Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes.

Plants that form root-nodule symbioses are within a monophyletic 'nitrogen-fixing' clade and associated signalling processes are shared with the arbuscular mycorrhizal symbiosis. Central to symbiotic signalling are nuclear-associated oscillations in calcium ions (Ca(2+) ), occurring in the root hairs of several legume species in response to the rhizobial Nod factor signal. In this study we expanded the species analysed for activation of Ca(2+) oscillations, including nonleguminous species within the nitrogen-fixing clade. We showed that Ca(2+) oscillations are a common feature of legumes in their association with rhizobia, while Cercis, a non-nodulating legume, does not show Ca(2+) oscillations in response to Nod factors from Sinorhizobium fredii NGR234. Parasponia andersonii, a nonlegume that can associate with rhizobia, showed Nod factor-induced calcium oscillations to S. fredii NGR234 Nod factors, but its non-nodulating sister species, Trema tomentosa, did not. Also within the nitrogen-fixing clade are actinorhizal species that associate with Frankia bacteria and we showed that Alnus glutinosa induces Ca(2+) oscillations in root hairs in response to exudates from Frankia alni, but not to S. fredii NGR234 Nod factors. We conclude that the ability to mount Ca(2+) oscillations in response to symbiotic bacteria is a common feature of nodulating species within the nitrogen-fixing clade.

Strigolactone biosynthesis in Medicago truncatula and rice requires the symbiotic GRAS-type transcription factors NSP1 and NSP2.

Legume GRAS (GAI, RGA, SCR)-type transcription factors NODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression after symbiotic signaling. However, legume NSP1 and NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved in higher plants. Here, we show that NSP1 and NSP2 are indispensable for strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice. Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is essential for conversion of orobanchol into didehydro-orobanchol, which is the main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds correlates with reduced expression of DWARF27, a gene essential for SL biosynthesis. Rice and M. truncatula represent distinct phylogenetic lineages that split approximately 150 million years ago. Therefore, we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. NSP1 and NSP2 are single-copy genes in legumes, which implies that both proteins fulfill dual regulatory functions to control downstream targets after rhizobium-induced signaling as well as SL biosynthesis in nonsymbiotic conditions.

Nonlegume Parasponia andersonii deploys a broad rhizobium host range strategy resulting in largely variable symbiotic effectiveness.

The non-legume genus Parasponia has evolved the rhizobium symbiosis independent from legumes and has done so only recently. We aim to study the promiscuity of such newly evolved symbiotic engagement and determine the symbiotic effectiveness of infecting rhizobium species. It was found that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to four different genera, and therefore, we conclude that this non-legume is highly promiscuous for rhizobial engagement. A possible drawback of this high promiscuity is that low-efficient strains can infect nodules as well. The strains identified displayed a range in nitrogen-fixation effectiveness, including a very inefficient rhizobium species, Rhizobium tropici WUR1. Because this species is able to make effective nodules on two different legume species, it suggests that the ineffectiveness of P. andersonii nodules is the result of the incompatibility between both partners. In P. andersonii nodules, rhizobia of this strain become embedded in a dense matrix but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia spp. Therefore, we argue that the Parasponia-rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.

A phylogenetic strategy based on a legume-specific whole genome duplication yields symbiotic cytokinin type-A response regulators.

Legumes host their Rhizobium spp. symbiont in novel root organs called nodules. Nodules originate from differentiated root cortical cells that dedifferentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole-genome duplication has occurred at the root of the legume Papilionoideae subfamily. We hypothesize that gene pairs originating from this duplication event and are conserved in distinct Papilionoideae lineages have evolved symbiotic functions. A phylogenetic strategy was applied to search for such gene pairs to identify novel regulators of nodulation, using the cytokinin phosphorelay pathway as a test case. In this way, two paralogous type-A cytokinin response regulators were identified that are involved in root nodule symbiosis. Response Regulator9 (MtRR9) and MtRR11 in medicago (Medicago truncatula) and an ortholog in lotus (Lotus japonicus) are rapidly induced upon Rhizobium spp. Nod factor signaling. Constitutive expression of MtRR9 results in arrested primordia that have emerged from cortical, endodermal, and pericycle cells. In legumes, lateral root primordia are not exclusively formed from pericycle cells but also require the involvement of the root cortical cell layer. Therefore, the MtRR9-induced foci of cell divisions show a strong resemblance to lateral root primordia, suggesting an ancestral function of MtRR9 in this process. Together, these findings provide a proof of principle for the applied phylogenetic strategy to identify genes with a symbiotic function in legumes.

Genetic Dissection of Apomixis in Dandelions Identifies a Dominant Parthenogenesis Locus and Highlights the Complexity of Autonomous Endosperm Formation.

Apomixis in the common dandelion (Taraxacum officinale) consists of three developmental components: diplospory (apomeiosis), parthenogenesis, and autonomous endosperm development. The genetic basis of diplospory, which is inherited as a single dominant factor, has been previously elucidated. To uncover the genetic basis of the remaining components, a cross between a diploid sexual seed parent and a triploid apomictic pollen donor was made. The resulting 95 triploid progeny plants were genotyped with co-dominant simple-sequence repeat (SSR) markers and phenotyped for apomixis as a whole and for the individual apomixis components using Nomarski Differential Interference Contrast (DIC) microscopy of cleared ovules and seed flow cytometry. From this, a new SSR marker allele was discovered that was closely linked to parthenogenesis and unlinked to diplospory. The segregation of apomixis as a whole does not differ significantly from a three-locus model, with diplospory and parthenogenesis segregating as unlinked dominant loci. Autonomous endosperm is regularly present without parthenogenesis, suggesting that the parthenogenesis locus does not also control endosperm formation. However, the high recovery of autonomous endosperm is inconsistent with this phenotype segregating as the third dominant locus. These results highlight the genetic complexity underlying apomixis in the dandelion and underline the challenge of introducing autonomous apomixis into sexual crops.

Rhizobium Lipo-chitooligosaccharide Signaling Triggers Accumulation of Cytokinins in Medicago truncatula Roots.

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

Evolutionary origin of rhizobium Nod factor signaling.

For over two decades now, it is known that the nodule symbiosis between legume plants and nitrogen fixing rhizobium bacteria is set in motion by the bacterial signal molecule named nodulation (Nod) factor. Upon Nod factor perception a signaling cascade is activated that is also essential for endomycorrhizal symbiosis (Fig. 1). This suggests that rhizobium co-opted the evolutionary far more ancient mycorrhizal signaling pathway in order to establish an endosymbiotic interaction with legumes. As arbuscular mycorrhizal fungi of the Glomeromycota phylum can establish a symbiosis with the fast majority of land plants, it is most probable that this signaling cascade is wide spread in plant kingdom. However, Nod factor perception generally is considered to be unique to legumes. Two recent breakthroughs on the evolutionary origin of Rhizobium Nod factor signaling demonstrate that this is not the case. The purification of Nod factor-like molecules excreted by the mycorrhizal fungus Glomus intraradices and the role of the LysM-type Nod factor receptor PaNFP in the non-legume Parasponia andersonii provide novel understanding on the evolution of rhizobial Nod factor signaling.

LysM-type mycorrhizal receptor recruited for rhizobium symbiosis in nonlegume Parasponia.

Rhizobium-root nodule symbiosis is generally considered to be unique for legumes. However, there is one exception, and that is Parasponia. In this nonlegume, the rhizobial nodule symbiosis evolved independently and is, as in legumes, induced by rhizobium Nod factors. We used Parasponia andersonii to identify genetic constraints underlying evolution of Nod factor signaling. Part of the signaling cascade, downstream of Nod factor perception, has been recruited from the more-ancient arbuscular endomycorrhizal symbiosis. However, legume Nod factor receptors that activate this common signaling pathway are not essential for arbuscular endomycorrhizae. Here, we show that in Parasponia a single Nod factor-like receptor is indispensable for both symbiotic interactions. Therefore, we conclude that the Nod factor perception mechanism also is recruited from the widespread endomycorrhizal symbiosis.