Analysis of Medicago truncatula polyribosomal mRNAs revealed selective translational regulation of early nodulation genes

REYNOSO, MAURICIO; BLANCO, FLAVIO; BAILEY-SERRES; CRESPI, MARTÍN; ZANETTI, MARÍA EUGENIA

Mar del Plata

Congreso; XXIX Reunión Argentina de Fisiología Vegetal; 2012

Selective recruitment of mRNAs and miRNAs to polysomes in response to rhizobia infection in Medicago truncatula Mauricio Reynoso1, Flavio Blanco1, Julia Bailey-Serres2, Martín Crespi3 and María Eugenia Zanetti1 1Instituto de Biotecnologia y Biologia Molecular, FCE, UNLP, CCT-La Plata, CONICET, 1900-La Plata, Argentina; 2Department of Botany and Plant Sciences, CEPCEB, University of California, Riverside, USA; 3Institut des Sciences du Végétal, CNRS, Gif sur Yvette, France. E-mail: reynosom@biol.unlp.edu.ar   Translation of mRNAs plays a crucial role in the regulation of gene expression during development or in response to environmental stimuli. Several studies have characterized the steady-state levels of mRNAs at different stages of root legume symbiosis. However, this data does not distinguish mRNAs that are targeted for degradation, sequestered in ribonucleoprotein complexes or undergoing translation. In addition to protein-coding genes, small RNAs (sRNAs) are major regulators of mRNA translation or stability. Here, we characterized changes in the association of mRNAs and sRNAs to polysomes in roots of the model legume Medicago truncatula during the symbiotic interaction with Sinorhizobium meliloti. Polysomes were isolated by immunopurification (IP) from root tissue expressing a FLAG-tagged ribosomal protein L18. Quantitative analysis of inoculated and control roots revealed differential translational status for a set of genes of the nodulation signalling pathway. Three categories were defined based on their regulation at translational level: 1) up-regulated, included three receptors and transcription factor (TFs) of the GRAS and NF-Y families 2) not regulated, included two TFs, a nuclear localized kinase and its interacting protein 3) down-regulated, represented only by a ion channel. Quantitative analysis of sRNAs associated with polysomes detected several mature miRNAs of 21 and 22 nt and a tasiRNA. Notably miR169d, which targets the A subunit of the NF-Y TF, significantly decreased its association with polysomes upon inoculation with S. meliloti in correlation with increased levels of NF-YA protein. More recently, polysomal mRNAs from root hairs, cortex and phloem were obtained by IP from roots expressing FLAG-L18 under the control of cell-specific promoters, showing enrichment of specific mRNAs in each cell type. Current experiments are being conducted to explore translational status at genome scale combining IP of polysomes and RNA sequencing technology.