Early responses of Phaseolus vulgaris to specific strains of Rhizobium etli: Roles of a CCAAT transcription factor and a monomeric GTPase

AGUILAR, OM, BLANCO F. ZANETTII MMEE PELTZER-MESCHINI I, E.L.

Curitiba

Simposio; First INCT Symposium on Biological Nitrogen Fixation; 2009

CNPq Universidad do Parana

Early responses of Phaseolus vulgaris to specific strains of Rhizobium etli: Roles of a CCAAT transcription factor and a monomeric GTPase   O. Mario Aguilar, María E. Zanetti, María P. Beker, Eitel Peltzer Meschini and Flavio A. Blanco Instituto de Biotecnología y Biología Molecular, Facultad de Ciencias Exactas, UNLP, CCT Conicet La Plata, CP1900, Argentina. aguilar@biol.unlp.edu.ar   We have previously reported the identification of genes differentially expressed in the strain specific response of P. vulgaris to its symbiotic partner Rhizobium etli . Transcripts from a group of these genes accumulate after infection of a Mesoamerican bean roots with a strain from the same geographic region, but not when roots were infected with a strain from the Andean region. Subtle or no differences were observed when an Andean bean cultivar was inoculated with its cognate strain, suggesting involvement of these genes in the cultivar-strain specific response (Peltzer Meschini et al, 2008). In order to assess the functionality of these genes in symbiosis, composite plants with increased (overexpression) or reduced (RNAi strategy) levels were generated by Agrobacterium rhizogenes mediated transformation.  Silencing of a monomeric G protein belonging to the Rab family produced a reduction in the number and length of the root hairs compared with control GUS RNAi plants. This gene is preferentially accumulated in root hairs and its transcript levels are increased after infection with a cognate strain of R. etli, with a maximum at 12 hs after inoculation. Nodulation was completely abolished in Rab RNAi plants, whereas overexpression did not have any noticeable effect on the phenotype. Bacterial attachment to the root was similar between Rab RNAi and control plants, but neither root hair curling nor infection thread formation was observed in roots were Rab transcrips were reduced. Furthermore, accumulation of early nodulins after rhizobial infection was severely compromised in Rab RNAi plants, suggesting this Rab protein to play a role in the early response of the root hair that leads to infection thread formation and induction of nodulin genes. PvHAP5 encodes a protein highly homologous to the C subunit of CCAAT heterotrimeric transcription factors.  PvHAP5 mRNA were found to accumulate at higher levels in P. vulgaris roots of the Mesoamerican cultivar at early time points, as well as in young and mature nodules, after inoculation with a R. etli strain from the same region than in roots inoculated with a strain from the Andean region.  RNAi mediated silencing of PvHAP5 dramatically reduced the number of nodules respect to control plants (GUS-RNAi) giving a similar number of nodules when inoculated with either the Mesoamerican or the Andean strain. On the other hand, in PvHAP5 overexpressing hairy roots, inoculation with a rhizobial strains from Mesoamerica did not significantly changed nodulation efficiency.  By contrast, inoculation with Andean strains resulted in formation of higher number of nodules in PvHAP5 overexpresing than in control roots (about 2 folds), reaching values that were comparable to the number of nodules per root developed upon inoculation with the Mesoamerican strains. These results suggest that PvHAP5 might mediate cultivar-strain specificity during the symbiotic association between P. vulgaris and R. etli     Peltzer Meschini, E., Blanco, F., Zanetti, M.E., Beker, M.P., Küster, H., Pühler, A., Aguilar, O.M. (2008). Host genes involved in nodulation preference in common beans (Phaseolus vulgaris)-Rhizobium etli symbiosis revealed by suppressive subtractive hybridization. Molecular Plant-Microbe Interactions 21(4): 459-468.