Host genes involved in nodulation preference in the common bean-Rhizobium etli symbiosis

AGUILAR; BEKER, MARÍA PÍA; BATTAGLIA, MARINA; ZANETTI, MARÍA EUGENIA; BLANCO, FLAVIO; NICOLETTI, K; SORIA DIAZ, ME; GIL SERRANO, A; MEGIAS, M

Zaragoza, Spain

Congreso; XIII SEFIN; 2010

Host genes involved in nodulation preference in the common bean-Rhizobium etli symbiosis Aguilar OM1*, Beker MP1, Battaglia ME1, Zanetti ME1, Blanco FA1, Nicoletti K1, Soria-Diaz ME2, Gil-Serrano A2, Megías M3 1Instituto de Biotecnología y Biología Molecular, CONICET-La Plata, Facultad Ciencias Exactas, Universidad Nacional de La Plata, 1900-La Plata, Argentina; 2Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, 41071 Sevilla, Spain; 3Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41071 Sevilla, Spain. *aguilar@biol.unlp.edu.ar   Summary Common beans (Phaseolus vulgaris) and rhizobia coevolved in the two main centres of host domestication. In order to characterize the symbiosis, the physiology of nodulation was examined in combinations of host x lineages of Rhizobium etli. Analysis of nodulation factors produced by strains of R. etli from both centres of diversification and competitiveness by nolL mutant, suggested that factors other than Nod factors (NFs) might be involved in strain preference. A host gene coding for a subunit of the NF-Y transcriptional factor was identified in the Mesomerican beans and found to be activated in response to inoculation with its cognate rhizobia   The common bean-rhizobia symbiosis in the Americas Common beans are believed to have been domesticated in the Americas with two centres of diversification, one of them in Meso-America and the other in the Southern Andes dispersed in Ecuador, Perú, Bolivia and Argentina. Beans are used for direct human nutrition all over the world particularly, in Latin American and African countries. We have demonstrated nodulation preference between beans and lineages of R. etli from the same host region. Our results invited us to speculate that the major genetic pools of beans and rhizobia coevolved independently after geographic separation (Aguilar et al., 2004). By applying a suppressive subtractive hybridization approach in which cDNA from a Mesoamerican cultivar inoculated either with the more or the less efficient strain was used as driver and tester, a set of genes were identified as being involved in the early preferential symbiotic interaction (Peltzer-Meschini et al., 2008). The role in nodulation, of one of these genes -coding for a monomeric GTPase with similarities to RabA2a- has been studied in bean composite plants by using a reverse genetic approach. Examination of root morphology in PvRabA2a RNAi plants revealed that the number and length of the root hairs were severely reduced. Upon inoculation with R. etli, nodulation was completely impaired and induction of early nodulation genes (ENODs) was undetectable in silenced hairy roots (Blanco et al., 2009). Early response to different lineages of R. etli We have further characterized the interaction between beans x lineage of R. etli. Inoculated roots showed earlier response to infection with cognate strains as compared to alopatric strains. These responses include root hair curling, formation of infection threads and number of nodule per plant. In order to gain insight on the differential response, the NF produced by strains from the Mesoamerican center of diversification and from the Andean center of diversification was analyzed. The molecule signal of one out of three Andean R. etli strains we examined showed a chitolipooligosaccharidic molecule that is devoid of the acetyl residue on the fucosyl moiety at the reducing end of the molecule. The structure of the other Andean strains as well as the Mesoamerican strains were found to be similar each other. We assessed the competitiveness for nodule occupancy of Mesoamerican beans, by an R. etli nolL mutant derived of the Mesoamerican strain CE3, which fails to acetylate the fucosyl residue. Results indicated that, although the nolL mutation affects negatively the competitiveness in co-inoculations with any of the two Mesoamerican R. etli wild-type strains that we assayed (CE3 and SC15), it does not affect the large nodule occupancy in co-inoculation experiments with the alopatric strain R. etli 55N1 from the Southern Andean region. These results suggest that factors other than NFs could play a role in determining affinity for nodulation by certain lineages of R. etli. Within this frame, we have performed competition experiments in beans from both centers of host diversification by using a combination of strains that included Rhizobium tropici CIAT899, a genotype other than R. etli. Nodules of the Mesoamerican cultivar occupied by strains CIAT899 and R. etli 55N1 were found to be comparable to each other, whereas in the Andean bean cultivar Alubia, occupancy by the sympatric strain 55N1 clearly overcomes strain CIAT899. This result indicated that competitiveness is expressed in the host x strain combination of the same centre of diversification. Identification of host genes involved in the efficient nodulation by specific R. etli strains A host gene was identified that is differently expressed in the Mesoamerican cultivar inoculated with its cognate strains. PvNF-YC, identified in a survey of genes that are differential expressed in the Mesoamerican cultivar with its cognate strains, encodes the C subunit of the nuclear factor Y (NF-Y) heterotrimeric transcription factor (Peltzer Meschini et al., 2008). RNA interference (RNAi)-mediated gene silencing of this gene in bean hairy roots led to a reduced nodulation phenotype and defects in initiation/progression of infection threads. Interestingly, expression of early nodulin genes such as RIP, ERN and ENOD40 was not affected in these roots, indicating that PvNF-YC might act independently or downstream of these early nodulins. By contrast, induction of G2/S transition cell cycle genes in response to rizobia was impaired in PvNF-YC RNAi roots, suggesting that this gene might be required for first cortical cell divisions that lead to the formation of initial primordia in determined nodules. In order to identify proteins that can physically interact with this transcription factor, a yeast two hybrid (Y2H) screening was performed using PvNF-YC as bait and a cDNA library from root tissue inoculated with R. etli. A total of eight clones that potentially interacts with NF-YC were isolated and sequenced. Among them, two encode receptor-like kinases and another clone encodes a putative transcriptional regulator. Interaction of PvNF-YC with these gene products was verified by retransformation of yeast and is being validated by co-immunoprecipitation assays and bimolecular fluorescent complementation in Agrobacterium-infiltrated Nicotiana benthamiana leaves. The function of these PvNF-YC interacting proteins in nodulation efficiency and bacterial infection is being evaluated by both RNAi and overexpression. This will help to elucidate the signal transduction pathway specifically activated in Mesoamerican beans in response to its cognate R. etli strain. Acknowledgments This work was supported by grants from the ANPCyT, PICT No. 2065/2006, and ICGEB, International Center for Genetic Engineering and Biotechnology, Trieste, Italy. References Aguilar OM, Riva O, Peltzer E (2004) Proc Nat Acad Sci USA 101: 13548-13553. Blanco FA, Peltzer Meschini E, Zanetti ME, Aguilar OM (2009) Plant Cell 21: 2797-2810. Peltzer-Meschini E, Blanco FA, Zanetti ME, Beker MP, Küster H, Pühler A, Aguilar OM (2008) Mol Plant-Microbe Interact 21: 459-468.