Symbiosis and immunity in Legumes

Team SYMUNITY/ Pascal Ratet

Characterization of symbiotic mutants affected in symbiotic immunity

DNF2 is necessary for bacteroid persistence in nodules (Bourcy et al., 2013b; Berrabah et al., 2014a)

We have identified and characterized the DNF2 gene that encodes a putative Phosphatidylinositol-Phospholipase C-XD-containing protein. The dnf2 mutant plants develop nodules which are correctly invaded during the first stages of the symbiosis but in which the symbiotic process aborts once bacteria are released into the plant cell. This arrest of the symbiotic process is accompanied by defense-like reactions and nodule necrosis with a fix^- phenotype (figure 3). The phenotype of this dnf2 mutant unravels a new aspect of the symbiotic interaction suggesting an active mechanism responsible for the repression of plant defense within the symbiotic nodule cells (figure 4).

The SymCRK gene is necessary for repression of immunity (Berrabah et al., 2014b; Berrabah et al., 2018b)

The SymCRK gene was isolated as a DNF2 co-regulated gene. The SymCRK gene encodes for a receptor-like kinase and was found to have a phenotype similar to dnf2 (i.e. necrotic fix^- nodules, figure 3). Our work shows that SymCRK is indeed also necessary to suppress defense reaction during symbiosis and might inhibit the defense ethylene signaling pathway in nodules (Berrabah et al., 2018b) (figure 4).

Using these two plant mutants in combination with rhizobium mutants, we have also shown that the rhizobium chronic infection is controlled at multiple steps during symbiosis (Berrabah et al., 2015; figure 4).

Figure 3. Aspects of M. truncatula Wild Type (WT), dnf2 and symCRK nodules. Top panel: Pictures show WT, dnf2 and symCRK nodules 4 weeks after inoculation with Sinorhizobium meliloti 2011. WT shows big, pink nodules, dnf2 and symCRK nodules are necrotic. Middle panel: longitudinal sections of WT, dnf2 and symCRK nodules showing the nodule ultrastructures. dnf2 and symCRK nodules display a reduced zone III (infection zone) and an increased senescing zone (IV). Lower panel: Staining of bacteria in one cell from the zone III. Green fluorescent structures: living bacteroids; red structures: dead bacteroids. From Bourcy et al. (2013a and 2013b).

In order to add more actors to this model (figure 4), additional necrotic mutants (such as those described above or in Pislariu et al., 2012; Domonkos et al., 2017) were identified. We are characterizing such mutants that probably represent new actors of the symbiotic immunity.

Figure 4. Model presenting the control of the plant immunity during symbiosis establishment, to allow chronic infection of nodule cells by rhizobia, their survival in cells and nitrogen fixation. Top and lower panels: Bacteroid death is prevented by multiple actors acting successively. DNF2 is the earliest actor identified as required for symbiotic suppression of immunity at the intracellular stage of the symbiosis. Its requirement is determined by environmental conditions that influence the development of defense-like reactions or senescence in nodules. After DNF2, the bacA bacterial gene prevents the NCR triggered bacteroid death. Later, SymCRK prevent defense-like reactions possibly triggered by massive intracellular invasion or initiation of bacteroid differentiation. Finally, nitrogen fixation is required to prevent the death of elongated bacteroids. Nodule senescence involves the activation of molecular actors associated to cell degradation as cysteine proteases. Some members of this gene family are also required for optimal defense responses against pathogenic organisms. Thus, nodule senescence and defenses that result both in bacterial death appear to share common elements. From Berrabah et al. (2015) and Gourion et al. (2014).

Role of defense hormones in symbiosis and legume immunity

The plant defense hormone salicylic acid (SA), jasmonic acid (JA) and ethylene are important hormones for elicitation of plant defense reactions in leaves and/or roots towards pathogenic microorganisms. Ethylene is implicated at various stages of the symbiotic interactions (figure 4). Some works also indicate that SA and JA signaling may also be fine-tuned to allow symbiosis. However, the exact actors of these signalling pathways as well as specific associated responses are still largely unknown in legumes and during symbiosis. We have undertaken a genetic approach to study the role of SA signalling during symbiosis in Medicago and pea. Similarly, we are isolating mutants for the JA signalling pathway in Medicago. These genetic tools will allow us to decipher the hormonal signalling pathways that might be induced in symbiotic and immune mutants such as dnf2 and symCRK mutants and understand how these important immune response are controlled during symbiosis. In the long term, this work will also help to understand how legume plants differentiate beneficial from pathogenic bacteria and how they adapt their responses to allow symbiosis.

Senescence and immunity

Senescence is part of the nodule developmental program (figure 4) but can also be controlled by external factors including abiotic stresses and nitrogen nutrition. Several studies indicate a link between senescence and immunity. The symCRK and dnf2 mutants trigger defense-like reactions and also display early senescence in nodules (Bourcy et al., 2013b; Berrabah et al., 2014b). It was proposed that suppression of plant innate immunity is reversed as nodules senesce.

We are addressing the question of the link between senescence and immunity during symbiosis, taking advantage of the mutant tools described above. We are for this characterizing new fix^- mutants developing or not defense reactions and showing accelerated senescence. A role of salicylic acid in the senescence process is also possible.

Endophytes and symbiosis

In the fields, the nodules of legumes not only harbour rhizobia but also other non-rhizobial endophytic bacteria. In some plants, there are more non-rhizobial endophytes in the nodules than rhizobia and there is a certain specificity of the nodule microbiome. This suggests a selection by the plant of hosted bacteria and/or compatibility with the rhizobial partner. We would like to understand the role of Nod factors signalling and of immunity in the recognition of these non-rhizobial endophytes. To this end, we have started the isolation of bacteria from Medicago nodules formed in the presence of non-sterile soil and the functional characterization of some of them.