CRISPR/Cas9-mediated knockout of a polyester synthase-like gene delays flowering time in alfalfa.

KEY MESSAGE: T-DNA and CRISPR/Cas9-mediated knockout of polyester synthase-like genes delays flowering time in Arabidopsis thaliana and Medicago sativa (alfalfa). Thus, we here present the first report of edited alfalfa with delayed flowering.

Exploring the Role of the NO-Detoxifying Enzyme HmpA in the Evolution of Domesticated Alfalfa Rhizobia.

We have previously shown the extensive loss of genes during the domestication of alfalfa rhizobia and the high nitrous oxide emission associated with the extreme genomic instability of commercial inoculants. In the present note, we describe the molecular mechanism involved in the evolution of alfalfa rhizobia. Genomic analysis showed that most of the gene losses in inoculants are due to large genomic deletions rather than to small deletions or point mutations, a fact consistent with recurrent DNA double-strand breaks (DSBs) at numerous locations throughout the microbial genome. Genetic analysis showed that the loss of the NO-detoxifying enzyme HmpA in inoculants results in growth inhibition and high DSB levels under nitrosative stress, and large genomic deletions in planta but not in the soil. Therefore, besides its known function in the effective establishment of the symbiosis, HmpA can play a critical role in the preservation of the genomic integrity of alfalfa rhizobia under host-derived nitrosative stress.

Whole-Genome Resequencing of Spontaneous Oxidative Stress-Resistant Mutants Reveals an Antioxidant System of Bradyrhizobium japonicum Involved in Soybean Colonization.

Soybean is the most inoculant-consuming crop in the world, carrying strains belonging to the extremely related species Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens. Currently, it is well known that B. japonicum has higher efficiency of soybean colonization than B. diazoefficiens, but the molecular mechanism underlying this differential symbiotic performance remains unclear. In the present study, genome resequencing of four spontaneous oxidative stress-resistant mutants derived from the commercial strain B. japonicum E109 combined with molecular and physiological studies allowed identifying an antioxidant cluster (BjAC) containing a transcriptional regulator (glxA) that controls the expression of a catalase (catA) and a phosphohydrolase (yfbR) related to the hydrolysis of hydrogen peroxide and oxidized nucleotides, respectively. Integrated synteny and phylogenetic analyses supported the fact that BjAC emergence in the B. japonicum lineage occurred after its divergence from the B. diazoefficiens lineage. The transformation of the model bacterium B. diazoefficiens USDA110 with BjAC from E109 significantly increased its ability to colonize soybean roots, experimentally recapitulating the beneficial effects of the occurrence of BjAC in B. japonicum. In addition, the glxA mutation significantly increased the nodulation competitiveness and plant growth-promoting efficiency of E109. Finally, the potential applications of these types of non-genetically modified mutant microbes in soybean production worldwide are discussed.

Generation of a multi-herbicide-tolerant alfalfa by using base editing.

KEYMESSAGE: We present the first report on base editing in alfalfa. Specifically, we showed edited alfalfa with tolerance to both sulfonylurea- and imidazolinone-type herbicides.

The Ammonium Channel NOD26 is the Evolutionary Innovation that Drives the Emergence, Consolidation, and Dissemination of Nitrogen-Fixing Symbiosis in Angiosperms.

Increasing evidence indicates that N-fixing symbiosis has evolved several times in the N-fixing clade of angiosperms and that this evolution is driven by a single evolutionary innovation. However, the genetics of this ancestral predisposition to N-fixing symbiosis remains unclear. A natural candidate for such molecular innovation is the ammonium channel NOD26, the main protein component of the symbiosome membrane, which facilitates the plant uptake of the nitrogen fixed by symbiotic bacteria. Here, in concordance with the emergence of N-fixing symbiosis in angiosperms but not in ancestral plants, phylogenetic analysis showed that NOD26 belongs to an angiosperm-exclusive subgroup of aquaporins. Integrated genomic, phylogenetic, and gene expression analyses supported NOD26 occurrence in the N-fixing clade, the increase in the NOD26 copy number by block and tandem duplications in legumes, and the low-copy number or even the loss of NOD26 in non-legume species of the N-fixing clade, which correlated with the possibility to lose N-fixing symbiosis in legume and non-legume lineages. Metabolic reconstructions showed that retention of NOD26 in N-fixing precursor could represent an adaptive mechanism to bypass energy crisis during anaerobic stress by ammonium detoxification. Finally, we discuss the potential use of NOD26 to transfer N-fixation to non-N-fixing crops as cereals.

Maximizing the expression of transgenic traits into elite alfalfa germplasm using a supertransgene configuration in heterozygous conditions.

KEY MESSAGE: A novel process for the production of transgenic alfalfa varieties. 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.

Absence of the Nitrous Oxide Reductase Gene Cluster in Commercial Alfalfa Inoculants Is Probably Due to the Extensive Loss of Genes During Rhizobial Domestication.

As other legume crops, alfalfa cultivation increases the emission of the greenhouse gas nitrous oxide (N2O). Since legume-symbiotic 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.

Microevolution Rather than Large Genome Divergence Determines the Effectiveness of Legume-Rhizobia Symbiotic Interaction Under Field Conditions.

Despite the vast screening for natural nitrogen-fixing isolates by public and private consortia, no significant progresses in the production of improved nitrogen-fixing 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.

Major cereal crops benefit from biological nitrogen fixation when inoculated with the nitrogen-fixing bacterium Pseudomonas protegens Pf-5 X940.

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. 15 N 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.

mRNA biogenesis-related helicase eIF4AIII from Arabidopsis thaliana is an important factor for abiotic stress adaptation.

Similar to other plant species, Arabidopsis has a huge repertoire of predicted helicases, including the eIF4AIII factor, a putative component of the exon junction complex related to mRNA biogenesis. In this article, we integrated evolutionary and functional approaches to have a better understanding of eIF4AIII function in plants. Phylogenetic analysis showed that the mRNA biogenesis-related helicase eIF4AIII is the ortholog of the stress-related helicases PDH45 from Pisum sativum and MH1 from Medicago sativa, suggesting evolutionary and probably functional equivalences between mRNA biogenesis and stress-related plant helicases. Molecular and genetic analyses confirmed the relevance of eIF4AIII during abiotic stress adaptation in Arabidopsis. Therefore, in addition to its function in mRNA biogenesis, eIF4AIII can play a role in abiotic stress adaptation.

Multiple ways to evade the bacteriostatic action of glyphosate in rhizobia include the mutation of the conserved serine 90 of the nitrogenase subunit NifH to alanine.

The genome resequencing of spontaneous glyphosate-resistant mutants derived from the soybean inoculant E109 allowed identifying genes most likely associated with the uptake (gltL and cya) and metabolism (zigA and betA) of glyphosate, as well as with nitrogen fixation (nifH). Mutations in these genes reduce the lag phase and improve nodulation under glyphosate stress. In addition to providing glyphosate resistance, the amino acid exchange Ser90Ala in NifH increased the citrate synthase activity, growth rate and plant growth-promoting efficiency of E109 in the absence of glyphosate stress, suggesting roles for this site during both the free-living and symbiotic growth stages.

Synthetic multi-antibiotic resistant plasmids in plant-associated bacteria from agricultural soils.

OBJECTIVES: Unlike higher organisms such as domestic animals and cultivated plants, which display a robust reproductive isolation and limited dispersal ability, microbes exhibit an extremely promiscuous gene flow and can rapidly disperse across the planet by multiple ways. Thus, microbial plasmids, including synthetic replicons, containing antibiotic resistance genes are a serious risk to public health. In this short communication, we explored the presence of synthetic elements in alfalfa symbionts (Ensifer meliloti strains) from agricultural soils. METHODS: A total of 148 E. meliloti isolates from alfalfa plants growing under field conditions were collected from January 2015 to June 2019. Antimicrobial susceptibility testing was performed under laboratory conditions. We identified five kanamycin-resistant E. meliloti strains (named K1-K5). Whole genome sequencing analysis and conjugations were used to identify and study the plasmids of K strains. RESULTS: We found that the genomes of K strains contain ampicillin, kanamycin and tetracycline resistance genes, the reporter gene lacZ from Escherichia coli and multiple cloning sites. These sequences were found within <58-kb plasmids related to the self-transmissible IncP plasmid RP4 from human pathogen Pseudomonas aeruginosa. Conjugation experiments confirmed the ability of K strains to transfer antibiotic resistance via conjugation to the Pseudomonas background. CONCLUSION: In addition to the traditional analysis of plant growth-promoting factors, the commercial deregulation of putative natural inoculants should also include genomic studies to ensure a reasonable balance between innovation and caution.

Elimination of GlnKAmtB affects serine biosynthesis and improves growth and stress tolerance of Escherichia coli under nutrient-rich conditions.

Nitrogen is a most important nutrient resource for Escherichia coli and other bacteria that harbor the glnKamtB operon, a high-affinity ammonium uptake system highly interconnected with cellular metabolism. Although this system confers an advantage to bacteria when growing under nitrogen-limiting conditions, little is known about the impact of these genes on microbial fitness under nutrient-rich conditions. Here, the genetically tractable E. coli BW25113 strain and its glnKamtB-null mutant (JW0441) were used to analyze the impact of GlnK-AmtB on growth rates and oxidative stress tolerance. Strain JW0441 showed a shorter initial lag phase, higher growth rate, higher citrate synthase activity, higher oxidative stress tolerance and lower expression of serA than strain BW25113 under nutrient-rich conditions, suggesting a fitness cost to increase metabolic plasticity associated with serine metabolism. The overexpression of serA in strain JW0441 resulted in a decreased growth rate and stress tolerance in nutrient-rich conditions similar to that of strain BW25113, suggesting that the negative influence on bacterial fitness imposed by GlnK-AmtB can be traced to the control of serine biosynthesis. Finally, we discuss the potential applications of glnKamtB mutants in bioproduction processes.

Stable symbiotic nitrogen fixation under water-deficit field conditions by a stress-tolerant alfalfa microsymbiont and its complete genome sequence.

We here characterized the stress-tolerant alfalfa microsymbiont Sinorhizobium meliloti B401. B401-treated plants showed high nitrogen fixation rates under humid and semiarid environments. The production of glycine betaine in isolated bacteroids positively correlated with low precipitation levels, suggesting that this compound acts as a critical osmoprotectant under field conditions. Genome analysis revealed that strain B401 contains alternative pathways for the biosynthesis and uptake of glycine betaine and its precursors. Such genomic information will offer substantial insight into the environmental physiology of this biotechnologically valuable nitrogen-fixing bacterium.