Sergio Svistoonoff

SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria

Root endosymbioses vitally contribute to plant nutrition and fitness worldwide. Nitrogen-fixing root nodulation, confined to four plant orders, encompasses two distinct types of associations, the interaction of legumes (Fabales) with rhizobia bacteria and actinorhizal symbioses, where the bacterial symbionts are actinomycetes of the genus Frankia. Although several genetic components of the host–symbiont interaction have been identified in legumes, the genetic basis of actinorhiza formation is unknown. Here, we show that the receptor-like kinase gene SymRK, which is required for nodulation in legumes, is also necessary for actinorhiza formation in the tree Casuarina glauca. This indicates that both types of nodulation symbiosis share genetic components. Like several other legume genes involved in the interaction with rhizobia, SymRK is also required for the interaction with arbuscular mycorrhiza (AM) fungi. We show that SymRK is involved in AM formation in C. glauca as well and can restore both nodulation and AM symbioses in a Lotus japonicus symrk mutant. Taken together, our results demonstrate that SymRK functions as a vital component of the genetic basis for both plant–fungal and plant–bacterial endosymbioses and is conserved between legumes and actinorhiza-forming Fagales.

cg12 expression is specifically linked to infection of root hairs and cortical cells during Casuarina glauca and Allocasuarina verticillata actinorhizal nodule development.

cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during mycorrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.

Casuarina glauca prenodule cells display the same differentiation as the corresponding nodule cells

Recent phylogenetic studies have implied that all plants able to enter root nodule symbioses with nitrogen-fixing bacteria go back to a common ancestor (D.E. Soltis, P.S. Soltis, D.R. Morgan, S.M. Swensen, B.C. Mullin, J.M. Dowd, and P.G. Martin, Proc. Natl. Acad. Sci. USA, 92:2647-2651, 1995). However, nodules formed by plants from different groups are distinct in nodule organogenesis and structure. In most groups, nodule organogenesis involves the induction of cortical cell divisions. In legumes these divisions lead to the formation of a nodule primordium, while in non-legumes they lead to the formation of a so-called prenodule consisting of infected and uninfected cells. Nodule primordium formation does not involve prenodule cells, and the function of prenodules is not known. Here, we examine the differentiation of actinorhizal prenodule cells in comparison to nodule cells with regard to both symbionts. Our findings indicate that prenodules represent primitive symbiotic organs whose cell types display the same characteristics as their nodule counterparts. The results are discussed in the context of the evolution of root nodule symbioses.

Heart of Endosymbioses: Transcriptomics Reveals a Conserved Genetic Program among Arbuscular Mycorrhizal, Actinorhizal and Legume-Rhizobial Symbioses

To improve their nutrition, most plants associate with soil microorganisms, particularly fungi, to form mycorrhizae. A few lineages, including actinorhizal plants and legumes are also able to interact with nitrogen-fixing bacteria hosted intracellularly inside root nodules. Fossil and molecular data suggest that the molecular mechanisms involved in these root nodule symbioses (RNS) have been partially recycled from more ancient and widespread arbuscular mycorrhizal (AM) symbiosis. We used a comparative transcriptomics approach to identify genes involved in establishing these 3 endosymbioses and their functioning. We analysed global changes in gene expression in AM in the actinorhizal tree C. glauca. A comparison with genes induced in AM in Medicago truncatula and Oryza sativa revealed a common set of genes induced in AM. A comparison with genes induced in nitrogen-fixing nodules of C. glauca and M. truncatula also made it possible to define a common set of genes induced in these three endosymbioses. The existence of this core set of genes is in accordance with the proposed recycling of ancient AM genes for new functions related to nodulation in legumes and actinorhizal plants.

The Independent Acquisition of Plant Root Nitrogen-Fixing Symbiosis in Fabids Recruited the Same Genetic Pathway for Nodule Organogenesis

Only species belonging to the Fabid clade, limited to four classes and ten families of Angiosperms, are able to form nitrogen-fixing root nodule symbioses (RNS) with soil bacteria. This concerns plants of the legume family (Fabaceae) and Parasponia (Cannabaceae) associated with the Gram-negative proteobacteria collectively called rhizobia and actinorhizal plants associated with the Gram-positive actinomycetes of the genus Frankia. Calcium and calmodulin-dependent protein kinase (CCaMK) is a key component of the common signaling pathway leading to both rhizobial and arbuscular mycorrhizal symbioses (AM) and plays a central role in cross-signaling between root nodule organogenesis and infection processes. Here, we show that CCaMK is also needed for successful actinorhiza formation and interaction with AM fungi in the actinorhizal tree Casuarina glauca and is also able to restore both nodulation and AM symbioses in a Medicago truncatula ccamk mutant. Besides, we expressed auto-active CgCCaMK lacking the auto-inhibitory/CaM domain in two actinorhizal species: C. glauca (Casuarinaceae), which develops an intracellular infection pathway, and Discaria trinervis (Rhamnaceae) which is characterized by an ancestral intercellular infection mechanism. In both species, we found induction of nodulation independent of Frankia similar to response to the activation of CCaMK in the rhizobia-legume symbiosis and conclude that the regulation of actinorhiza organogenesis is conserved regardless of the infection mode. It has been suggested that rhizobial and actinorhizal symbioses originated from a common ancestor with several independent evolutionary origins. Our findings are consistent with the recruitment of a similar genetic pathway governing rhizobial and Frankia nodule organogenesis.

Symbiotic Signaling in Actinorhizal Symbioses

Actinorhizal symbioses are mutualistic associations between plants belonging to eight angiosperm families and soil bacteria of the genus Frankia. These interactions lead to the formation of new root organs, actinorhizal nodules, where the bacteria are hosted and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. It involves an elaborate signaling between both partners of the symbiosis. In recent years, our knowledge of this signaling pathway has increased tremendously thanks to a series of technical breakthroughs including the sequencing of three Frankia genomes [1] and the implementation of RNA silencing technology for two actinorhizal species. In this review, we describe all these recent advances, current researches on symbiotic signaling in actinorhizal symbioses and give some potential future research directions.

Infection-Related Activation of the cg12 Promoter Is Conserved between Actinorhizal and Legume-Rhizobia Root Nodule Symbiosis

Two nitrogen-fixing root nodule symbioses between soil bacteria and higher plants have been described: the symbiosis between legume and rhizobia and actinorhizal symbioses between plants belonging to eight angiosperm families and the actinomycete Frankia. We have recently shown that the subtilisin-like Ser protease gene cg12 (isolated from the actinorhizal plant Casuarina glauca) is specifically expressed during plant cell infection by Frankia. Here we report on the study of C. glauca cg12 promoter activity in the transgenic legume Medicago truncatula. We found that cg12 promoter activation is associated with plant cell infection by Sinorhizobium meliloti. Furthermore, applications of purified Nod factors and mycorrhizal inoculation failed to trigger expression of the cg12-reporter gene construct. This indicates that at least part of the transcriptional environment in plant cells infected by endosymbiotic nitrogen-fixing bacteria is conserved between legume and actinorhizal plants. These results are discussed in view of recent data concerning molecular phylogeny that suggest a common evolutionary origin of all plants entering nitrogen-fixing root nodule symbioses.

Infection-specific activation of the Medicago truncatula Enod11 early nodulin gene promoter during actinorhizal root nodulation.

The MtEnod11 gene from Medicago truncatula is widely used as an early infection-related molecular marker for endosymbiotic associations involving both rhizobia and arbuscular mycorrhizal fungi. In this article, heterologous expression of the MtEnod11 promoter has been studied in two actinorhizal trees, Casuarina glauca and Allocasuarina verticillata. Transgenic C. glauca and A. verticillata expressing a ProMtEnod11::beta-glucuronidase (gus) fusion were generated and the activation of the transgene investigated in the context of the symbiotic associations with the N-fixing actinomycete Frankia and both endo- and ectomycorrhizal fungi (Glomus intraradices and Pisolithus albus, respectively). ProMtEnod11::gus expression was observed in root hairs, prenodules, and nodules and could be correlated with the infection of plant cells by Frankia spp. However, no activation of the gus reporter gene was detected prior to infection or in response to either rhizobial Nod factors or the wasp venom peptide MAS-7. Equally, ProMtEnod11::gus expression was not elicited during the symbiotic associations with either ecto- or endomycorrhizal fungi. These observations suggest that, although there is a conservation of gene regulatory pathways between legumes and actinorhizal plants in cells accommodating endosymbiotic N-fixing bacteria, the events preceding bacterial infection or related to mycorrhization appear to be less conserved.

Actinorhizal root nodule symbioses: what is signalling telling on the origins of nodulation?

Two groups of bacteria are able to induce the formation of nitrogen-fixing nodules: proteobacteria called rhizobia, which associate with Legumes or Parasponia and actinobateria from the genus Frankia which are able to interact with ∼220 species belonging to eight families called actinorhizal plants. Legumes and different lineages of actinorhizal plants differ in bacterial partners, nodule organogenesis and infection patterns and have independent evolutionary origins. However, recent technical achievements are revealing a variety of conserved signalling molecules and gene networks. Actinorhizal interactions display several primitive features and thus provide the ideal opportunity to determine the minimal molecular toolkit needed to build a nodule and to understand the evolution of root nodule symbioses.

Casuarina Root Exudates Alter the Physiology, Surface Properties, and Plant Infectivity of Frankia sp. Strain CcI3

ABSTRACT The actinomycete genus Frankia forms nitrogen-fixing symbioses with 8 different families of actinorhizal plants, representing more than 200 different species. Very little is known about the initial molecular interactions between Frankia and host plants in the rhizosphere. Root exudates are important in Rhizobium-legume symbiosis, especially for initiating Nod factor synthesis. We measured differences in Frankia physiology after exposure to host aqueous root exudates to assess their effects on actinorhizal symbioses. Casuarina cunninghamiana root exudates were collected from plants under nitrogen-sufficient and -deficient conditions and tested on Frankia sp. strain CcI3. Root exudates increased the growth yield of Frankia in the presence of a carbon source, but Frankia was unable to use the root exudates as a sole carbon or energy source. Exposure to root exudates caused hyphal “curling” in Frankia cells, suggesting a chemotrophic response or surface property change. Exposure to root exudates altered Congo red dye binding, which indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate changes. Frankia cells preexposed to C. cunninghamiana root exudates for 6 days formed nodules on the host plant significantly earlier than control cells. These data support the hypothesis of early chemical signaling between actinorhizal host plants and Frankia in the rhizosphere.