Symbiosis and immunity in legume plants

SYMUNITY team/ Pascal Ratet

Characterization of symbiotic mutants affected in immunity

DNF2 is required for bacterial survival in nodules (Bourcy et al., 2013b; Berrabah et al., 2014a)

We previously identified and characterized the DNF2 gene that encodes an X-domain phosphatidylinositol -phospholipase C protein. The dnf2 mutant plants develop nodules that are properly invaded by rhizobia during the early stages of symbiosis. However, in these mutant nodules the symbiotic process stops as soon as the bacteria are released into the plant cells. This termination of the symbiotic process is accompanied by defense reactions in the nodules, which are then no longer able to fix nitrogen (nodule fix-; Figure 3). This pioneering work revealed a new aspect of the symbiotic interaction involving a control of the plant's defenses in the late stages of symbiosis (Figure 4). DNF2 is thus involved in an active process of repression of plant cell immunity within the nodules to allow the establishment and survival of symbiosomes.

SymCRK is required for the repression of immunity (Berrabah et al., 2014b; Berrabah et al., 2018b)

The SymCRK gene was isolated because it has a very similar transcriptional profile to the DNF2 gene, suggesting possible co-regulation and similar function encoded by these two genes. The SymCRK gene encodes a receptor-like kinase (RLK). The nodules of the symCRK mutant have a phenotype similar to that of dnf2, with necrotic fix- nodules (Figure 3). Our work shows that SymCRK is also required to suppress defense responses in nodules, including inhibiting the ethylene signaling pathway, a major phytohormone of immunity (Berrabah et al., 2018b) (Figure 4).

Figure 3. Aspects of M. truncatula wild-type (WT), dnf2 and symCRK mutantnodules . Top panel: photos show WT, dnf2 and symCRK nodules 4 weeks after inoculation with Sinorhizobium meliloti 2011. The WT plant shows functional pink nodules while the nodules of dnf2 and symCRK mutants are necrotic. Middle panel: longitudinal sections of WT, dnf2 and symCRK nodules showing nodule ultrastructures. The dnf2 and symCRK nodules show a reduced zone III (infection zone) and a zone IV of increased senescence. Lower panel: fluorescence labeling of bacteria in a zone III cell. Green fluorescent structures: live bacteroids; red structures: dead bacteroids. Adapted from Bourcy et al (2013a and 2013b).

Using these two mutant lines of M. truncatula in combination with rhizobium mutants, we were able to show that chronic rhizobium infection is controlled at multiple stages during symbiosis (Berrabah et al., 2015; Figure 4).

In order to complete this model (Figure 4), other M. truncatula mutants, also showing necrotic nodules, (similar to those described in the literature by other research teams: Pislariu et al., 2012; Domonkos et al., 2017) were identified in the laboratory. We are currently characterizing these mutants to discover new actors of immunity or its control during the establishment of symbiosis.

Figure 4: Model showing the control of immunity during the establishment of symbiosis to allow chronic infection of nodule cells by rhizobia and their survival in the cells for nitrogen fixation. Bacterial death is prevented by multiple actors acting successively. DNF2 is the first identified actor, which intervenes by repressing the immune response after the internalization of bacteria into the cells. Its action is controlled by the environmental conditions influencing the development of defense or senescence reactions in the nodules. After DNF2, the bacterial bacA gene prevents bacterial death triggered by NCR peptides. Then, SymCRK prevents defense reactions that can be triggered by massive intracellular invasion or initiation of bacterial differentiation into bacteroids. Finally, nitrogen fixation is required to prevent bacteroid death. Nodule senescence involves the activation of molecular players, such as genes encoding cysteine proteases, associated with cellular degradation. Some members of this gene family are also required for the activation of optimal defense responses against pathogens. Thus, the processes of senescence and defense in nodules, both of which resulting in bacterial death, appear to have common elements. Adapted from Berrabah et al. (2015) and Gourion et al. (2014).

Role of defense hormones in immunity and symbiosis

Salicylic acid (SA), jasmonic acid (JA) and ethylene are important phytohormones, particularly involved in triggering plant defense reactions at the root and/or leaf level against pathogenic microorganisms. Ethylene is also involved at different stages of symbiotic interactions with rhizobia. Some works indicate that signaling involving SA and JA would be finely controlled to allow the establishment of symbiosis. However, the actors of these signaling pathways as well as the specific defense responses associated with them are still very poorly understood in legumes. We have undertaken a genetic approach to study the role of SA signaling during symbiosis in Medicago and pea. Similarly, we are isolating mutants of the JA signaling pathway in Medicago. These genetic tools will allow us to better understand the involvement of these hormonal pathways in the induction of defenses observed in symbiotic mutants, such as dnf2 and symCRK, and thus to better understand the importance of their control for the establishment of symbiosis. Ultimately, this work may provide information on how legumes differentiate and adapt their responses to symbiotic versus pathogenic microorganisms.

Senescence and immunity

Senescence is part of the nodule developmental program (Figure 4) and can be controlled by external factors, such as abiotic stresses and nitrogen nutrition. Several studies indicate a link between senescence and immunity. Nodules of symCRK and dnf2 mutants show significant defense responses but also early senescence (Bourcy et al., 2013b; Berrabah et al., 2014b). It has been proposed that the suppression of immunity in nodules can be triggered by the establishment of the senescence program in nodule cells.

We are addressing the link between senescence and immunity during symbiosis, taking advantage of the mutant tools previously described. We are currently characterizing new fix-, Medicago mutants that do or do not develop defense responses and show accelerated nodule senescence. A role of salicylic acid in the senescence process is also possible.

Endophytes and symbiosis

In the field, legume nodules host not only 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 some specificity in the nodule microbiome. This suggests a choice by the plant of the endophytic bacteria and/or compatibility with the rhizobial partner. The symbiotic and immune determinants governing these interactions are not well understood.

We recently isolated from nodules an atypical bacterium (Ensifer adhaerens T4) that behaves as a pathogen or induces nodules in the same species, Medicago truncatula. The outcome of the interaction depends on the age of the host. T4 can also co-colonize nodules induced by another strain.

In the framework of an ANR project, in collaboration with B. Gourion and F. Vailleau (INRAE Toulouse) and the startup iMean (Toulouse), we want to better describe the pathogen and symbiotic-like interactions of this system, and then by integrating genomic (the T4 genome sequence is known) and physiological data of the strain, build a model of T4 genome functionning. This model will be confronted with the omics data produced to predict the key actors of the T4-Medicago interaction. We will also study the response of the plant during symbiotic-like and pathogenic interactions with the T4 strain. This project (PATHOSYM) will provide a better understanding of how the plant differentiates pathogens and symbionts.