control root transformed with pRedRoot lacking the silencing cassette (middle), and non-nodulated DgRNAi knockdown root (right) (A) under white light and (B) with transgenic roots showing DsRED1 fluorescence.
(C–H) AM phenotype of
Scale bars: (A and B) 2 mm; (C, E, and G) 0.1 mm; (D, F, and H) 0.02 mm.
Transgenic roots were identified via fluorescence of eGFP encoded on the transfer-DNA.
(A–D)
(E–AB)
(A, B, E, F, K, L, Q, R, W, and X) Roots co-cultivated with
(C, D, G–J, M–P, S–V, and Y–AB) Root systems inoculated with
Scale bars: (A, E, K, Q, and W) 0.1 mm; (B, F, L, R, and X) 0.02 mm; (C, D, G, H, M, N, S, T, Y, and Z) 2 mm; (I, J, O, P, U, V, AA, and AB) 0.5 mm.
To test whether
To determine whether
Restoration of Root Symbioses in
To analyze the symbiotic capabilities of “full-length” eurosid
SYMRK from the non-nodulating eudicots
A reduced
In legumes,
The ability of different
Our survey of
A common feature associated with endosymbiotic bacterial infection in both actinorhizal [
Repetitive LRR modules have been implicated in the determination and evolution of novel recognition specificities of receptor proteins [
At an overall structural level, exon acquisition from other genes encoding LRR or NEC-like domains [
The NEC domain encoded by
It will be a future challenge to determine the contribution of individual SYMRK LRR units as well as of the NEC domain and to resolve at the amino acid level the features of SYMRK proteins involved in conferring endosymbiotic nodulation capacity.
The diversity and scattered occurrence of nodulation symbioses within the eurosid lineage suggest multiple independent origins [
We used a PCR strategy employing degenerate primers to obtain
λ Zap cDNA libraries were available for isolation of
For rapid amplification of cDNA ends (RACE) reactions, total RNA was extracted from roots of uninoculated seedlings or young plants and DNaseI treated. RT and 5′/3′RACE reactions were done using the SMART RACE kit (Clontech), following nested degenerate PCR reactions ([10 s 94 °C, 10 s 52 °C, 30 s 72 °C] × 35, 5 min 72 °C) to obtain initial sequence information.
For hairy root complementation assays,
For
Transgenic roots on
Twelve-wk-old
Databases used for BLAST sequence search and analysis included
Black shading indicates amino acid residues identical in all sequences, residues found in at least 50% of the sequences are shaded gray. Bars delimit predicted SYMRK protein domains. Dark blue, conserved extracellular region (CEC); black, LRRs; gray, imperfect LRR; white, juxtamembrane regions; brown, transmembrane region; green, protein kinase domain. Light blue bars with stars mark some of the regions conserved among SYMRK candidates, but not in other homologous sequences in rice and
(96 KB DOC)
Transformation assay and selection were as in
(C–F, M, and N)
(A–J) Roots inoculated with
(K–P) Roots co-cultivated with
Scale bars: (A–D and G–H) 2 mm; (E–F and I–J) 0.5 mm; (K, M, and O) 0.1 mm; (L, N, and P) 0.02 mm.
(1.8 MB PDF)
Transformation assay and selection were as in
(A, B, E, F, I, J, M, N, S, and T) Roots co-cultivated with
(C, D, G, H, K, L, O–R, and U–Z) Root systems inoculated with
Scale bars: (A, E, I, M, and S) 0.1 mm; (B, F, J, N, and T) 0.02 mm; (C, D, G, H, K, L, O, P, U, V) 2 mm; (Q, R, and W–Z) 0.5 mm.
(3.8 MB PDF)
(106 KB DOC)
Sequences of
We are grateful to K. Pawlowski (Department of Botany, Stockholm University, Sweden) for supplying
Arbuscular Mycorrhiza
leucine-rich repeat
N-terminal extracellular region of predicted SYMRK proteins
pre-infection thread
pre-penetration apparatus
root nodule symbiosis
rapid amplification of cDNA ends
RNA interference