Development of synthetic methods, namely oligonucleotide aptamers, in order to stimulate the plant defense response in Arabidopsis thaliana

 Supervisor: Prof. Giuseppe Firrao 

Aptamers are small single stranded synthetic nucleic acids that fold into a well-defined three-dimensional structure to bind to a variety of diverse molecular targets specifically and tightly, ranging from small molecules to whole cells. Aptamers evolve from random oligonucleotide pools by an in vitro selection process called Systemic Evolution of Ligands by EXponential enrichment (SELEX). SELEX is structured in four essential steps: incubation of random oligonucleotide pools with the molecular target, separation of bound from unbound sequences, elution and amplification of bound nucleic acids.

The aim of the PhD is selecting specific DNA aptamers which target the plant cell and a plant pathogen biomarker. These two aptamers are combined to originate a synthetic biosensor, which properly stimulated by the pathogen, readily activates in vivo the plant defense response.

Cell-SELEX methodology will be performed on mesophyll protoplasts isolated from Arabidopsis thaliana and, to promote the interaction between aptamer-defense membrane receptor, plants will be treated with an inducer of the plant immune state. At the same time, specific DNA aptamers which internalized into the cell cytoplasm are also selected for potential applications in plant biotechnology, such as transfection and carrying.

SELEX methodology will be performed to select chemically modified DNA aptamers which are highly specific for the different variable regions of flagellin of Pseudomonas syringae pv. tomato DC3000, the best candidates will be integrated into the synthetic biosensor for the plant defense activation.

A combined strategy dependent on quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) amplification curve and High-Resolution Melting (HRM) curve analysis is employed to monitor the convergence of the aptamer species during Cell-SELEX and SELEX procedures.

Thanks to the designed synthetic biosensor, plants could readily counteract the pathogen spreading as soon as they are threatened.

Biography and Contacts

Federico Bosetto was born in Arzignano (VI), Italy, on 30/12/1992. He graduated in Biological Sciences in 2014 with a final grade of 104/110 and in Molecular and Cellular Biology in 2017 with a final grade of 110/110, both at the University of Bologna (Italy). In 2016 he performed a master’s degree internship of ten months at the University of Copenhagen (Denmark), submitting a master thesis on “Study on effects of cytokinins on Nicotiana tabacum - Pseudomonas syringae pv. tabaci interaction”. From November 2017, he is attending the PhD school in Agricultural Sciences and Biotechnology at the University of Udine. From November 2019, he is performing a research period abroad at the Institut Pasteur of Paris (France) to work about the in vitro expression and purification of flagellin of Pseudomonas syringae pv. tomato DC3000 and to use it as target for SELEX with chemically modified aptamers.

Institutional e-mail: bosetto.federico@spes.uniud.it