A novel RIVET system suitable for the study of plant bacteria interactions

LOZANO, M.J.; GIUSTI, M.A.; DRAGHI, W.O.; TORRES TEJERIZO, G.A.; DEL PAPA, M.F.; PISTORIO, M.; LAGARES, A.

Mar del Plata, Argentina

Congreso; XLIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Bioquímica y Biología Molecular

In cases where bacterial gene expression specifically occurs in biological environments of difficult access, RIVET (Recombination-based In Vivo Expression Technology) can be a suitable approach to search for markers that are differentially expressed only under those particular conditions.We developed a new RIVET variant based on the appearance of a gentamycin (Gm)resistance upon expression of genes of interest. The system was designed to be suitable for the study of plant-bacteria interactions where other selection markers such as sacB may be useless due to the presence of sucrose in several plant compartments. The expression of the gamma-delta-Tn-resolvase, TnpR, results in: a) the excision of an nptII (Nm resistance) cassette flanked by res sites, and b) the simultaneous appearance of a transcriptional fusion aacCI-gfp leading to a Gm resistant-green fluorescent phenotype.Using the N2-fixing legume-symbiont Sinorhizobium melil we tested the new RIVET tool with two promoters. While the expression of tnpR from the constitutive promoter p-nptII resultedin the excision of the nptII cassette, expression of tnpR from the symbiotic p-nifH promoter generated Nm(s)-Gm(r) clones only when rhizobia where recovered from 3-week old (N2-fixing) alfalfa nodules. The validated system will now be used to try unraveling new rhizobial markers expressed early in infection thread formation.