The auxin-inducible degron system in Trypanosoma cruzi.

GONZALO PERUGORRIA; MARISOL CANTALUPI; LEÓN ALBERTO BOUVIER; GABRIELA TERESA NIEMIROWICZ

La Plata

Congreso; XXXIV REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE PROTOZOOLOGÍA; 2023

Genetic manipulation of Trypanosoma cruzi has significantly improved since the implementation of the CRISPR/Cas9 system. Although this technology has successfully been used to knockout genes as well as to perform endogenous gene tagging, functional analysis of essential genes remains impaired due to the absence of tools for generating conditional mutant strains. In this context, degron-based strategies for controlling protein expression have become a popular approach for the rapid and reversible depletion of target proteins. Among these methods, auxin-inducible degron (AID) technology – which utilizes plant auxin signaling components to modulate protein degradation in non-plant species – is a widely employed small-molecule-controlled degradation methodology in yeasts, animals, and apicomplexa. In this study, we explore the AID system for regulating protein expression in T. cruzi. To evaluate this approach, we developed a vector containing a single transcription unit that includes the auxin receptor F-box protein TIR1 from Oryza sativa (OsTIR1), a mEGFP reporter fused to the miniAID (AID-mEGFP) sequence and a downstream G418 resistance (Neo) coding sequence connected through two 2A peptides. Overexpression of OsTIR1 protein did not affect T. cruzi doubling time nor produce any noticeable phenotype. Addition of the natural plant hormone indole acetic acid (IAA) to transgenic epimastigote cultures had no effect on AID-mEFP protein levels. However, when using the synthetic auxin naphthalene acetic acid (NAA), we observed a rapid depletion of the reporter with a 70% reduction after 4 hours. Our preliminary findings suggest that the AID may serve as an alternative tool for studying essential genes in trypanosomatids.