?Towards an inducible system of heterologous protein expression in chloroplast using bacterial quorum sensing regulatory elements?

BOCCARDO, NOELIA; SEGRETIN, MARÍA EUGENIA; MIRKIN, FEDERICO GABRIEL; BRAVO-ALMONACID, FERNANDO FELIX

Foz de Iguazú

Congreso; 11th International Plant Molecular Biology Congress IPMB2015; 2015

Plant diseases affect most staple crops decreasing productivity worldwide and severely compromising food security. Several attempts have been done to obtain disease resistant crops through transgenesis, with variable results. For many strategies to get resistance to pathogens, it is required to achieve high levels of protein accumulation in order to be successful, although this could result in lesion-mimic phenotypes. To overcome some of these limitations, we decided to evaluate an inducible expression system combined with plastid transformation. Plastome transformation advantages nuclear transformation because of the higher genome copy number, absence of positional effects and silencing and maternal inheritance of transgenes. We are developing an inducible expression system inspired by the bacterial Quorum Sensing (QS) mechanism. We assembled a plastid transformation vector with the following QS elements from Pseudomonas aeruginosa: lasB promoter sequence was cloned upstream uidA reporter gene, and CDS corresponding to regulatory proteins LasR and RhlR were included alone or combined as a polycistron downstream psbA promoter sequence. These regulatory proteins LasR and RhlR interact and respond to different homoserine lactones (HSL), like 3-oxo-C12-HSL and C4-HSL respectively. With these constructs we transformed Nicotiana tabacum cv. Petit Havana plants by the biolistic approach and obtained transplastomic plants with no phenotypic differences compared to non-transformed tobacco plants. We are characterizing these transplastomic lines at the molecular level and performing GUS activity assays to determine the inducibility of the reporter protein expression after treatment with HSLs.