MELONOD

Plants belonging to the legume family are able to interact with nitrogen-fixing soil bacteria collectively named rhizobia. This symbiotic interaction referred to as the legume-rhizobium symbiuos (RLS) leads to the formation of a facultative organ called the root nodule, inside which atmospheric nitrogen is fixed by the bacteria to the benefit of the plant. In the soil, legumes also face threats from root-knot nematodes (RKN) that induce the formation of a new root organ called a gall containing hypermetabolic, multinucleate and giant feeding cells that serve as unique source of nutrients. These seemingly very different interactions nevertheless share some common genetic pathways. Nodule development is specifically controlled by the CCAAT box-binding transcription factor NF-YA1. Interestingly, we have recently shown that NF-YA1 is also strongly upregulated in RKN-induced galls and that nf-ya1 mutants are affected in both interactions. NF-Ys are considered pioneer transcription factors modulating local chromatin accessibility and hence developmental switches. In addition, we showed that NF-YA1 regulates the expression of many genes in response to rhizobia, including emerging actors of chromatin-based gene regulatory mechanisms, the long non-coding RNAs (lncRNAs). The objective of the MELONOD project is to decipher the common and specific mechanisms by which the NF-YA1 pioneer transcription factor regulates both the beneficial rhizobial symbiosis and the pathogenic interactions with RKN in the model legume Medicago truncatula. MELONOD will be subdivided in three axes: (i) MELONOD will deliver a list of direct NF-YA1 target genes during RLS and RKN infection, as well as a list of common target genes during both interactions. This will be achieved by a combination of RNA-seq and ChIP-seq approaches ; (ii) MELONOD will characterize the chromatin landscape at NF-YA1 target sites through four complementary approaches. Nucleosome occupancy especially at promoters of direct targets defined in (i) will be investigated using ATAC-seq. Whole-genome bisulfite-sequencing will be performed to identify differentially methylated regions in the promoters of NF-YA1 targets in nodules and galls. We will perform comparative ChIP-seq analyses in WT and nf-ya1-1 mutant to investigate if NF-YA1 binding during RLS and RKN infection influences repressive or permissive histone modifications at target gene promoters. To probe for the presence of NF-YA1-dependent chromatin loops, we will use Hi-C on WT or nf-ya1-1 mutant nodules and galls; (iii) LncRNAs have known roles in chromatin-based gene regulatory mechanisms and are thus promising candidates to explain the pioneer role of NF-YA1 during both interactions. We will characterize four NF-YA1-regulated lncRNAs including NANO1, which is strongly upregulated at the onset of both interactions. We will generate M. truncatula knock-down (KD) and knockout (KO) lines using RNA interference, CRISPR-Cas9, and Tnt1 transposon insertion lines. Overexpression constructs will also be tested. To investigate the underlying mode of action of these selected lncRNAs, target chromatin regions will be identified genome-wide by Chromatin isolation by RNA purification and proteins bound by these lncRNAs will be identified using RNA immunoprecipitation followed by mass-spectrometry. Finally, the potential regulatory role of these lncRNAs on chromatin loop formation and target gene expression will be investigated using Hi-C in transgenic lines with modified lncRNAs levels. These approaches should allow a better understanding within the same host plant of a fascinating case of convergence between two plant development programs, one in mutualistic and the other in parasitic interactions. This knowledge will allow further development of applied innovative strategies for increasing nematode resistance without altering symbioses, or even promoting symbiosis in legume crops.