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

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

Mar del Plata, Buenos Aires, Argentina, 17 - 20 de noviembre de 2007

Congreso; XLIII Reunión Nacional de la sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2007

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, guided by promoters of interest 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 meliloti we tested the new RIVET tool with two promoters. While the expression of tnpR from the constitutive p-nptII promoter resulted in the excision of the nptII cassette, expression of tnpR from the symbiotic p-nifH promoter generated Nms-Gmr 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 during infection thread formation.