Post transcriptional silencing of RNases during phosphate starvation in Nicotiana

ESCOBAR, ELIANA GISELLE; HERNÁN ROJAS; ARIEL GOLDRAIJ

Mar del Plata

Congreso; LI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2015

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Ribonucleases (RNases) T2 are endonucleolytic enzymes thatcatalyze the cleavage of single-strand RNA producing mononucleotides with aterminal 3´ phosphate (Pi). In plants, different classes of RNases T2 are inducedin a variety of stress scenarios, including Pi starvation. The induction of these RNases contributes to Pimobilization from nucleic acid sources. In Nicotianaalata, class III NnSR1 (Nicotiananon S-RNase1) and class I S-like-RNase NE are,thus far, the only RNases induced for Pi mobilization. NnSR1 is localized inendoplasmic reticulum of root cells while RNase NE is secreted to therizosphere to scavenge extracellular RNA sources. To discern whether theinduction of these two RNases is simultaneous or sequential, as well as the Pithreshold triggering such induction, plants were cultivated under Pi limitingconcentrations. Early induction of NnSR1, assayed by western blot and in gelRNase activity, suggested that the intracellular RNA may be the first source ofPi used by the cell under Pi stress. To understand  the functional role of these RNases, a posttranscriptional silencing strategy was developed. An hypervariable region of bothRNase NE and NnSR1was selected to clone into a binary vector. Preliminar PCR,restriction analysis and subsequent plasmid DNA sequencing indicated successfulcloning to express the hairpin RNA required for silencing RNase expression.