CE20 - Biologie des animaux, des organismes photosynthétiques et des microorganismes

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

Effectors triggering nodulation of legumes

Legumes play a major agronomical and ecological role due to their ability to fix atmospheric nitrogen during symbiosis with rhizobia. In the Frame of the SymEffectors ANR project (2016-2020), we revealed an alternative symbiotic process that does not rely on the Nod factor synthesis by the rhizobia but by secretion of effectors translocated by the T3SS machinery. Among the effectors required for symbiosis, we identified the nuclear-targeted ErnA effector as a key actor for nodule organogenesis.

Deciphering the molecular mechanisms by which ErnA and other effectors (ET-Nod) activate nodulation in legumes

Our main goal in this project is to decipher the molecular mechanisms by which ErnA activates nodulation in legumes and characterize the importance of this effector family (ET-Nod) in the symbiotic efficiency of agronomically important legumes.<br />This project is divided in 3 parallel tasks : in Task 1, we will characterize the ErnA nucleic acid target(s) and the ErnA proximal proteome by combining biochemical, genetic and omic approaches. This information will permit to characterize the molecular mechanisms by which ErnA can activate the nodulation program in Aeschynomene. In Task 2, we will explore, at the level of the Bradyrhizobium genus, the prevalence to use the NF-independent and T3SS-dependent nodulation process by examining the ability of a very large diversity of sequenced strains to nodulate A. indica. Furthermore, by comparative genomic analysis coupled to a mutagenesis approach, we will identify effectors (hereinafter referred to as ET-Nod effectors) that trigger nodulation independently of NFs, like ErnA. In Task 3, we will investigate the importance of ErnA and ET-Nods during symbiosis of agronomically important legumes. For this purpose, the ernA and et-nod genes identified during Task 2 will be mutated in several representative strains and the symbiotic properties of the mutants will be tested on soybean, peanut, cowpea, mungbean and black mungbean. In parallel, transgenic lines of crop legume species overexpressing ernA or et-nod will be generated and analysed for their ability to induce nodule-like structures or to activate canonical symbiosis marker genes.<br />The knowledge acquired during this project could be used in agronomy to improve yield of several legume crops and to design new strategies aimed at transferring nitrogen-fixing symbiosis to cereals.

For the different purposes of the project , our consortium, involving specialists in plant symbiosis and pathogenesis will i) combine biochemical, genetic and omic approaches (RNAseq, ChIP-seq RIP-seq, DAP-seq, Co-IP and proximity labeling using Turbo-ID) 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 on the emerging candidates 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 interaction with legume crops (soybean, peanut, cowpea …) by analysing the symbiotic properties of various mutants in nod genes or ET-Nod.

For the development of the different approaches aiming to identify ErnA protein or nucleic acid targets (ChIP-seq, DAP-seq, Co-IP, proximity labeling), it is necessary to have tagged forms of ErnA. Unfortunately, we have observed that the addition of a tag at the C-ter of ErnA often compromises its functionality. To overcome this problem, it is necessary to work with transgenic lines of A. evenia overexpressing the N-ter tagged proteins although this makes the generation of the biological material more laborious. By this approach, we validated that ErnA is still functional (able to trigger pseudonodules on transgenic roots) after the addition of N-ter 3xHA or 3xFlag epitopes allowing the development of ChIP-seq, DAP-seq, and Co-IP, which are in progress. Constructs containing N-ter Turbo biotin ligase are currently tested and proximity labeling will be initiated if the data are positive.
For the task 2 and 3, we assembled a collection of 200 Bradyrhizobium strains whose genome is available or that we sequenced in the frame of the ET-Nod project (for 9 strains). The symbiotic properties of most of these strains was tested on Aeschynomene indica and more than one third was found able to induce nodules, although with variable efficiency. Except for the photosynthetic Bradyrhizobia, all these strains do possess a T3SS, reinforcing the idea that these strains use a T3SS-dependent process to induce nodules. Interestingly, among these strains, 12 do not possess an ErnA homolog, suggesting the involvement of novel ET-Nod(s) to trigger nodulation. Effectome prediction of these strains and their comparison with the ones of neighboring strains unable to nodulate, highlighted some T3E candidates that we have started to test by mutagenesis. By this approach, we identified a new effector, named Sup3, in strains Nas96-2 and WSM1744 encoding a putative SUMO-Protease that is required for Aeschynomene nodulation. Furthermore, introduction of the sup3 gene from NAS96-2 or WSM1744 into the ORS3257?ernA mutant restores the ability to nodulate A. indica, which validates Sup3 as an ET-Nod. On the other hand, among the above group of 12 nodulating strains, some, including strain LMTR13, do not have ErnA nor Sup3 homologs indicating the existence of other ET-Nods to be discovered.
We focused for the moment our research on the LMTR13 strain and showed that the mutation of two previously unknown effectors (named U1 and U3) had a considerable impact on the ability of the strain to nodulate (each mutant induced 70% fewer nodules). It is therefore possible that these two new effectors act in synergy to activate the nodulation process.

Despite a slow start, important advances were made during this period. The biological material for the execution of Task 1 has been largely constructed and functionally validated.
The development of the planned approaches (ChIP-seq, DAP-seq, and Co-IP) to advance in the characterization of ErnA targets (proteins and nucleic acids) is therefore under progress. If we do not validate the possibility of using the Turbo-ID labeling approach to identify the ErnA interactome, we plan to develop a yeast two-hybrid (Y2H) approach by generating a Y2H cDNA library from A. evenia roots.
For the development of the RNA-seq approach, we decided to use a dexamethasone-inducible expression system (pDEX) to identify early genes under the control of ErnA. The generated construct provides in the Aeschynomene transgenic lines a high level of ernA induction within hours of addition of dexamethasone validating its use. RNA-seq analysis in multiple short time points after the inducer treatment will be performed in the next months. In a targeted approach (with a priori), we will test if ErnA interacts with that proteins of the common symbiosis pathway that are present in A. evenia, including Pollux, CaCMK, cyclops and NIN. The putative interactions of ErnA with these signaling proteins will be tested by Co-IP and yeast two-hybrid. Currently, the constructs required for these experiments are being prepared.

Concerning the new putative ET-nods (U1 and U3) identified, we will validate them by testing their ability to complement for nodulation the ORS3257?ernA mutant. Very interestingly, we identified that the C-ter region of ErnA is well conserved in both U1 and U3 effectors. In particular, this region contains the HILV motif predicted as a SUMO-interactive motif (SIM). By directed mutagenesis, we confirmed that this motif is required for the functionality of ErnA. It is therefore possible that ErnA and the U1 and U3 effectors, as well as the SUMO-proteases Sup3 and Bel-2-5, interact with SUMOylated partner(s) of the host plant during nodulation signaling. This hypothesis, which would indicate a possible functional link between these different ET-Nods, is currently being evaluated with biochemical approaches.
For the Task 3, the first data obtained on the ORS3257 strain did not confirm a role for ErnA during the symbiotic interaction of ORS3257 with these 3 Vigna species but this does not allow us to rule that the ET-Nods are dispensable in all the symbioses involving NFs. It is possible that this class of effectors strengthens or replaces the NF signal in certain conditions or during interactions with particular hosts. Thus, as initially planned, we will continue to study the symbiotic properties of the various strains and mutants that emerged during the task 2 on various legumes crops.

• 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.

Project coordination

Eric GIRAUD (Laboratoire des Symbioses Tropicales et Méditerranéennes)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LSTM Laboratoire des Symbioses Tropicales et Méditerranéennes
I2BC Institut de Biologie Intégrative de la Cellule
LIPM Laboratoire des Interactions Plantes - Microorganismes

Help of the ANR 589,914 euros
Beginning and duration of the scientific project: December 2020 - 48 Months

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