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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Nod2Root (Keeping nodules in check: Interplay of rhizobial and host factors controlling nodule morphogenesis and identity in legume plants.)

Teaser

Summary

As sessile organisms, plants are directly challenged by fluctuating environments that usually lead to limited growth. As all living organisms, plants require for growth the basic chemical elements on top of which appear: carbon (C), hydrogen (H), oxygen (O), and nitrogen (N). Plants are capable of acquiring C, H and O elements via photosynthesis while N availability is one of the crucial factors limiting plant yield in agriculture. Consequently, external supply of N fertilizers has been key to improve plant yield in extensive farming systems. However, two main costs arise from this practice: (i) the significant and high energetic demand of the industrial synthesis of N fertilizers, and (ii) the environmental costs of releasing N compounds into the environment resulting in e.g., water pollution, algal bloom, and eutrophication.
To reduce the impact of agricultural fertilization on ecosystems, naturally occurring processes such as biological nitrogen fixation could be viewed as a promising and sustainable strategy for plant N supply. Indeed, a limited portion of flowering plants can symbiotically interact with nitrogen-fixing soil bacteria collectively known as rhizobia. This interaction takes place in specialized, relatively recently evolved and facultative plant organs termed as â€œsymbiotic nodulesâ€. In these symbiotic organs, a bacterial enzyme, the nitrogenase, is converting gaseous dinitrogen (N2) into ammonium that is provided to the host plant. As N2 accounts for approximately 78% of atmospheric gases and is recycled, this form of nitrogen represents a huge and potentially unlimited pool available to plants forming a nitrogen-fixing symbiosis. In particular, this group of plants encompasses the agronomical relevant Legume family (e.g. garden pea, common bean, soybean, chickpea, alfalfa and lentil). Soybean (Glycine max [L.] Merrill) is the most extensively cultivated legume worldwide and accounts for 8.77% of world total harvested area in 2016, ranking it fourth after wheat, maize and rice (fao.org).
Understanding the molecular mechanisms controlling the robustness of nodule development and functioning in soybean is crucial to improve symbiotic efficiency and to alleviate environmental costs of chemical N fertilisation. Exploiting the potential of several documented examples of nodule-to-root conversion, we propose to identify plant and bacterial factors required for an efficient symbiosis and nodule maintenance. It has been shown that both the bacterial general stress response (GSR) system and the plant NBCL gene products are required for nodule maintenance. However, this has been done in two different biological systems, namely the G. max - Bradyrhizobium diazoefficiens and Medicago truncatula - Sinorhizobium meliloti models. Using the B. diazoefficiens - soybean system, we propose to decipher (i) the symbiotic roles of the three NBCL proteins in soybean, (ii) which cells are involved in nodule to root conversion in soybean nodules induced by B. diazoefficiens mutants impaired in the GSR, and (iii) the molecular determinants and mechanisms which respond to the lack of the bacterial GSR-dependant signals and subsequently lead to nodule-to-root conversion.

Work performed

1/ The symbiotic functions of NBCL proteins in soybean have been analysed mainly via two approaches. We first analysed the expression pattern of soybean NBCL using promoter reporter gene fusions. We showed that the soybean NBCLb and the M. truncatula NOOT promoters are driving very similar nodule expression patterns, despite nodule organogenesis is different in these two legume species. Additionally, we down-regulated NBCL expression using an artificial microRNA system. Collectively, these results strongly suggest a conserved role of NBCLs in regulating nodule development, irrespectively of the legume species (at least in the legume lineage common to G. max and M. truncatula) and the nodule type (determinate vs. indeterminate).
2/ To identify the first cells that are switching to root identity in the soybean nodules elicited by B. diazoefficiens GSR mutants, we expressed root-specific marker genes in soybean and compared their expression pattern in WT- and mutant-elicited nodules. Specifically, we used a soybean PT7::GUS stable transgenic line that is characterised by the expression of the GUS reporter gene in root tips and lateral root primordia (Inoue et al., Plant Cell Physiol, 55:2102-11). Interestingly, the PT7::GUS fusion is also expressed in WT-inoculated soybean nodules but displays a different expression pattern than in GSR mutant-induced nodules, where it highlights the cell territories switching to root identity. In addition, the induction of PT7 (PT: phosphate transporter) during nodulation suggests that PT7 is participating in P homeostasis during the formation of N2-fixing organs as it was shown for another member of the PT family in soybean (Qin et al., Plant Phys, 159(4):1634-43).
3/ We next aimed to apply comparative metabolomics and transcriptomics in order to identify genes and/or metabolites which are potentially associated with the nodule-to-root conversion in mutant-induced nodules. As the Nod2Root project was early terminated, this work package was not fully implemented. However, we collected samples for future analysis of the gene expression networks and metabolites related to the nodule-to-root conversion.

Final results

This project allowed generating a promising tool for root transformation of various legume species using a versatile Agrobacterium rhizogenes strain - binary vector combination. The results obtained with the PT7::GUS soybean line point to a particular regulation of PT7 during nodulation, linking nodulation, nitrogen and phosphate homeostasis. It should be noted here that P availability is also a limiting factor for crop productivity. Interestingly, PT7 is also expressed in root cells hosting symbiotic soil fungi (Inoue et al., Plant Cell Physiol, 55:2102-11, 2014). This plant-fungus symbiosis, known as the arbuscular mycorrhization, provides benefits to the plant in terms of P nutrition. Additionally, the distinct PT7::GUS expression patterns during lateral root and nodule formation could be used as an easy-to-use tool for distinguishing these two types of root-derived organs.
The analysis of the soybean NBCL genes provides strong support for conserved symbiotic functions of this plant gene family and hence reinforces the hypothesis that members of NBCL family exert conserved functions in the developmental control of plant organs. Our work highlighted a tight link between bacterial factors necessary for pervasive infection and robust nodule development and/or maintenance. It also shows that the plant NBCL gene family may be crucial in determining the symbiotic cell fate, which is crucial for efficient symbiotic nitrogen fixation. In conclusion, our work contributed to expand the scientific knowledge in the field of legume-rhizobia symbiosis, which is essential for further development of environment-friendly, economic N fertilization in sustainable agriculture.