Project description:Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N2) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N2 fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N2 fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different Rhizobium leguminosarum strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ?20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple Rhizobium-legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.
Project description:Following the initial discovery of two legume-nodulating Burkholderia strains (L. Moulin, A. Munive, B. Dreyfus, and C. Boivin-Masson, Nature 411:948-950, 2001), we identified as nitrogen-fixing legume symbionts at least 50 different strains of Burkholderia caribensis and Ralstonia taiwanensis, all belonging to the beta-subclass of proteobacteria, thus extending the phylogenetic diversity of the rhizobia. R. taiwanensis was found to represent 93% of the Mimosa isolates in Taiwan, indicating that beta-proteobacteria can be the specific symbionts of a legume. The nod genes of rhizobial beta-proteobacteria (beta-rhizobia) are very similar to those of rhizobia from the alpha-subclass (alpha-rhizobia), strongly supporting the hypothesis of the unique origin of common nod genes. The beta-rhizobial nod genes are located on a 0.5-Mb plasmid, together with the nifH gene, in R. taiwanensis and Burkholderia phymatum. Phylogenetic analysis of available nodA gene sequences clustered beta-rhizobial sequences in two nodA lineages intertwined with alpha-rhizobial sequences. On the other hand, the beta-rhizobia were grouped with free-living nitrogen-fixing beta-proteobacteria on the basis of the nifH phylogenetic tree. These findings suggest that beta-rhizobia evolved from diazotrophs through multiple lateral nod gene transfers.
Project description:Establishment and maintenance of mutualistic plant-microbial interactions in the rhizosphere and within plant roots involve several root cell types. The processes of host-microbe recognition and infection require complex signal exchange and activation of downstream responses. These molecular events coordinate host responses across root cell layers during microbe invasion, ultimately triggering changes of root cell fates. The progression of legume root interactions with rhizobial bacteria has been addressed in numerous studies. However, tools to globally resolve the succession of molecular events in the host root at the cell type level have been lacking. To this end, we aimed to identify promoters exhibiting cell type enriched expression in roots of the model legume Lotus japonicus, as no comprehensive set of such promoters usable in legume roots is available to date.Here, we use promoter:GUS fusions to characterize promoters stemming from Arabidopsis, tomato (Lycopersicon esculentum) or L. japonicus with respect to their expression in major cell types of the L. japonicus root differentiation zone, which shows molecular and morphological responses to symbiotic bacteria and fungi. Out of 24 tested promoters, 11 showed cell type enriched activity in L. japonicus roots. Covered cell types or cell type combinations are epidermis (1), epidermis and cortex (2), cortex (1), endodermis and pericycle (2), pericycle and phloem (4), or xylem (1). Activity of these promoters in the respective cell types was stable during early stages of infection of transgenic roots with the rhizobial symbiont of L. japonicus, Mesorhizobium loti. For a subset of five promoters, expression stability was further demonstrated in whole plant transgenics as well as in active nodules.11 promoters from Arabidopsis (10) or tomato (1) with enriched activity in major L. japonicus root and nodule cell types have been identified. Root expression patterns are independent of infection with rhizobial bacteria, providing a stable read-out in the root section responsive to symbiotic bacteria. Promoters are available as cloning vectors. We expect these tools to help provide a new dimension to our understanding of signaling circuits and transcript dynamics in symbiotic interactions of legumes with microbial symbionts.
Project description:Manganese (Mn) toxicity is a very common soil stress around the world, which is responsible for low soil fertility. This manuscript evaluates the effect of the endophytic bacterium <i>Pseudomonas</i> sp. Q1 on different rhizobial-legume symbioses in the absence and presence of Mn toxicity. Three legume species, <i>Cicer arietinum</i> (chickpea), <i>Trifolium subterraneum</i> (subterranean clover), and <i>Medicago polymorpha</i> (burr medic) were used. To evaluate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase produced by strain Q1 in these interactions, an ACC deaminase knockout mutant of this strain was constructed and used in those trials. The Q1 strain only promoted the symbiotic performance of <i>Rhizobium leguminosarum</i> bv. trifolii ATCC 14480<sup>T</sup> and <i>Ensifer meliloti</i> ATCC 9930<sup>T</sup>, leading to an increase of the growth of their hosts in both conditions. Notably, the <i>acdS</i> gene disruption of strain Q1 abolished the beneficial effect of this bacterium as well as causing this mutant strain to act deleteriously in those specific symbioses. This study suggests that the addition of non-rhizobia with functional ACC deaminase may be a strategy to improve the pasture legume-rhizobial symbioses, particularly when the use of rhizobial strains alone does not yield the expected results due to their difficulty in competing with native strains or in adapting to inhibitory soil conditions.
Project description:Malonyl-coenzyme A (CoA) decarboxylase, malonyl-CoA synthetase, and malonate transporter mutants of Rhizobium leguminosarum bv. viciae and trifolii fixed N2 at wild-type rates on pea and clover, respectively. Thus, malonate does not drive N2 fixation in legume nodules.
Project description:The establishment of nitrogen-fixing rhizobium-legume symbioses requires a highly complex cascade of events. In this molecular dialogue the bacterial NodD transcriptional regulators in conjunction with plant inducers, mostly flavonoids, are responsible for the biosynthesis and secretion of Nod factors which are key molecules for successful nodulation. Other transcriptional regulators related to the symbiotic process have been identified in rhizobial genomes, including negative regulators such as NolR. Rhizobium tropici CIAT 899 is an important symbiont of common bean (Phaseolus vulgaris L.), and its genome encompasses intriguing features such as five copies of nodD genes, as well as other possible transcriptional regulators including the NolR protein. Here we describe and characterize a new regulatory gene located in the non-symbiotic plasmid pRtrCIAT899c, that shows homology (46% identity) with the nolR gene located in the chromosome of CIAT 899. The mutation of this gene, named nrcR (nolR-like plasmid c Regulator), enhanced motility and exopolysaccharide production in comparison to the wild-type strain. Interestingly, the number and decoration of Nod Factors produced by this mutant were higher than those detected in the wild-type strain, especially under salinity stress. The nrcR mutant showed delayed nodulation and reduced competitiveness with P. vulgaris, and reduction in nodule number and shoot dry weight in both P. vulgaris and Leucaena leucocephala. Moreover, the mutant exhibited reduced capacity to induce the nodC gene in comparison to the wild-type CIAT 899. The finding of a new nod-gene regulator located in a non-symbiotic plasmid may reveal the existence of even more complex mechanisms of regulation of nodulation genes in R. tropici CIAT 899 that may be applicable to other rhizobial species.
Project description:The discovery that the actinobacterium Micromonospora inhabits nitrogen-fixing nodules raised questions as to its potential ecological role. The capacity of two Micromonospora strains to infect legumes other than their original host, Lupinus angustifolius, was investigated using Medicago and Trifolium as test plants. Compatible rhizobial strains were used for coinoculation of the plants because Micromonospora itself does not induce nodulation. Over 50% of nodules from each legume housed Micromonospora, and using 16S rRNA gene sequence identification, we verified that the reisolated strains corresponded to the microorganisms inoculated. Entry of the bacteria and colonization of the plant hosts were monitored using a GFP-tagged Lupac 08 mutant together with rhizobia, and by using immunogold labeling. Strain Lupac 08 was localized in plant tissues, confirming its capacity to enter and colonize all hosts. Based on studying three different plants, our results support a non-specific relationship between Micromonospora and legumes. Micromonospora Lupac 08, originally isolated from Lupinus re-enters root tissue, but only when coinoculated with the corresponding rhizobia. The ability of Micromonospora to infect and colonize different legume species and function as a potential plant-growth promoting bacterium is relevant because this microbe enhances the symbiosis without interfering with the host and its nodulating and nitrogen-fixing microbes.
Project description:For identification of plant gene networks which interact to initiate and support both rhizobial nodulation and AM fungal colonization, the transcription profiles of soybean genes induced during rhizobial, AM and their dual symbioses. Overall design: Four different treatments were used: an uninoculated control, inoculation with rhizobium alone (rhizobial symbiosis), inoculation with AM fungus alone (AM symbiosis), and inoculation with rhizobium and AM fungus together (dual symbiosis). After the soybeans inoculated were grown in a glasshouse for 6 weeks, the transcriptome of host genes were analyzed using a cDNA microarray
Project description:Horizontal gene transfer (HGT) is an important mode of adaptation and diversification of prokaryotes and eukaryotes and a major event underlying the emergence of bacterial pathogens and mutualists. Yet it remains unclear how complex phenotypic traits such as the ability to fix nitrogen with legumes have successfully spread over large phylogenetic distances. Here we show, using experimental evolution coupled with whole genome sequencing, that co-transfer of imuABC error-prone DNA polymerase genes with key symbiotic genes accelerates the evolution of a soil bacterium into a legume symbiont. Following introduction of the symbiotic plasmid of Cupriavidus taiwanensis, the Mimosa symbiont, into pathogenic Ralstonia solanacearum we challenged transconjugants to become Mimosa symbionts through serial plant-bacteria co-cultures. We demonstrate that a mutagenesis imuABC cassette encoded on the C. taiwanensis symbiotic plasmid triggered a transient hypermutability stage in R. solanacearum transconjugants that occurred before the cells entered the plant. The generated burst in genetic diversity accelerated symbiotic adaptation of the recipient genome under plant selection pressure, presumably by improving the exploration of the fitness landscape. Finally, we show that plasmid imuABC cassettes are over-represented in rhizobial lineages harboring symbiotic plasmids. Our findings shed light on a mechanism that may have facilitated the dissemination of symbiotic competency among ?- and ?-proteobacteria in natura and provide evidence for the positive role of environment-induced mutagenesis in the acquisition of a complex lifestyle trait. We speculate that co-transfer of complex phenotypic traits with mutagenesis determinants might frequently enhance the ecological success of HGT.
Project description:The evolutionary stability of mutualistic interactions involving multiple partners requires "sanctioning"-the ability to influence the fitness of each partner based on its respective contribution. Sanctions must be sensitive to even small differences if even slightly less-beneficial partners could gain a fitness advantage by diverting resources away from the mutualistic service toward their own reproductive fitness. Here, we test whether legume hosts sanction even mediocre N2-fixing rhizobial strains by influencing either nodule growth (which limits rhizobial cell numbers) or carbon accumulation (polyhydroxybutryate or PHB) per rhizobial cell. We also test whether sanctions depend on the availability of less-expensive nitrogen alternatives, either as nitrate or coinoculation with a more-efficient isogenic strain. We found that nitrate eliminated differences in nodule size between the mediocre and more-efficient strains, suggesting that host sanctions were compromised. However, nitrate additions also decreased PHB accumulation by the mediocre strain, which may eliminate any fitness advantages of diverting resources from N2 fixation. Coinoculation with a more-efficient strain could also compromise host sanctions if reduction in fitness from smaller nodules does not offset the potential fitness gain from greater PHB accumulation that we observed in the mediocre strain. Hence, a host's ability to sanction mediocre strains depends not only on alternative sources of nitrogen but also the relative importance of different components of rhizobial fitness.