Divergence of rhizobium-induced cytokinin signalling in nodulating species

Research output: Thesisinternal PhD, WU


Nitrogen-fixing symbioses provide a beneficial platform for plants to sustain the growth and development under the nitrogen-limiting environment. The nitrogen-fixing symbioses occur exclusively in a subset of genera of 10 taxonomic families. These nodulating plant genera form a paraphylitic clade in the orders Fabales, Fagales, Cucurbitales and Fagales, which collectively are known as ‘Nitrogen Fixing Clade’. Nodulating species of eight families establish the symbiotic interaction with the filamentous bacteria Frankia, and therefore are classified as actinorhizal plants. Nodulating species of two other families interact with the nitrogen-fixing rhizobium; namely legumes (Fabaceae, Fabales) and Parasponia (Cannabaceae, Rosales). Both nitrogen-fixing microbes induce the formation of a niche organ, so-called root nodules. Despite varieties in nodule morphology across nodulating species, the root nodules serve a general purpose to accommodate the bacteria intracellularly and to provide a suitable physiological environment for the bacteria to convert the atmospheric nitrogen (N2) into ammonium. A major aim in nitrogen-fixing nodulation research is to unravel the signalling pathways required for a successful mutualism. Recently, phylogenomic comparisons of nodulating and non-nodulating species postulated that the nodulation trait likely originated from a common ancestor of the Nitrogen-Fixing Clade. This implies that the nodulation trait encountered massive parallel loss during the divergence of this clade. Considering the contribution of nitrogen-fixing symbioses to sustainable plant growth under low nitrogen input farming, it leads to a question whether it is possible to extend the host range of nitrogen-fixing symbionts to non-nodulating crops (such as wheat, maize, or rice). To this end, it is important to identify the key genetic adaptations underlying these successful symbioses. To date, a massive body of knowledge on nodulation has been generated in molecular genetic studies on two model species; Medicago truncatula and Lotus japonicus which belong to the legume subfamily of Papilionoideae. In accordance with the single gain hypothesis, it implies that the core genetic adaptations underlying the nodulation trait are conserved in all nodulating plant species and orchestrated via a shared genetic network. Therefore, expanding the nodulation research outside the Papilionoideae clade is highly relevant to pinpoint the core set of nodulation genes, and to reveal the lineage-specific genetic adaptations that have shaped the symbiotic interactions.

In this PhD thesis, I utilise two alternative species to study the nodulation trait, namely Chamaecrista mimosoides and Parasponia andersonii. C. mimosoides belongs to the legume subfamily of Caesalpinoideae, in which the occurrence of nodulating species is far less frequent than in the subfamily of Papilionoideae. Caesalpinoideae also represents a basal node in the divergence of the legume family, and therefore functional studies Chamaecrista species may offer insights in basal adaptations in the legume-rhizobium interactions. The second species, P. andersonii is a most distantly related nodulating species to legumes. As both lineages diverged at the root of the nitrogen-fixing clade, and therefore studying Parasponia in the context of rhizobial symbiosis may provide a platform to identify the core genetic adaptations underlying the nodulation trait. Genetic screens in M. truncatula and L. japonicus uncovered synchronised processes leading to the formation of functional nodules. Rhizobial chitin oligomer-based signal molecules (namely, LCOs or Nod-Factors) are perceived by the plant by specific LysM-type receptors, which initiate signalling cascade that sets in motion nodule organogenesis and rhizobium intracellular infection. It was found that cytokinin signalling is an integral part of Nod-Factors induced signalling, and essential for nodule organogenesis. Cytokinin signalling is tightly linked to the functional activity of transcription factor NIN. The perception of cytokinin is mainly regulated by histidine kinase cytokinin receptor MtCRE1 in M. truncatula and LjLHK1 in L. japonicus. Knockout mutations in these receptors result in a dramatic reduction of nodule numbers, whereas a dominant-active mutant MtCRE1/LjLHK1 form can induce spontaneous nodule formation. This underlines the importance of cytokinin signalling in nodulation of M. truncatula and L. japonicus. However, it remains elusive whether symbiotic cytokinin signalling is conserved in nodulating species outside the legume Papilionoideae subfamily.

Prior genetic studies in either C. mimosoides or P. andersonii, it is very crucial to establish the genetic tools allowing plant transformation and the corresponding phenotypic analyses in both species. In Chapter 2 and Chapter 3-4, I provided the established transformation protocols that can be used for performing functional studies in C. mimosoides or P. andersonii, respectively. Transformation protocol in C. mimosoides was based on the transient hairy root transformation mediated by Agrobacterium rhizogenes (Chapter 2). In P. andersonii, the plant transformation was mediated by A. tumefaciens (Chapter 3-4).

Based on study in C. mimosoides (Chapter 2), I observed that both NIN genes (CmNIN1 and CmNIN2) and the Type ARR cytokinin responsive genes (CmARR4 and CmARR9) are transcriptionally activated upon rhizobial inoculation and the exogenous cytokinin (benzylaminopurine, BAP) treatment. This suggests that the cytokinin signalling and NIN expression in C. mimosoides are Nod-Factors responsive. This finding is consistent with the observations in the model legumes. Functional studies in C. mimosoides mediated by RNA interference (RNAi) targeting either single CmNIN1 or CmNIN2, and double CmNIN1;CmNIN2 resulted in a significant reduction in nodule numbers. In particular silencing of CmNIN2, resulted also in nodules devoid of a fixation zone. This suggests that CmNIN2 may have an important function not only in nodule organogenesis, but also for successful release of bacteria from infection threads. A similar RNAi strategy was applied to target C. mimosoides cytokinin histidine kinase receptor CmHK4. Also, CmHK4 RNAi lines possess less functional nodules when compared to wild type control plants. This suggests that nodule organogenesis is HK4-dependent. Therefore, I hypothesize that the MtCRE1/LjLHK1/CmHK4-mediated cytokinin and NIN signalling is a conserved mechanism in nodule formation of Papilionoideae and Caesalpinoideae legumes. However, further studies in C. mimosoides are still required to substantiate the interconnection between the cytokinin signalling and NIN.

In Chapter 4, I present a proof of concept of the reverse genetic analyses in P. andersonii, by targeting four putative symbiosis genes; namely PanHK4, PanEIN2, PanNSP1, or PanNSP2. These genes are known to be involved in the cytokinin and ethylene signalling, and the regulation of strigolactone biosynthesis, respectively. Additionally, these genes also exhibit essential functions in root nodulation of legumes. Loss-of-functions of Pannsp1 and Pannsp2 revealed a conserved role for NSP1 and NSP2 to regulate nodule organogenesis. Also, both mutants are affected in the expression of the strigolactone biosynthesis genes D27 and MAX1. The Panein2 mutant also displayed a symbiotic phenotype, though clearly distinct from what is found in legumes. Instead of displaying a hypernodulation phenotype as seen in legume ein2 mutants, Panein2 mutants developed nodules without fixation zones. Analyses of Panhk4 mutants revealed no symbiotic phenotypes. However, the Panhk4 mutants, as well as Panein2 showed non-symbiotic phenotypes that correlate with the reduced cytokinin and ethylene signalling, respectively. Altogether, this study provides a valuable example of utilising P. andersonii as an experimental system to study the rhizobial-induced symbiotic interaction, alongside the model legumes.

In Chapter 5, I present an in-depth analysis on the role of cytokinin signalling in the P. andersonii-rhizobium interaction. Utilising the cytokinin synthetic reporter TCSn, it was shown that the cytokinin signalling induced upon rhizobium and Nod factor application. TCSn remained active during nodule organogenesis in cells that become infected by rhizobium. By analysing the spatiotemporal expression of the three P. andersonii cytokinin HK genes, we found an expression pattern of PanHK3 that largely overlaps with TCSn during nodulation. RNAi knockdown studies targeting different cytokinin receptors simultaneously (PanHK2, PanHK3) suggests some functional redundancy among three of the cytokinin receptors in regulating nodule organogenesis. Additionally, mutagenesis of PanHK3 resulted in nodules that are devoid of fixation threads. Taken together, I argue that the cytokinin signalling is integrated in the rhizobial-induced nodulation of P. andersonii.

In summary, this thesis provides an example of comparative genetic analyses of the nodulation trait by utilising two species outside the legume Papilionoideae subfamily. Results that are presented in this thesis illustrate the integration of cytokinin signalling in the rhizobium-induced nodulation. This suggests that the Nod factors-induced cytokinin signalling represents a conserved mechanism in nodulation, which has been recruited in the common ancestor of the Nitrogen-Fixing Clade.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Bisseling, Ton, Promotor
  • Geurts, Rene, Co-promotor
Award date16 Dec 2020
Place of PublicationWageningen
Print ISBNs9789463955768
Publication statusPublished - 16 Dec 2020


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