Abstract:

As other legume crops, alfalfa cultivation increases the emission of the greenhouse gas nitrous oxide (N2O). Since legumesymbiotic nitrogen-fixing bacteria play a crucial role in this emission, it is important to understand the possible impacts of rhizobial domestication on the evolution of denitrification genes. In comparison with the genomes of non-commercial strains, those of commercial alfalfa inoculants exhibit low total genome size, low number of ORFs and high numbers of both frameshifted genes and pseudogenes, suggesting a dramatic loss of genes during bacterial domestication. Genomic analysis focused on denitrification genes revealed that commercial strains have perfectly conserved the nitrate (NAP), nitrite (NIR) and nitric (NOR) reductase clusters related to the production of N2O from nitrate but completely lost the nitrous oxide (NOS) reductase cluster (nosRZDFYLX genes) associated with the reduction of N2O to gas nitrogen. Based on these results, we propose future screenings for alfalfa-nodulating isolates containing both nitrogen fixation and N2O reductase genes for environmental sustainability of alfalfa production.

Instituto de Genética

Author affiliation: Brambilla, Silvina Maricel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Frare, Romina Alejandra. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Jozefkowicz, Cintia. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Ayub, Nicolás Daniel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

Background: The production of antimicrobial peptides is a common defense strategy of living cells against a wide range of pathogens. Plant snakin peptides inhibit bacterial and fungal growth at extremely low concentrations. However, little is known of their molecular and ecological characteristics, including origin, evolutionary equivalence, specific functions and activity against beneficial microbes. The aim of this study was to identify and characterize snakin-1 from alfalfa (MsSN1). Results: Phylogenetic analysis showed complete congruence between snakin-1 and plant trees. The antimicrobial activity of MsSN1 against bacterial and fungal pathogens of alfalfa was demonstrated in vitro and in vivo. Transgenic alfalfa overexpressing MsSN1 showed increased antimicrobial activity against virulent fungal strains. However, MsSN1 did not affect nitrogen-fixing bacterial strains only when these had an alfalfa origin. Conclusions: The results reported here suggest that snakin peptides have important and ancestral roles in land plant innate immunity. Our data indicate a coevolutionary process, in which alfalfa exerts a selection pressure for resistance to MsSN1 on rhizobial bacteria. The increased antimicrobial activity against virulent fungal strains without altering the nitrogen-fixing symbiosis observed in MsSN1-overexpressing alfalfa transgenic plants opens the way to the production of effective legume transgenic cultivars for biotic stress resistance.

Instituto de Genética

Author affiliation: Garcia, Araceli Nora. INTA. Instituto de Genética; Argentina

Author affiliation: Ayub, Nicolás Daniel. INTA. Instituto de Genética; Argentina

Author affiliation: Fox, Ana Romina. INTA. Instituto de Genética; Argentina.

Author affiliation: Gomez, Maria Cristina. INTA. Instituto de Genética; Argentina

Author affiliation: Dieguez, María José. INTA. Instituto de Genética; Argentina

Author affiliation: Pagano, Elba Maria. INTA. Instituto de Genética; Argentina

Author affiliation: Berini, Carolina Andrea. Instituto de Investigaciones Biomédicas en Retrovirus y SIDA; Argentina

Author affiliation: Muschietti, Jorge Prometeo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, “Dr. Hector Torres”; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética; Argentina

Abstract:

One of the most intriguing properties of plasma membrane intrinsic protein (PIP) aquaporins (AQPs) is their ability to modulate water transport by sensing different levels of intracellular pH through the assembly of homo‐ and heterotetrameric molecular species in the plasma membrane. In this work, using a phenomenological modeling approach, we demonstrate that cooperativity in PIP biological response cannot be directly attributed to a cooperative proton binding, as it is usually considered, since it could also be the consequence of a cooperative conformation transition between open and closed states of the channel. Moreover, our results show that, when mixed populations of homo‐ and heterotetrameric PIP channels are coexpressed in the plasma membrane of the same cell, the observed decrease in the degree of positive cooperativity would result from the simultaneous presence of molecular species with different levels of proton sensing. Indeed, the random mixing between different PIP paralogues as subunits in a single tetramer, plus the possibility of mixed populations of homo‐ and heterotetrameric PIP channels widen the spectrum of cooperative responses of a cell membrane. Our approach offers a deep understanding of cooperative transport of AQP channels, as members of a multiprotein family where the relevant proton binding sites of each member have not been clearly elucidated yet.

Instituto de Genética

Author affiliation: Vitali, Victoria Andrea. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Ciudad Universitaria. Instituto de Química y Fisicoquímica Biológica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Fisicoquímica Biológica; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática; Argentina

Author affiliation: Jozefkowicz, Cintia. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Canessa Fortuna, Agustina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Ciudad Universitaria. Instituto de Química y Fisicoquímica Biológica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Fisicoquímica Biológica; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Ciudad Universitaria. Instituto de Química y Fisicoquímica Biológica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Fisicoquímica Biológica; Argentina.

Author affiliation: Alleva, Karina Edith. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Ciudad Universitaria. Instituto de Química y Fisicoquímica Biológica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Fisicoquímica Biológica; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática; Argentina

Abstract:

Nitrogen is the second most critical factor for crop production after water. In this study, the beneficial rhizobacterium Pseudomonas protegens Pf-5 was genetically modified to fix nitrogen using the genes encoding the nitrogenase of Pseudomonas stutzeri A1501 via the X940 cosmid. Pf-5 X940 was able to grow in L medium without nitrogen, displayed high nitrogenase activity and released significant quantities of ammonium to the medium. Pf-5 X940 also showed constitutive expression and enzymatic activity of nitrogenase in ammonium medium or in nitrogen-free medium, suggesting a constitutive nitrogen fixation. Similar to Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii and Pseudomonas taetrolens but not Pseudomonas balearica and Pseudomonas stutzeri transformed with cosmid X940 showed constitutive nitrogenase activity and high ammonium production, suggesting that this phenotype depends on the genome context and that this technology to obtain nitrogen-fixing bacteria is not restricted to Pf-5. Interestingly, inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-deficient conditions. In conclusion, these results open the way to the production of effective recombinant inoculants for nitrogen fixation on a wide range of crops.

Author affiliation: Setten, Lorena. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Mozzicafreddo, Matteo. Università di Camerino. School of Biosciences and Biotechnology; Italia

Author affiliation: Fox, Ana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Lisi, Christian Daniel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Cuccioloni, Massimiliano. Università di Camerino. School of Biosciences and Biotechnology; Italia

Author affiliation: Angeletti, Mauro. Università di Camerino. School of Biosciences and Biotechnology; Italia

Author affiliation: Pagano, Elba Maria. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Diaz Paleo, Antonio Horacio. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Ayub, Nicolás Daniel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

The vast majority of Pseudomonas species are unable to fix atmospheric nitrogen. Although several studies have demonstrated that some strains belonging to the genus Pseudomonas sensu stricto do have the ability to fix nitrogen by the expression of horizontally acquired nitrogenase, little is known about the mechanisms of nitrogenase adaptation to the new bacterial host. Recently, we transferred the nitrogen fixation island from Pseudomonas stutzeri A1501 to the non-nitrogen-fixing bacterium Pseudomonas protegens Pf-5, and interestingly, the resulting recombinant strain Pf-5 X940 showed an uncommon phenotype of constitutive nitrogenase activity. Here, we integrated evolutionary and functional approaches to elucidate this unusual phenotype. Phylogenetic analysis showed that polyhydroxybutyrate (PHB) biosynthesis genes from natural nitrogen-fixing Pseudomonas strains have been acquired by horizontal transfer. Contrary to Pf-5 X940, its derived PHB-producing strain Pf-5 X940-PHB exhibited the inhibition of nitrogenase activity under nitrogen-excess conditions, and displayed the typical switch-on phenotype observed in natural nitrogen-fixing strains after nitrogen deficiency. This indicates a competition between PHB production and nitrogen fixation. Therefore, we propose that horizontal transfer of PHB biosynthesis genes could be an ancestral mechanism of regulation of horizontally acquired nitrogenases in the genus Pseudomonas.

Instituto de Genética

Author affiliation: Pascuan, Cecilia Gabriela. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Fox, Ana Romina. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Ayub, Nicolás Daniel. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

Hygienic behavior is an economically beneficial, heritable trait, which has evolved to limit the impact of honeybee pathogens. Selecting and breeding colonies with high levels of hygienic behavior has become a feasible and environmentally friendly strategy to control brood diseases in honeybee colonies worldwide. The identification of genes involved in the expression of this character may not only unravel molecular and biochemical pathways underlying hygienic behavior, but also serve as a practical approach to select disease resistance biomarkers useful for honeybee breeding programs. In the present work, we evaluated, at genetic level, Apis mellifera stocks selected for hygienic behavior, widely used for commercial apiculture in Argentina. We analyzed the expression profiles of five genes previously identified as candidates associated with hygienic behavior both in QTL and global gene expression studies in honeybees, more precisely, involved in perception and processing of olfactory information. We validated the differential expression of these genes as potentially responsible for behavioral differences in our selected stocks. Our results indicate that four of them (octopamine receptor, smell-impaired, odorant-binding protein 3, and odorant-binding protein 4) were differentially expressed between hygienic and non-hygienic bees within our highly hygienic colonies. The present findings improve our understanding of the molecular mechanisms underlying the differentiation of middle-age worker bees in their genetic propensity to perform hygienic behavior. This progress towards the genetic characterization of highly hygienic colonies that are commercially used in Argentine apiculture lays the groundwork for future development of targets for marker-assisted selection of disease-resistant honeybee stocks.

Inst. de Genética "Ewald A. Favret"- IGEAF

Author affiliation: Scannapieco, Alejandra Carla. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Mannino, Constanza. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Palacio, María Alejandra. INTA. Estación Experimental Agropecuaria Balcarce; Argentina. Unidad Integrada. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina

Author affiliation: Cladera, Jorge Luis. INTA. Instituto de Genética; Argentina

Author affiliation: Lanzavecchia, Silvia Beatriz. INTA. Instituto de Genética; Argentina

Abstract:

Alfalfa, usually known as the “Queen of Forages”, is the main source of vegetable protein to meat and milk production systems worldwide. This legume is extremely rich in proteins due to its highly efficient symbiotic association with nitrogen-fixing strains. In the last years, alfalfa culture has been displaced to saline environments by other important crops, including major cereals, a fact that has reduced its biomass production and symbiotic nitrogen fixation. In this short communication, we report the high forage production and nutrient quality of alfalfa under saline conditions by alfalfa transformation with the AtNHX1 Na+/H+ antiporter and inoculation with the stress-resistant nitrogen-fixing strain Sinorhizobium meliloti B401. Therefore, the incorporation of transgenic traits into salt-sensitive legumes in association with the inoculation with natural stress-resistant isolates could be a robust approach to improve the productivity and quality of these important nitrogen-fixing crops

Journal of biotechnology 276–277 : 42-45. (20 June 2018)

Instituto de Genética

Author affiliation: Stritzler, Margarita. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Pagano, Elba Maria. INTA. Instituto de Genética; Argentina

Author affiliation: Berini, Carolina Andrea. Instituto de Investigaciones Biomédicas en Retrovirus y SIDA; Argentina

Author affiliation: Gomez, Maria Cristina. INTA. Instituto de Genética; Argentina

Author affiliation: Ayub, Nicolás Daniel. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops.

Inst. de Genética "Ewald A. Favret"- IGEAF

Author affiliation: Fox, Ana Romina. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina

Author affiliation: Russo, Daniela Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Lagares, Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina

Author affiliation: Zorreguieta, Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Alleva, Karina Edith. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Pascuan, Cecilia Gabriela. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Frare, Romina Alejandra. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Mercado Blanco, Jesús. Consejo Superior de Investigaciones Científicas. Instituto de Agricultura Sostenible. Departamento de Protección de Cultivos; España

Author affiliation: Dixon, Ray. John Innes Centre. Department of Molecular Microbiology; Reino Unido

Author affiliation: Ayub, Nicolás Daniel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

Numerous species of legumes, including alfalfa, are critical factors for agroecosystems due to their ability to grow without nitrogen fertilizers derived from non-renewable fossil fuels, their contribution of organic nitrogen to the soil, and their increased nutritional value. Alfalfa is the main source of vegetable proteins in meat and milk production systems worldwide. Despite the economic and ecological importance of this autotetraploid and allogamous forage crop, little progress has been made in the incorporation of transgenic traits into commercial alfalfa. This is mainly due to the unusually strong transgene silencing and complex reproductive behavior of alfalfa, which limit the production of events with high transgene expression and the introgression of selected events within heterogeneous synthetic populations, respectively. In this report, we describe a novel procedure, called supertransgene process, where a glufosinate-tolerant alfalfa variety was developed using a single event containing the BAR transgene associated with an inversion. This approach can be used to maximize the expression of transgenic traits into elite alfalfa germplasm and to reduce the cost of production of transgenic alfalfa cultivars, contributing to the public improvement of this legume forage and other polyploid and outcrossing crop species.

Instituto de Genética

Author affiliation: Jozefkowicz, Cintia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. INTA. Instituto de Genética; Argentina.

Author affiliation: Frare, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. INTA. Instituto de Genética; Argentina.

Author affiliation: Odorizzi, Ariel. INTA. Estación Experimental Agropecuaria Manfredi; Argentina

Author affiliation: Arolfo, Valeria. INTA. Estación Experimental Agropecuaria Manfredi; Argentina

Author affiliation: Pagano, Elba Maria. INTA. Instituto de Genética; Argentina

Author affiliation: Basigalup, Daniel Horacio. INTA. Estación Experimental Agropecuaria Manfredi; Argentina

Author affiliation: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. INTA. Instituto de Genética; Argentina

Author affiliation: Soto, Gabriela Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. INTA. Instituto de Genética; Argentina

Abstract:

Despite the vast screening for natural nitrogenfixing isolates by public and private consortia, no significant progresses in the production of improved nitrogenfixing inoculants for alfalfa production have been made in the last years. Here, we present a comprehensive characterization of the nitrogen-fixing strain Ensifer meliloti B399 (originally named Rhizobium meliloti 102F34), probably the inoculant most widely used in alfalfa production since the 1960s. Complete nucleotide sequence and genome analysis of strain B399 showed that the three replicons present in this commercial strain and the model bacterium Ensifer meliloti 1021 are extremely similar to each other in terms of nucleotide identity and synteny conservation. In contrast to that observed in B399-treated plants, inoculation of plants with strain 1021 did not improve nitrogen content in different alfalfa cultivars under field conditions, suggesting that a small genomic divergence can drastically impact on the symbiotic phenotype. Therefore, in addition to the traditional screening of natural nitrogen-fixing isolates, the genome engineering of model strains could be an attractive strategy to improve nitrogen fixation in legume crops.

Inst. de Microbiología y Zoología Agrícola IMyZA

Author affiliation: Jozefkowicz, Cintia. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Brambilla, Silvina Maricel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Frare, Romina Alejandra. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Stritzler, Margarita. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Puente, Mariana Laura. INTA. Instituto de Microbiología y Zoología Agrícola; Argentina

Author affiliation: Piccinetti, Carlos Fabian. INTA. Instituto de Microbiología y Zoología Agrícola; Argentina

Author affiliation: Soto, Gabriela Cinthia. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Ayub, Nicolás Daniel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina