Characterization of NF-Y heterotrimeric transcription factor complexes involved in the symbiotic interaction between M. truncatula and S. meliloti

BAUDIN, MAEL; LALOUM, T; LEPAGE, A; CARVALHO-NIEBEL, F; RÍPODAS, CAROLINA; BLANCO, FLAVIO; ZANETTI, MARÍA EUGENIA; SINHAROY, S; UDVARDI, M; NIEBEL, ANDREA

Tenerife

Congreso; 11th European Nitrogen Fixation Conference; 2014

The root endosymbiotic association between legumes and soil bacteria known as rhizobia leads to the formation of root nodules in which the differentiated bacteria convert atmospheric nitrogen into a form that can be assimilated by the host plant. Several specialized NF-Y (Nuclear Factor Y) transcription factors (TFs) are described to be involved in this symbiotic interaction (1-3). Among them, MtNF-YA1 was first shown to be a key regulator of nodule development in Medicago truncatula (1) and more recently, also of rhizobial infection (4). The NF-Y proteins are part of a transcriptional complex also called CBF (CCAAT-box Binding Factor) or HAP (Heme Activator Protein) present in all eukaryotic systems (5). This complex is composed by three unrelated proteins (NF-YA, NF-YB and NF-YC) which assemble together to bind DNA at CCAAT-boxes, a motif present in most eukaryotic promoters. Interestingly, the trimeric form of the NF-Y complex is required for DNA binding, the NF-YA subunit can thus not act as a TF alone (5). However, in most plant reports, NF-Y subunits are not studied as a trimeric complex thereby neglecting an important aspect of this regulator. In plants, NF-Y proteins are encoded by small multigene families with sometimes specialized expression patterns and functions, and show a certain degree of interaction specificity. In this study, we characterized the NF-YB and NF-YC proteins interacting with MtNF-YA1 during the rhizobium?legume symbiosis. For this purpose, we combined blind protein/protein interaction approaches in yeast and analysis of transcriptomic data to identify the most relevant symbiotic NF-Y complex. Two by two interactions and trimeric complex formation were confirmed using yeast-two and -three hybrid, BiFC and Co-IP experiments. In addition functional studies using RNAi approaches revealed partial redundancy among certain NF-Y subunits and similar functions to MtNF-YA1 for others. The identification and characterization of trimeric NF-Y complexes around MtNF-YA1 reveal an additional layer of regulation of this important symbiotic TF.