Combining immunopurification of ribonucleoprotein complexes and RNA-seq to characterize postranscripcional gene regulation in the symbiotic interaction Medicago truncatula- Sinorhizobium meliloti

LANCIA, MARCOS; REYNOSO, MAURICIO; BLANCO, FLAVIO; ZANETTI, MARÍA EUGENIA

Chascomús

Congreso; XV Jornadas anuales de la Sociedad Argentina de Biología; 2013

Several studies have characterized the steady-state levels of transcripts at different stages of root legume symbiosis. However, this appoach does not distinguish transcripts that are targeted for degradation, sequestered in ribonucleoprotein complexes or undergoing translation. The distribution of mRNAs on these cytoplasmic complexes plays a crucial role in the regulation of gene expression during development or in response to environmental stimuli. Here, we characterized changes in the association of transcripts 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. This methodology was combined with Illumina RNA sequencing technology (RNA-seq) allowing a quantitative analysis at genome scale of inoculated and control roots. Approximately 3500 transcripts were classified as up-regulated at translational level, whereas 5770 transcripts were found as down-regulated. A comparison of fold change levels in response to rhizobia of 12 transcripts encoding proteins involved in the nodulation signalling pathway obtained previously by qRT-PCR showed a good correlation with data generated by RNA-seq. Current experiments are being conducted to immunopurify other cytoplasmic complexes involved in storage or degradation of mRNAs,  such as storage granules or P Bodies. This experimental approach constitutes a valuable tool to shed light on the mechanisms and significance of post-transcriptional regulation in M. truncatula roots during its association with S. meliloti.