COMBINING IMMUNOPURIFICATION OF POLYRIBOSOMES AND DEEP SEQUENCING TECHNOLOGIES TO CHARACTERIZE GLOBAL CHANGES IN THE TRANSLATOME OF NODULATING ROOTS OF Medicago truncatula

MAURICIO REYNOSO, FLAVIO A BLANCO, MARIO AGUILAR, MARTIN CRESPI AND MARIA EUGENIA ZANETTI

La Plata

Congreso; Reunio Anual de Fisiología Vegetal; 2010

Sociedas Argentina de Fisiología Vegetal

Legume plants establish a symbiotic association with bacteria from the genus Rizhobium, resulting in the formation of a new organ, the root nodule, where the bacteria is allocated and fix atmospheric nitrogen. This developmental process is accompanied by a reprogramming of gene expression in the host plant. Since abundance of a particular mRNA may not reflect the level of its translation, we aim to characterize the mRNAs actively translated into polyribosomes (translatome) of Medicago truncatula roots at early time points of the association with its symbiotic partner Shinorhizobium meliloti. We used the immunopurification (IP) of polyribosome technology (Zanetti et al, Plant Phys 138:624) to isolate mRNAs actively translated in M. truncatula roots. A 60S ribosomal protein, MtRPL18, was expressed as a translation fusion to the FLAG epitope in M. truncatula roots. Western blot analysis revealed that the FLAG-MtRPL18 protein was expressed and incorporated into ribosomes. Polyribosomes were immunopurified from root tissue using anti-FLAG conjugated agarose beads. The polypeptide composition of IP complexes was similar in electrophoretic mobility and stoichiometric intensity to those of conventional purified ribosomes. IP of ribosomes was confirmed by the presence of the 28S and 18S rRNAs in the IP fraction of roots. This fraction also contained mRNAs associated with ribosome, as evidenced by the detection of transcripts of ubiquitin in the IP RNA fraction from roots that express FLAG- MtRPL18, but not from control roots transformed with the empty vector. These results revealed that co-IP of actively translated mRNAs with FLAG-MtRPL18 is feasible in M. truncatula. This methodology, combined with the new generation sequencing technologies, constitutes a valuable tool for quantification of mRNAs differentially translated in M. truncatula roots during its association with S. meliloti.