The common symbiosis pathway (CSP), comprising the receptor-like kinase SYMRK, the calcium/calmodulin-dependent protein kinase CCaMK, and the transcription factor CYCLOPS, has been conserved since the most recent common ancestor of land plants, where it serves arbuscular mycorrhizal symbiosis. Comparative phylogenomics indicates that this ancient pathway was subsequently co-opted to support other intracellular symbioses, most prominently the root-nodule symbiosis with nitrogen-fixing bacteria that arose in the nitrogen-fixing clade of flowering plants, but the mechanism of this co-option is not understood. The open problem is to determine, at gene and cis-regulatory-element resolution, which changes downstream of the CCaMK-CYCLOPS module redirected the conserved symbiosis signalling output to drive rhizobial infection and de novo nodule organogenesis, and to establish which of those changes were necessary and sufficient for the evolutionary transition between symbiotic types. Major recent advances have resolved components of this transition - the cis-regulatory element PACE in the NIN promoter, specific to the nitrogen-fixing clade, connecting the nodulation master regulator NIN to the CSP; the ancestral functionality and subsequent protein-level refinement of NIN; and a phylogenomic reconstruction of the nodulation gene regulatory network - but a complete mechanistic account of how the conserved signalling output was redirected to drive both the infection and organogenesis branches of the nodulation program remains lacking.

Background

Arbuscular mycorrhizal (AM) symbiosis is a mutualistic association between most land plants and fungi of the Glomeromycotina, in which the fungus colonizes root cells intracellularly and forms branched feeding structures called arbuscules; fossil and genetic evidence indicates this symbiosis facilitated the colonization of land by plants roughly 450 million years ago. In angiosperms the best-studied intracellular symbioses - AM symbiosis and the root-nodule symbioses with nitrogen-fixing bacteria (rhizobia in legumes, or Frankia in actinorhizal plants) - share a signal transduction cascade called the common symbiosis pathway (CSP, also called CSSP or SYM pathway). Its core components are the plasma-membrane receptor-like kinase SYMRK (also named DMI2, from 'does not make infections', the mutant phenotype by which the gene was first identified in Medicago truncatula), the nuclear-localized calcium and calmodulin-dependent protein kinase CCaMK (DMI3), which decodes oscillations of nuclear calcium concentration (calcium spiking) triggered by microbial signals, and the CCaMK-interacting transcription factor CYCLOPS (IPD3), together with the nuclear ion channel DMI1. The CSP activates downstream transcriptional programs through cis-regulatory elements - short DNA sequence motifs in gene promoters that control where and when a gene is expressed - bound by CYCLOPS, such as the CYCLOPS response element (CYC-RE) found in promoters of symbiosis genes. Root-nodule symbiosis is phylogenetically restricted to the nitrogen-fixing clade (NFC) of flowering plants (rosids including legumes, Parasponia - the only non-legume genus known to form nodules with rhizobia - and actinorhizal lineages) and requires, in addition to CSP activity, the transcription factor NODULE INCEPTION (NIN), a member of the NIN-LIKE PROTEIN (NLP) nitrate-response transcription factor family, as well as a symbiotic cytokinin signalling branch; bacteria enter via infection threads, plant-made intracellular tunnels that deliver rhizobia into root cortical cells, where nodules form as new organs. Comparative phylogenomics showed that the three core CSP genes are conserved specifically in plant lineages forming intracellular symbioses (Radhakrishnan et al. 2020) and that non-nodulating lineages have repeatedly lost symbiosis-related genes (Griesmann et al. 2018), supporting the hypothesis that nodulation evolved by co-opting the ancient AM-serving CSP rather than by inventing a new pathway. Functional evidence for deep conservation came from the liverwort Marchantia paleacea: Vernié et al. (2024) demonstrated that SYMRK, CCaMK, and CYCLOPS are each essential for AM symbiosis in this bryophyte, that the CYC-RE reports CYCLOPS activation during intracellular colonization, and that gain-of-function CCaMK/CYCLOPS versions induce overlapping transcriptomic responses - establishing that the CSP and its molecular function have been maintained for roughly 450 million years of plant evolution. That work ended by formulating the unresolved question examined here: by what mechanism was this ancient pathway co-opted to support other intracellular symbioses, and how do evolutionary transitions from one symbiotic type to another occur through such co-option?

Problem Statement

Determine the mechanism by which the ancient common symbiosis pathway (CSP), conserved since the most recent common ancestor of land plants to serve arbuscular mycorrhizal symbiosis, was co-opted to support root-nodule symbiosis with nitrogen-fixing bacteria in the nitrogen-fixing clade of flowering plants; the root-nodule case is the concrete instance of the more general question of how evolutionary transitions from one symbiotic type to another occur by co-opting this pathway, and a complete mechanistic account of the root-nodule case is what resolves that general question. Concretely: identify the set of genetic changes downstream of the CCaMK-CYCLOPS module - gains, losses, and modifications of cis-regulatory elements in promoters of nodulation genes, and protein-level adaptations of pathway components and their targets such as NIN and SYMRK - that redirected the conserved symbiosis-signalling output so as to drive both branches of the nodulation program, namely rhizobial uptake through infection threads and de novo nodule organogenesis in the root cortex; date and place these changes on the phylogeny relative to the origin(s) of nodulation; and establish which of them were necessary and sufficient, for instance by reconstituting the transition in a plant lineage outside the nitrogen-fixing clade.

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