A Set of Lotus japonicus Gifu × Lotus burttii Recombinant Inbred Lines Facilitates Map-based Cloning and QTL Mapping

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype ‘Gifu’ was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation

Sinorhizobium fredii Strains HH103 and NGR234 Form Nitrogen Fixing Nodules With Diverse Wild Soybeans (Glycine soja) From Central China but Are Ineffective on Northern China Accessions

Sinorhizobium fredii indigenous populations are prevalent in provinces of Central China whereas Bradyrhizobium species (Bradyrhizobium japonicum, B. diazoefficiens, B. elkanii, and others) are more abundant in northern and southern provinces. The symbiotic properties of different soybean rhizobia have been investigated with 40 different wild soybean (Glycine soja) accessions from China, Japan, Russia, and South Korea. Bradyrhizobial strains nodulated all the wild soybeans tested, albeit efficiency of nitrogen fixation varied considerably among accessions. The symbiotic capacity of S. fredii HH103 with wild soybeans from Central China was clearly better than with the accessions found elsewhere. S. fredii NGR234, the rhizobial strain showing the broadest host range ever described, also formed nitrogen-fixing nodules with different G. soja accessions from Central China. To our knowledge, this is the first report describing an effective symbiosis between S. fredii NGR234 and G. soja. Mobilization of the S. fredii HH103 symbiotic plasmid to a NGR234 pSym-cured derivative (strain NGR234C) yielded transconjugants that formed ineffective nodules with G. max cv. Williams 82 and G. soja accession CH4. By contrast, transfer of the symbiotic plasmid pNGR234a to a pSym-cured derivative of S. fredii USDA193 generated transconjugants that effectively nodulated G. soja accession CH4 but failed to nodulate with G. max cv. Williams 82. These results indicate that intra-specific transference of the S. fredii symbiotic plasmids generates new strains with unpredictable symbiotic properties, probably due to the occurrence of new combinations of symbiotic signals.España, Junta de Andalucía P11-CVI-7500España Ministerio de Economía y Competitividad BIO2016-78409-

The Sinorhizobium fredii HH103 Lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules

In this work we have characterised the Sinorhizobium fredii HH103 greA lpsB lpsCDE genetic region and analysed for the first time the symbiotic performance of Sinorhizobium fredii lps mutants on soybean. The organization of the S. fredii HH103 greA, lpsB, and lpsCDE genes was equal to that of Sinorhizobium meliloti 1021. S. fredii HH103 greA, lpsB, and lpsE mutant derivatives produced altered LPS profiles that were characteristic of the gene mutated. In addition, S. fredii HH103 greA mutants showed a reduction in bacterial mobility and an increase of auto-agglutination in liquid cultures. RT-PCR and qPCR experiments demonstrated that the HH103 greA gene has a positive effect on the transcription of lpsB. Soybean plants inoculated with HH103 greA, lpsB or lpsE mutants formed numerous ineffective pseudonodules and showed severe symptoms of nitrogen starvation. However, HH103 greA and lps mutants were also able to induce the formation of a reduced number of soybean nodules of normal external morphology, allowing the possibility of studying the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis. The infected cells of these nodules showed signs of early termination of symbiosis and lytical clearance of bacteroids. These cells also had very thick walls and accumulation of phenolic-like compounds, pointing to induced defense reactions. Our results show the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis and their role in preventing host cell defense reactions. S. fredii HH103 lpsB mutants also showed reduced nodulation with Vigna unguiculata, although the symbiotic impairment was less pronounced than in soybean

Sinorhizobium fredii Strains HH103 and NGR234 Form Nitrogen Fixing Nodules With Diverse Wild Soybeans (Glycine soja) From Central China but Are Ineffective on Northern China Accessions

Sinorhizobium fredii indigenous populations are prevalent in provinces of Central China whereas Bradyrhizobium species (Bradyrhizobium japonicum, B. diazoefficiens, B. elkanii, and others) are more abundant in northern and southern provinces. The symbiotic properties of different soybean rhizobia have been investigated with 40 different wild soybean (Glycine soja) accessions from China, Japan, Russia, and South Korea. Bradyrhizobial strains nodulated all the wild soybeans tested, albeit efficiency of nitrogen fixation varied considerably among accessions. The symbiotic capacity of S. fredii HH103 with wild soybeans from Central China was clearly better than with the accessions found elsewhere. S. fredii NGR234, the rhizobial strain showing the broadest host range ever described, also formed nitrogen-fixing nodules with different G. soja accessions from Central China. To our knowledge, this is the first report describing an effective symbiosis between S. fredii NGR234 and G. soja. Mobilization of the S. fredii HH103 symbiotic plasmid to a NGR234 pSym-cured derivative (strain NGR234C) yielded transconjugants that formed ineffective nodules with G. max cv. Williams 82 and G. soja accession CH4. By contrast, transfer of the symbiotic plasmid pNGR234a to a pSym-cured derivative of S. fredii USDA193 generated transconjugants that effectively nodulated G. soja accession CH4 but failed to nodulate with G. max cv. Williams 82. These results indicate that intra-specific transference of the S. fredii symbiotic plasmids generates new strains with unpredictable symbiotic properties, probably due to the occurrence of new combinations of symbiotic signals

Legumes in biodiversity-based farming systems in Mediterranean basin

Legumes belong to the Leguminosae family and have been known for their use since ancient times as a source of food and as fodder plants. Many legume genera establish a symbiotic relationship with soil bacteria called rhizobia. This relationship gives rise to the formation in the roots of specialized structures called nodules, where atmospheric nitrogen is fixed. The agricultural advantages of this symbiosis are numerous. The aim of this work is to select Rhizobium strains on previously selected chickpea and lentil cultivars to determine their efficiency on BNF and, to assess the size of native lentil-specific Rhizobium populations in agricultural soils using the most probable number (MPN) technique [1].Methods: Four chickpea and four lentil cultivars were used for the greenhouse trials and eight Rhizobium strains, four specific for chickpea and four for lentil, plus a control treatment (uninoculated). A nutrient solution lacking combined nitrogen was used for plant growth. The seeds, once germinated, were transferred to a sterile system [2]. Each seed was inoculated with 1 mL of the corresponding legume-specific Rhizobium cultures.Results: In the case of chickpea, the cultivar with a tan color (5-RIL-33), unlike the rest with a cream color, has a higher number of nodules and, consequently, a greater dry weight of nodules than the rest of the cultivars, resulting in a bigger accumulation of biomass in the plants. Strain ISC11 shows the highest symbiotic efficiency with three (BT6-19, 5-RIL-33 and 5-RIL-98) of the four cultivars used in the greenhouse trials, being better, in the case of cultivar RR-98 "Kasin", strain ISC15. With respect to the MPN technique [3], in the case of chickpea, a native population of 26 rhizobia/g soil was estimated. For lentils, a native population of 1100 rhizobia/g soil was determined.Conclusions: From these results, we can conclude that the use of inoculants considerably increases legume crop yields, avoiding the increasing and harmful use of chemical fertilizers. Moreover, in soils with native rhizobia populations higher than 100 rhizobia/g soil, the application of these inoculants is unnecessary due to the high competitiveness produced and, therefore, the inefficiency and economic loss that their use implie

Sinorhizobium fredii HH103 nolR and nodD2 mutants gain capacity for infection thread invasion of Lotus japonicus Gifu and Lotus burttii

Sinorhizobium fredii HH103 RifR, a broad-host-range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen-fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 RifR nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen-fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod-factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 RifR. Complementation of the nodD2 and nolR mutants provoked a decrease in Nod-factor production, the incapacity to form nitrogen-fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 RifR nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod-factor overproduction, although other bacterial symbiotic signals could also be involvedUnión Europea. Ministerio de Economía y Competitividad. BIO2016-78409-

Sinorhizobium fredii HH103 invades lotus burttii by crack entry in a nod factor- and surface polysaccharide-dependent manner

Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rif r surface-polysaccharide mutants gained effective nodulation with L. japonicus.Junta de Andalucía P11-CVU-7500Ministerio de Ciencia e Innovación BIO2011-30229-C0