P. RATET - Ankirin and BTB/POZ domain proteins: roles in symbiosis, development and immunity.

Genetic control of the symbiosis

Establishment of the symbiotic interaction between legume plants and Rhizobia occurs when the soil nitrogen source is limiting and results in the de-novo formation of symbiotic nodules, generally formed on roots of the plant and hosting the symbiotic bacteria. The symbiotic association allows the plant to overcome nitrogen limitation taking advantage of the bacterial nitrogenase activity. In return, the plant furnishes carbon derivatives to its hosts. The first steps of this symbiotic association and the formation of the symbiotic organ have been described in detail (Ferguson et al., 2010; Oldroyd et al., 2011) but organ identity and later steps of the interaction related to bacterial accommodation, and immunity are less understood. Rhizobia often invade the plant root using specialized symbiotic structures called infection threads. In the mature, nitrogen fixing nodule, the rhizobia reside in symbiotic nodule cells within organelle-like structures, called symbiosome. In contrast to what is generally observed during microbial invasion, legumes do not elicit defense reactions during symbiosis despite that the bacterial population reaches massive densities in nodules.

In the Medicago truncatula legume model plant most of the tools necessary for a deeper understanding of legume biology are now available for the scientific legume community. These tools have greatly facilitated the molecular identification of the first legume genes via positional cloning. The development of an efficient insertion mutagenesis technique for legumes using the Tnt1 and MERE1 retro-elements was pioneered by our laboratory (d’Erfurth et al. 2003a; Tadege et al., 2008) and has allowed the creation of a large Tnt1 mutant collection principally at the Noble Foundation (21 000 mutant lines, www.noble.org).

We use these tools to understand the molecular basis of the Medicago truncatula-Sinorhizobium symbiotic interaction. Our team is mainly interested in the plant functions which are: (i) involved in determining the identity of the symbiotic organ; and, (ii) required to reach a homeostatic interaction between the host and its symbiotic microbial partner.

(i) The NOOT like genes are necessary for nodule identity:

The Medicagonoot mutant isolated in our laboratory develops roots in apical position of the symbiotic organ (Couzigou et al., 2012). The coch mutant from pea has a symbiotic phenotype similar to noot but also shows stipule to leaf conversion and flower developmental modifications (Ferguson et al., 2005). We have isolated the NOOT and COCH genes and shown that they are BTB/POZ and Ankyrin domain containing proteins necessary for the robust persistence of the nodule. NOOT and COCH areArabidopsis BOP orthologs and their functions in the identity of the lateral aerial organs (stipule and flower development) are conserved in M. truncatula and P. sativum (Couzigou et al., 2012; Norberg et al., 2005; Hepworth et al., 2005). In the noot and coch mutants the ectopic roots do not derive from the nodule meristem but originate from the vascular initials. This suggests that the nodule vascular strands are ontologically related to roots.

We have shown that a second NOOT gene exists in Medicago that also plays a role in nodule identity. In addition we are now studying the role of NOOT-like genes in other plants

(ii) Characterization of symbiotic mutants affected in symbiotic immunity:

Role of the Salycilic acid pathway in Medicago:

The NOOT genes belong to the same class of protein that the NPR genes controlling the salycilic signalling in plants. This pathway is involved in the responses to different stresses and in the control of immunity. In order to understand the role of the salycilic acid pathway during symbiosis and other plant-microbe interactions we are now studying the role of the NPR gene family composed of the NPR1, NPR2 and NPR3 genes in Medicago. We have isolated the Tnt1 insertion-mutants for these three genes and are now characterizing the npr1, npr2 and npr3 mutants for their symbiotic phenotypes. We will also characterize their responses to known pathogens.

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

We have identified and characterized the DNF2 gene (Starker et al., 2006) that encodes a PhosphoInositol-PhosphoLipaseC-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. We have shown that the dnf2 phenotype is environment dependant.  

The SYMCRK gene is also necessary for repression of immunity (Berrabah et al., 2014b; 2015):

The SYMCRK symbiotic specific cysteine rich receptor kinase gene is also necessary to supress defense reactions during symbiosis and defines a second signalling pathway necessary to accommodate the symbiotic partner. The symRK mutant plants develop fix^- nodules that accumulate phenolic compounds. We have shown that bacterial differentiation and not the environement is necessary to trigger this phenotype.

Literature cited

• Bourcy et al, (2013) New Phytologist, 197(4), 1250-61.
• Berrabah et al., (2014a) PLoS ONE 9(3): e91866.
• Berrabah et al., (2014b) New Phytologist, 203(4): 1305-14.
• Berrabah et al., (2015) J Exp Bot. 66(7): 1977-85.
• Couzigou et al., (2012) Plant Cell, 24(11), 4498-4510.
• d’Erfurth et al. (2003) Plant J., 34, 95-106.
• Ferguson et al., (2005) Plant Cell Physiol., 46, 1583-1589.
• Hepworth et al., (2005) Plant Cell, 17, 1434-1448
• Norberg et al., (2005) Development, 132, 2203-2213
• Oldroyd et al., (2011) Annu. Rev. Genet., 45, 119–144.
• Starker et al., (2006) Plant Physiology, 140, 671–680.
• Tadege et al., (2008) Plant J.,54 (2), 335–347.41.

En savoir plus

• http://www.ips2.u-psud.fr/spip.php?article46
• http://www.isv.cnrs-gif.fr/veranglais/research/pra/pra.html
• https://www.researchgate.net/profile/Pascal_Ratet/

Contact :Fabienne.Vailleau@toulouse.inra.fr