When grown in nitrogen limited conditions, legume plants establish a symbiotic association with soil nitrogen fixing bacteria collectively named rhizobia. This symbiosis leads to the formation on the roots of the legume plant of new organs termed root nodules. In these nodules, bacteria find a favorable environment to fix atmospheric nitrogen and supply it in a reduced form to their host. Cytokinins are key regulators of nodulation. The aim of the CytoSYM project is to understand how a cytokinin-signaling transcription factor, RRB3, can function as a positive but also as a negative regulator in the early stages of nodulation. The proposed project comprises 3 Work Packages (WP). The WP1 will identify by combined single cell RNAseq (rrb3 mutant vs wild-type) and ChIPseq approaches the genes directly controlled by RRB3 in the different cell types during the response to rhizobia. To determine whether RRB3 transcriptional function involves modulating chromatin accessibility, chromatin compaction will be followed in response to rhizobium by ATACseq and histone acetylation by ChiPseq comparatively in the WT and in rrb3 mutant plants. WP2 will study the role of target genes selected from WP1 in the control of nodulation using loss-of-function approaches, in particular to identify new actors with positive or negative regulatory roles.This WP2 will also focus on two already known key players in nodulation to explain the dual role of RRB3 in the regulation of nodulation: NIN which is a transcription factor essential for the initiation of nodules, and TML2 which is a F-Box protein inhibiting nodulation. Finally, the WP3 aims to identify the partners of RRB3 capable of modulating its transcription activity by an immunoprecipitation approach associated with mass spectrometry. The role of these partners in the initiation of nodulation will then be investigated, in particular by loss-of-function approaches. Ultimately, this project will provide a better understanding of how a transcription factor, RRB3, can exert opposite regulations of nodulation over time, depending on the root tissues, and in relation to chromatin or transcriptional interactors modulating its activity, and to reveal the spatiotemporal dynamics of gene expression and chromatin remodeling during the early stages of nitrogen-fixing symbiotic nodulation in legumes.

Due to climate change, heat stress is going to become a major source of yield loss in Europe in the coming years. There is thus urgent need for the elucidation of cellular mechanisms involved in heat stress response to be able to produce new varieties with improved tolerance. Although the mechanisms involved in heat tolerance have been previously explored, little attention has been given to the contribution of chromatin dynamics to this process, particularly in crops. In this context, in the 3Dwheat project, we propose to monitor changes in the transcriptome, epigenome and 3D-chromatin structure in wheat in order to obtain a global picture of how changes at the chromatin level are associated with changes in gene expression during heat stress response. Therefore, to go beyond the mere description of heat stress response we propose to study the molecular mechanisms controlling the bivalent chromatin state observed on heat responsive genes.

The chloroplast is the plant compartment in which light energy is captured and transformed into chemical energy, ultimately making life on Earth possible. They are descendants of free-living photosynthetic bacteria and maintain a small but essential genome. Most genes are transcribed into polycistrons that are then heavily processed by a combination of RNA-binding proteins and ribonucleases to produce the functional RNA population. One poorly understood aspect of chloroplast gene expression and regulation is the functions of asRNAs and more globally double-stranded RNA-related processes. One of the key enzymes in these processes is RNase J, whose characteristic is to be both a 5’-3’ exoribonuclease and an endoribonuclease. It trims RNA termini 5’ ends and eliminate deleterious antisense RNA that could harm translation. The goal of the JOAQUIN project is to gain a better understanding of chloroplast RNA quality control by (i) identifying the full set of chloroplast RNA isoforms and RNA-RNA duplexes and (ii) and understand how this transcriptome is shaped by RNase J. The substrate specificity of RNase J will be deciphered focusing on the land plant’s specific C-terminal GT-1 domain and by the identification of the protein and nucleic acids that co-purify with the enzyme.

The maintenance of genome integrity is essential in meristems that generate new organs and tissues throughout the life of the plant. Several transcription factors controlling the DNA damage response (DDR) have been identified. However, how and in what order they act to induce different responses in different cell types remains an open question. The coordinated activation of successive levels of response, and the differential control of cell division and cell death in different cell types, are essential to preserve meristematic function in response to DNA damage. In this project, we propose to spatially and temporally resolve the regulatory network associated with DDR in the root meristem. The root meristem is generally the first to be exposed to potential toxic substances in the environment, and is also essential for root growth, and thus for the whole plant. By combining the most recent genomic techniques (snRNAseq and snATACseq), cell biology (live-imaging) and biochemistry (proximity labelling), we will reconstruct the regulatory networks involved in the DDR in a cell-specific manner. This model will be validated by reverse genetics and molecular biology (ChIP) approaches. Beyond the spatio-temporal resolution of the DDR, which is essential to understand how meristematic function is preserved in response to stress, the implementation of these integrative approaches will pave the way for similar studies applied to other physiological contexts such as heat stress or drought, thus addressing the major challenges associated with plant growth in the context of climate change.

Plant-parasitic nematodes have a major impact on global food production with an estimated annual cost of about €100 billion worldwide. Root-knot nematodes (Meloidogyne spp.) alone are responsible for 5% of the global crop losses. These soilborne root pathogens have a worldwide distribution, can infect most all cultivated plants, and are particularly damaging for vegetables (tomato, pepper and cucumber). For decades, wide-spectrum agrochemical control agents (nematicides) have been used. Because of their toxicity and harmful impact on the environment, most of these pesticides have been or will be banned worldwide. New control strategies must be developed to meet consumers demand for safer food production and more durable environmental approaches. Plant natural resistances to control nematodes is an efficient alternative to pesticides. However, few resistance sources were identified in a limited number of crops and an increasing number of field nematode populations have already overcome these host resistances. The MASH project would provide a comprehensive knowledge of the biological processes manipulated by root-knot nematodes that are essential to the development of the disease, and could lead to the development of new control strategies. During infection, root-knot nematodes secrete a cocktail of molecules, named effectors, to induce specialised multinucleate feeding cells, named giant cells, essential for the nematode development and reproduction. Transcriptome analyses revealed a major reprogramming of gene expression during giant cell formation, associated with tight regulation of nuclear division, cell growth and plant hormone pathways. Recently, root-knot nematodes have been shown to modulate a process named alternative splicing in giant cells in the model plant Arabidopsis. Alternative splicing enables a precursor messenger RNA (mRNA) to generate not only one, but two or several mature mRNAs, giving rise to a set of mRNA sequences from a single parental gene. It occurs by rearranging the pattern of intron and exon elements. Alternative splicing largely contributes to gene expression regulation and proteome diversity, in particular during plant adaptation to stresses. MASH aims at a better understanding of plant responses to root-knot nematode infection focusing on the third most consumed vegetable worldwide, tomato. Our objectives are to (i) study the prevalence of genome-wide alternative splicing changes during root-knot nematode infection, (ii) characterize conserved root-knot nematode nuclear secreted effectors modulating alternative splicing, and (iii) identify and functionally characterize plant targets of splicing regulatory effectors and key alternative splicing-regulated genes. MASH is a multidisciplinary project including bioinformatics, RNAomics and phytopathology. MASH should significantly improve our knowledge of the role of alternative splicing in plant responses to parasitic nematodes. The characterisation of target plant genes essential for root-knot nematode infection and the identification of mutants/variants reducing nematode infection, may provide innovative control strategies against these pests.