(I) Microbial Signaling and Host Cellular Dynamics Associated with Endosymbiotic Infection
In the case of the legume M. truncatula, root entry by both rhizobia and AM fungi is characterized by the formation of specialized intracellular compartments within host cells through which the microbes are transported towards inner root tissues where the respective symbioses are established. Our team has developed live-cell confocal imaging approaches for M. truncatula aimed at studying the in vivo cellular dynamics of this particular mode of apoplastic infection (Genre et al., 2008; Fournier et al., 2008). For example, fluorescence-tagged proteins are being used to monitor cell wall/extracellular matrix remodeling during different stages of rhizobial root hair infection (Fournier et al. 2015). In addition, fluorescence-based Ca2+ reporters (cameleons) allow the direct visualisation of host cell-specific responses to rhizobial/AM signals and hence the study of microbial/host communication throughout microbial infection (Sieberer et al., 2009; Chabaud et al., 2011). These approaches have led to the identification of short-chain chitin oligomers as novel symbiotic AM fungal signals (Genre et al. 2013), and their activity/role is now being studied in a variety of non-legume hosts including rice and actinorhizal plants. We have also discovered that strikingly similar cellular and Ca2+ signaling responses are associated with both rhizobial and AM root infection (Sieberer et al., 2012), thus underlining the evolutionary conservation of endosymbiotic infection mechanisms. Finally, in collaboration with colleagues at the IRD, these studies are now being extended to symbioses involving ecologically-important actinorhizal plant species in association with both AM fungi and filamentous Frankia N-fixing soil bacteria. Experiments using cameleon reporters have been decisive in revealing that Frankia symbiotic factors are biochemically distinct from the chitin-based signals secreted by rhizobia and AM fungi (Chabaud et al., 2016).
Project leaders: M. Chabaud, J. Fournier & D.G. Barker
Collaborations: A. Genre & P. Bonfante (Univ. Torino, Italy); E. Limpens & coll. (Univ. Wageningen, Holland); S. Svistoonoff, H. Gherbi & coll. (IRD, Montpellier); G. Bécard & coll. (LRSV, Toulouse); J. Murray & coll. (JIC, Norwich, UK); T. Nakagawa, N. Shibuya & coll. (Univ. Meiji, Japan); A. Jauneau & coll., FRAIB imaging platform, Toulouse.
Principal Project Funding: ANR SYMDYNAMICS (2009-2011; coordinator: D.G. Barker), ANR SYMActino (2013-2017; coordinators: H. Gherbi & D.G. Barker).
(II) Tissue-Specific Regulation of Host Root Reprogramming for Infection
The first steps of the nitrogen fixing legume-rhizobia symbiotic association involve precise signaling and reprogramming of the host to allow intracellular accommodation of the bacterial partner within newly formed nodules. We have focused on understanding how the host plant reprograms and regulates the expression of genes during these early stages of rhizobial root entry using the model legume M. truncatula. After defining host cis-regulatory promoter sequences responsive to rhizobial Nod factor signals, we identified closely-related ERF transcription factors (ERN-type) as key regulators of symbiotic gene expression (Andriankaja et al., 2007). We further studied the interconnection of these factors with other symbiotic regulators (see Laloum et al., 2014 in section III and Vernié et al., 2015) and demonstrated by genetic approaches their key roles to coordinate early steps of rhizobial root nodulation (Cerri et al., 2016). These studies also revealed that functional redundancy and precise spatio-temporal regulation of expression are key aspects related to the functioning of these factors (Cerri et al, 2012). The development of a number of fluorescent-based constructs, allowed us to study the dynamics of these regulators and of related symbiotic components in vivo, that revealed a precise spatio-temporal regulation of symbiotic signaling in host cells undergoing infection (Cerri et al, 2012; Fournier et al., 2015). We are now focusing on the understanding of this infection reprogramming with a spatio-temporal resolution, using a variety of molecular and cell-imaging strategies.
Project leader: F. de Carvalho-Niebel (CR CNRS)
Team members directly involved: A. Kelner (PhD), L. Frances (TR INRA), F. Guerrero-Molina (Post-doc), M. Beck (Post-doc), J. Fournier (CR CNRS).
Collaborations: G. Oldroyd, J. Murray, JIC, Norwich (UK); M. Parniske, LMU, Munich (Germany), C. Mazars (LRSV, Toulouse), S. Svistoonoff (IRD, Montpellier), F. Maillet and C. Gough (LIPM) and FRAIB imaging platform (Toulouse).
Project Funding: ANR « COME-IN» project (2015-2019), FRAIB; INRA-CNRS, LABEX TULIP, Agrenskills.
(III) NF-Y TFs: Key Regulators of Infection & Nodule Development
NF-YA1 (previously called MtHAP2a) has been identified in our group as a gene strongly and specifically up-regulated during nodule development in M. truncatula (El Yahyaoui et al., 2004; Combier et al., 2006). This gene belongs to the NF-Y (nuclear factor Y) or CCAAT box-binding family of transcription factors (TFs) comprising heterotrimers of NF-YA, B and C sub-units (reviewed in Laloum et al. 2013). NF-YA is upregulated only a few hours after Rhizobial inoculation is highly expressed in young developing nodules and in the infection and meristematic zones of mature nodules. Functional analyses showed that MtNF-YA1 is first required during the initial stages of Nod Factor signaling and rhizobial infection preceding nodule formation by targeting the ERN1 TF (shown using transactivation and chromatin immunoprecipitation assays) but also for nodule development (Combier et al., 2006, Laporte et al., 2013). A nodule fate map approach developed in collaboration with Ton Bisseling’s team (Wageningen University, The Netherlands) using the nf-ya1-1 null mutant has in addition validated the importance of NF-YA1 for nodule meristem development and function (Xiao et al., 2014). Studies are currently directed towards understanding the precise mechanism(s) by which NF-Y regulates rhizobial infection thread progression and nodule development by the identification and characterization of proteins interacting with NF-YA1 transcriptional complexes and regulated targets. In addition, in animal systems NF-Y has been reported to be a “pioneer TF” locally modifying chromatin structure. We are currently testing the epigenetic potential of MtNF-YA1 using ChIP-Seq, ATAC-Seq and Mnase experiments in collaboration with Moussa Benhamed (IPS2, Orsay, France).
Project leader: A. Niebel
Collaborations: LIPM CBI platform; Martin Crespi, Florian Frugier and Moussa Benhamed’s groups, IPS2, Orsay, France; F. Blanco & E. Zanetti, University of La Plata, and Federico Ariel University of Santa Fe, Argentina; D. Cook’s team, UC Davis, USA; Estibaliz Larrainzar, University of Navarra, Pamplona, Spain; Jeremy Murray’s team JIC Norwich, U.K.; M. Udvardi’s team, Noble foundation, Ardmore, USA; M. Parniske’s team, LMU, Munich; S. Goormachtig, VIB, Gent, Belgium; T. Bisseling’s team, U. Wageningen, Netherlands; Roberto Mantovani’s team at University of Milan, Italy.
Project Funding: ANR HAPIHUB (2009-2013; coordinator: Andreas Niebel), CNRS-CONICET PICS project in collaboration with Eugenia Zanetti (La Plata, Argentina) (2015-2017) and ANR NODCCAAT (2016-2019) coordinator: Andreas Niebel
(IV) Control of Nodule Differentiation
A key process in nodule development is the developmental transition involving coordinated differentiation of plant and bacterial cells. This step is critical in generating the appropriate micro-environment for symbiotic nitrogen fixation, and involves a massive reprogramming of gene expression, with thousands of genes affected in successive waves. We are investigating how these late stages of nodule development are controlled, both by genome-wide approaches and in depth functional analyses of specific transcriptional regulators.
Indeterminate nodules are formed in M. truncatula and related legume species via persistent apical meristematic activity. The spatial zonation of subtending nodule cells corresponds to successive developmental stages, leading ultimately to the nitrogen-fixation zone. A particularly fruitful approach conducted by our team in the framework of a collaborative project involving all the LIPM teams working on the rhizobium-legume symbiosis (“SYMbiMICS” ANR-funded project), has coupled laser capture microdissection (LCM) of several of the nodule zones and RNAseq analysis of both the plant and rhizobial transcriptomes (Sallet et al., 2013; Roux et al., The Plant J. 2014; https://iant.toulouse.inra.fr/symbimics). This has provided a valuable resource to investigate the dynamics of gene expression in the nodule and identify novel regulators, notably epigenetic regulators, that we are now characterizing in detail (“EPISYM” ANR-funded project). We have used a similar LCM-RNAseq approach to analyze the root epidermal responses to Nod factors, which involve a number of genes also expressed during nodule development, as well as the MtCRE1/cytokinin signaling pathway (Jardinaud et al., 2016). As an essential resource for these genome-wide analyses, we are also contributing to the completion of the full genome sequence of M. truncatula (Roux et al., 2014; unpublished data) in addition to the “Legoo” knowledge base elaborated by the LIPM bioinformatics platform under J. Gouzy’s supervision.
Several transcriptional regulators identified by transcriptomics have been demonstrated to play important roles in nodule development, including NF-YA1 (see previous section), MtbHLH1 (Godiard et al., 2011) and EFD (Ethylene Response Factor involved in nodule Differentiation; Vernié et al., 2008). Intriguingly, EFD is in addition a positive factor for bacterial wilt caused in M. truncatula by the bacterial pathogen Ralstonia solanacearum, and is upregulated by R.solanacearum via the MtCRE1/cytokinin signaling pathway (Moreau, Fromentin et al., 2014). We are now pursuing the characterization of MtEFD to better understand its mode of action and its evolution within the extensive ERF family.
Project leader: P. Gamas
Team members directly involved: Carine Satgé (PhD student), Sandra Moreau (IE CNRS), Marie-Françoise Jardinaud (MC ENSAT), P. Gamas
Collaborations: LIPM bioinformatics platform, FR AIB imagery platform, LIPM symbiosis teams; M. Crespi’s, F. Frugier’s, M. Benhamed’s and A. Bendhamane’s teams (IPS2, Orsay); J. Burstin and K. Gallardo (INRA Dijon); M. Lepetit (INRA Montpellier); J. Buitink (INRA Angers); B. Schmitz (University of Georgia, USA).
Project Funding: ANR SYMbiMICS (2009-2012; coordinator: P. Gamas); ANR Genopea (2010-2013; coordinator J. Burstin, Agroécologie INRA Dijon); EPINOD project (2015-2016; INRA SPE and Labex TULIP; coordinator: P. Gamas); ANR EPISYM (2016-2020; coordinator: M. Crespi, IPS2, Orsay).