ET-Nod : Effectors Triggering Nodulation In legumes – ET-Nod

To address these various objectives, the ET-Nod project was structured into three tasks:

Task 1: Mode of action of ErnA

To identify genes regulated by ErnA and Sup3, we performed RNA-seq analyses on transformed roots of A. evenia expressing these genes under an inducible promoter. Some genes commonly found induced by ErnA and Sup3 were validated through transactivation assays in tobacco.

The characterization of ErnA’s proximal proteome was carried out using a proximity-labeling approach (TurboID) in vivo, with ErnA fused at its N-terminus to a Biotin ligase under the control of an inducible promoter. This analysis was performed in both N. benthamiana leaves and transformed A. evenia roots. Interactions of ErnA with candidate proteins identified in this analysis were further validated using split-luciferase complementation and FRET-FLIM assays.

The conservation of signaling pathways triggered by ET-Nod effectors, relative to the classical NF-dependent pathway or the pathway activated by photosynthetic bradyrhizobia, was assessed using A. evenia mutants for key symbiotic genes (generated within the AeschyNod ANR project) and by analyzing the ability of various Bradyrhizobium strains with different ET-Nod repertoires to nodulate these mutant lines.

Finally, rather than focusing on ErnA DNA targets as initially planned, we shifted our attention to the potential interaction of ErnA and Sup3 with the host SUMOylome. This decision was prompted by the identification of the SUMO-protease domain or the SUMO Interacting Motif in several ET-Nod effectors. To investigate these interactions, we employed yeast two-hybrid assays, co-immunoprecipitation (Co-IP), and targeted mutagenesis of these functional domains to assess the binding of ErnA and Sup3 to SUMO peptides from A. evenia.

Task 2: Identification of New ET-Nod

We collected approximately 200 Bradyrhizobium strains from different countries, all with sequenced genomes. Analysis of their ability to nodulate A. indica, combined with in silico analyses of their predicted effectomes, allowed us to identify several strains capable of nodulating A. indica but lacking the ernA gene. Using mutagenesis of candidate genes and analysis of the resulting mutants, we identified the effector(s) responsible for nodulation. One of these effectors, Sup3, was validated as an ET-Nod effector by testing its ability to induce the formation of nodules when overexpressed in transformed Aeschynomene roots in the absence of bacteria.

Task 3: Role of ET-Nod during symbiosis with legume crops.

Based on the collection established in Task 2, we selected seven Bradyrhizobium strains with different ET-Nod repertoires. For each strain, mutants were generated in the nod genes, the T3SS, and the identified ET-Nod effectors. The symbiotic properties of these mutants were then analyzed on three cultivated Vigna species. This work was carried out in close collaboration with Pr Neung Teaumrong’s group at Suranaree University of Thailand.

Analysis of a collection of Bradyrhizobium strains revealed that one-third of them can induce nodule formation on Aeschynomene indica through a T3SS-dependent mechanism. Twelve nodulating strains lacking ernA enabled the identification of four new ET-Nod genes: sup3, coding for a SUMO protease related to Bel2-5, and Ubi1, Ubi2 and Ubi3, which likely act cooperatively in nodulation. The ET-Nod family now includes six members sharing conserved domains such as a nuclear localization signal (NLS) or SUMO-related motifs, suggesting a common mode of action. These findings highlight an unexpected diversity of Bradyrhizobium strains capable of inducing nodulation through various ET-Nods, emphasizing the flexibility of the T3SS-dependent symbiotic mechanism (Camuel et al., ISME J, 2023).

On the plant side, certain Bradyrhizobium strains combining distinct ET-Nods can bypass symbiotic genes previously considered essential, such as AePOLLUX, AeCCaMK, and AeCYCLOPS. In contrast, the transcription factors NSP2 and NIN remain indispensable for nodulation (Camuel et al., New Phytologist, 2024). Functional analysis of the SIM and SUMO protease domains of ErnA and Sup3 demonstrated their direct interaction with SUMO proteins. Mutation of these domains abolishes their ability to trigger nodulation, indicating that T3SS-dependent symbiosis is regulated by post-translational SUMOylation, and that ErnA and Sup3 modulate this pathway to initiate nodule formation (Fazal et al., New Phytologist, 2025).

Analysis of the proxyome of ErnA revealed an interaction with several subunits of the Mediator complex, a central component of transcriptional regulation (Carcagno et al., in preparation). RNA-seq analyses confirmed that ErnA and Sup3 induce the expression of hundreds of genes, including several transcription factor genes (NFY-C, WRKY, bHLH) These results suggest that ET-Nods act within a regulatory complex involving the Mediator to activate key nodulation genes.

Finally, the study of seven Bradyrhizobium strains carrying different ET-Nods and tested on three cultivated Vigna species (V. mungo, V. unguiculata,V. radiata) confirmed the essential role of Nod factors (NFs), as their mutation completely abolished nodulation. In contrast, mutation of the T3SS produced variable effects depending on the strain and legume species, either inhibiting or, conversely, enabling nodulation. ET-Nod mutants showed no significant effect under these NF-dependent symbiotic conditions. However, analysis of T3E mutants from strain ORS3257 revealed that certain effectors specifically modulate symbiosis: NopP2 blocks nodulation in V. radiata, while NopT and NopAB enhance it in V. mungo and V. unguiculata. Other T3Es carrying SUMO protease domains can inhibit symbiosis with specific cultivated legumes. Thus, although ET-Nods do not appear to play a major role in these NF-dependent interactions under the tested conditions, other T3 effectors act as key modulators of symbiotic efficiency.

The ET-Nod project has significantly advanced our understanding of this protein family. It has led to the identification of new members, revealed their interactions with SUMOylated proteins and the Mediator complex, and demonstrated their ability to activate numerous plant genes. These findings suggest that ET-Nods act as central regulators of plant–microbe interactions. However, their molecular mode of action remains poorly understood, and several key questions remain open.

Future research will need to identify the SUMOylated protein targets of ET-Nods and determine whether they function as transcription factors or as epigenetic regulators modulating chromatin accessibility and transcriptional activity. These studies will require integrated approaches combining cell biology, biochemistry, and genetics to precisely define the molecular mechanisms underlying their nodulation-inducing activity.

So far, no specific role of ET-Nods has been demonstrated in Nod factor (NF)-dependent symbioses with cultivated legumes. However, this conclusion remains partial, as only a limited number of bacterial strains and host species have been studied. Exploring a broader diversity of Bradyrhizobium strains and legume species—particularly soybean, where Bel2-5 can trigger nodulation without Nod factors—may reveal wider functions. Most studies have also been conducted under axenic in vitro conditions, neglecting the complexity of soils and their microbiota. ET-Nods may provide subtle, context-dependent advantages. Investigating their roles under more realistic conditions will help clarify their ecological significance and contributions to bacterial competitiveness, host specificity, and adaptation, providing a more integrated view of rhizobium–plant communication.

To date, functional analyses have primarily relied on loss-of-function mutants, offering an incomplete perspective. Future studies should emphasize gain-of-function approaches, combining different ET-Nod variants or expressing them heterologously to test redundancy and synergy. These experiments will benefit from new genetic tools, including RepABC-type shuttle vectors stable in both Bradyrhizobium and E. coli.

Overall, this research will establish a mechanistic and evolutionary framework for ET-Nods, clarifying their role in symbiosis and their potential for sustainable agriculture. Understanding how these effectors influence host transcriptional and epigenetic networks will help optimize biological nitrogen fixation and improve legume productivity while reducing reliance on chemical fertilizers and minimizing environmental impact.

• Busset N, Gully D, Teulet A, Fardoux J, Camuel A, Cornu D, Severac D, Giraud E, Mergaert P. (2021) The Type III Effectome of the Symbiotic Bradyrhizobium vignae Strain ORS3257. Biomolecules. 11(11):1592. doi: 10.3390/biom11111592.
• Teulet A, Camuel A, Perret X, Giraud E. (2022) The Versatile Roles of Type III Secretion Systems in Rhizobia-Legume Symbioses. Annu Rev Microbiol. 2022 Apr 8. doi: 10.1146/annurev-micro-041020-032624.
• Songwattana P, Chaintreuil C, Wongdee J, Teulet A, Mbaye M, Piromyou P, Gully D, Fardoux J, Zoumman AMA, Camuel A, Tittabutr P, Teaumroong N, Giraud E. (2021) Identification of type III effectors modulating the symbiotic properties of Bradyrhizobium vignae strain ORS3257 with various Vigna species. Sci Rep. 11(1):3266. doi: 10.1038/s41598-021-82751-x.
• Tighilt L, Boulila F, De Sousa BFS, Giraud E, Ruiz-Argüeso T, Palacios JM, Imperial J, Rey L. (2021)The Bradyrhizobium Sp. LmicA16 Type VI Secretion System Is Required for Efficient Nodulation of Lupinus Spp. Microb Ecol. 2021 Oct 25. doi: 10.1007/s00248-021-01892-8. Online ahead of print.
• Nouwen N, Chaintreuil C, Fardoux J, Giraud E. (2021) A glutamate synthase mutant of Bradyrhizobium sp. strain ORS285 is unable to induce nodules on Nod factor-independent Aeschynomene species. Sci Rep. 2021 Oct 22;11(1):20910. doi: 10.1038/s41598-021-00480-7.
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Legumes play a major agronomical and ecological role due to their ability to fix atmospheric nitrogen during symbiosis with rhizobia. The main legume crops are tropical species (soybean, peanut, mungbean, …) that represent more than 85% of the grain legume production. These species are all nodulated by Bradyrhizobium strains which contain nodulation genes (nod genes) necessary for the synthesis of key symbiotic signals, named Nod factors (NFs), but also T3SS genes that encode the Type 3 Secretion System. This secretory machinery, initially identified in animal and plant bacterial pathogens, permits the delivery of effector proteins inside the host cells where they interfere with various host processes including suppression of immune responses and favour the infection. For a long time, it was assumed that nodulation absolutely required NFs to trigger nodule organogenesis and infection. The T3SS machinery on the other hand was viewed as an accessory equipment, which modulates the efficiency and the host spectrum of the bacteria. However, it has been shown that some legume species of the Aeschynomene genus but also the cultivar Glycine max cv. Enrie are nodulated by Bradyrhizobium strains even if NF synthesis is disrupted. In this case, the establishment of the interaction requires that the bacteria has a functional T3SS indicating that specific Type 3 effectors can directly activate the nodulation signalling pathway in legumes, bypassing the perception of NFs.
Recently, we have demonstrated that in the Bradyrhizobium strain ORS3257 this T3SS-dependent symbiosis relies on a cocktail of at least five effectors playing synergistic and complementary roles in nodule organogenesis, infection and repression of plant immune responses. Among them, we identified the nuclear-targeted ErnA effector, which is highly conserved among bradyrhizobia, as a key actor for nodule organogenesis. Furthermore, preliminary data indicate that other Bradyrhizobium strains can use other Type 3 effectors, distinct of ErnA, to trigger nodulation in legumes.
Our discovery that a single effector protein is sufficient to induce nodule organogenesis without the need of NFs is a paradigm shift in the field and indicates that legume nodulation programs are not exclusively controlled by NFs.
Our main goals in the current ET-Nod project are: i) to decipher the molecular mechanisms by which ErnA activates nodulation in Aeschynomene, ii) to identify new effectors (named ET-Nods) behaving like ErnA in the triggering of nodulation and iii) to characterize the importance of this effector family in the symbiotic efficiency of agronomically important legumes.
For these purposes, our consortium, involving specialists in plant symbiosis and pathogenesis will i) combine biochemical, genetic and omic approaches to characterize the molecular target(s) and interactome of ErnA, ii) develop at the level of the Bradyrhizobium genus a comparative genomic analysis coupled with a mutagenesis approach to identify new ET-Nod effectors and iii) investigate, using bacterial and plant genetics, the role played by ErnA and ET-Nod effectors in various Bradyrhizobium strains during symbioses with legume crops (soybean, peanut, cowpea …).
The knowledge acquired during this project could be exploited in agronomy to improve yield of several legume crops and to design new strategies aimed at transferring nitrogen-fixing symbiosis to cereals.