Plant-Microbe Symbiosis

Although positive effects of arbuscular mycorrhizal (AM) fungi on plant performance under drought have been well documented, how AM fungi regulate soil functions and multifunctionality requires further investigation. In this study, we first performed a meta-analysis to test the potential role of AM fungi in maintaining soil functions under drought. Then, we conducted a greenhouse experiment, using a pair of hyphal ingrowth cores to spatially separate the growth of AM fungal hyphae and plant roots, to further investigate the effects of AM fungi on soil multifunctionality and its resistance against drought. Our meta-analysis showed that AM fungi promote multiple soil functions, including soil aggregation, microbial biomass and activities of soil enzymes related to nutrient cycling. The greenhouse experiment further demonstrated that AM fungi attenuate the negative impact of drought on these soil functions and thus multifunctionality, therefore, increasing their resistance against drought. Moreover, this buffering effect of AM fungi persists across different frequencies of water supply and plant species. These findings highlight the unique role of AM fungi in maintaining multiple soil functions by mitigating the negative impact of drought. Our study highlights the importance of AM fungi as a nature-based solution to sustaining multiple soil functions in a world where drought events are intensifying.

pon first glance, plant roots seem distinct from animal guts. However, like the intestines, roots are responsible for nutrient acquisition, they interact with robust microbial communities, and both are split into segments with different functions.1-3 As roots wind their way through the soil, they create an underground network responsible for anchoring a plant’s above-ground portion, absorbing water, and communicating with the environment. Different root segments, including the differentiation zone, elongation zone, and root tip, are heterogeneous in their architecture, gene expression, immune state, and metabolic profiles, all of which may influence the root microbiota. 

Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.

The necessity for sustainable agricultural practices has propelled a renewed interest in legumes such as faba bean (Vicia faba L.) as agents to help deliver increased diversity to cropped systems and provide an organic source of nitrogen (N). However, the increased cultivation of faba beans has proven recalcitrant worldwide as a result of low yields. So, it is hoped that increased and more stable yields would improve the commercial success of the crop and so the likelihood of cultivation. Enhancing biological N fixation (BNF) in faba beans holds promise not only to enhance and stabilize yields but also to increase residual N available to subsequent cereal crops grown on the same field. In this review, we cover recent progress in enhancing BNF in faba beans. Specifically, rhizobial inoculation and the optimization of fertilizer input and cropping systems have received the greatest attention in the literature. We also suggest directions for future research on the subject. In the short term, modification of crop management practices such as fertilizer and biochar input may offer the benefits of enhanced BNF. In the long term, natural variation in rhizobial strains and faba bean genotypes can be harnessed. Strategies must be optimized on a local scale to realize the greatest benefits. Future research must measure the most useful parameters and consider the economic cost of strategies alongside the advantages of enhanced BNF.

Biological nitrogen fixation requires substantial metabolic energy in form of ATP as well as low-potential electrons that must derive from central metabolism. During aerobic growth, the free-living soil diazotroph Azotobacter vinelandii transfers electrons from the key metabolite NADH to the low-potential ferredoxin FdxA that serves as a direct electron donor to the dinitrogenase reductases. This process is mediated by the RNF complex that exploits the proton motive force over the cytoplasmic membrane to lower the midpoint potential of the transferred electron. Here we report the cryogenic electron microscopy structure of the nitrogenase-associated RNF complex of A. vinelandii, a seven-subunit membrane protein assembly that contains four flavin cofactors and six iron–sulfur centers. Its function requires the strict coupling of electron and proton transfer but also involves major conformational changes within the assembly that can be traced with a combination of electron microscopy and modeling.

The Green Revolution transformed agriculture with high-yielding, stress-resistant varieties. However, the urgent need for more sustainable agricultural development presents new challenges: increasing crop yield, improving nutritional quality, and enhancing resource-use efficiency. Soil plays a vital role in crop-production systems and ecosystem services, providing water, nutrients, and physical anchorage for crop growth. Despite advancements in plant and soil sciences, our understanding of belowground plant–soil interactions, which impact both crop performance and soil health, remains limited. Here, we argue that a lack of understanding of these plant–soil interactions hinders sustainable crop production. We propose that targeted engineering of crops and soils can provide a fresh approach to achieve higher yields, more efficient sustainable crop production, and improved soil health.

Background and aims
Sinorhizobium fredii HH103 is a broad host-range rhizobial strain able to induce the formation of nitrogen-fixing nodules in dozens of legumes, including soybean. S. fredii HH103 exhibits genistein-induced surface motility. The aim of this work has been to determine whether the flgJ gene, which is inducible by genistein and codes for a flagellar protein, is involved in this motility and is relevant for symbiosis with soybean.

Methods
We have generated two independent mutants in the flgJ gene of HH103 and analysed their phenotypes in motility, exopolysaccharide production, biofilm formation, soybean root colonization, symbiosis with soybean, and secretion of effector proteins. We have also further studied the regulation of the expression of flgJ.

Results
We show that the expression of flgJ is driven by a tts box previously not detected, which accounts for its induction by flavonoids and the NodD1 and TtsI transcriptional activators. Inactivation of flgJ led to severe impairments in bacterial motility (swimming and genistein-induced surface motility) as well as to a significant reduction in symbiotic performance with soybean when bacteria are not directly inoculated onto the seedling roots. However, the absence of a functional FlgJ protein did not affect the bacterial ability to colonize soybean roots.

Conclusion
The flgJ gene of S. fredii HH103 connects the nod regulon with the genistein-induced surface motility exhibited by this rhizobial strain.

The apocarotenoid strigolactones (SLs) facilitate pre-symbiotic communication between arbuscular mycorrhizal (AM) fungi and plants. Related blumenol-C-glucosides (blumenols), have also been associated with symbiosis, but the cues that are involved in the regulation of blumenol accumulation during AM symbiosis remain unclear. In rice, our analyses demonstrated a strict correlation between foliar blumenol abundance and intraradical fungal colonisation. More specifically, rice mutants affected at distinct stages of the interaction revealed that fungal cortex invasion was required for foliar blumenol accumulation. Plant phosphate status and D14L hormone signalling had no effect, contrasting their known role in induction of SLs. This a proportion of the SL biosynthetic enzymes, D27 and D17, are equally required for blumenol production. These results importantly clarify that, while there is a partially shared biosynthetic pathway between SL and blumenols, the dedicated induction of the related apocarotenoids occurs in response to cues acting at distinct stages during the root colonisation process. However, we reveal that neither SLs nor blumenols are essential for fungal invasion of rice roots.

Bacteria have an extensive adaptive ability to live in close association with eukaryotic hosts, exhibiting detrimental, neutral or beneficial effects on host growth and health. However, the genes involved in niche adaptation are mostly unknown and their functions poorly characterized. Here, we present bacLIFE (https://github.com/Carrion-lab/bacLIFE) a streamlined computational workflow for genome annotation, large-scale comparative genomics, and prediction of lifestyle-associated genes (LAGs). As a proof of concept, we analyzed 16,846 genomes from the Burkholderia/Paraburkholderia and Pseudomonas genera, which led to the identification of hundreds of genes potentially associated with a plant pathogenic lifestyle. Site-directed mutagenesis of 14 of these predicted LAGs of unknown function, followed by plant bioassays, showed that 6 predicted LAGs are indeed involved in the phytopathogenic lifestyle of Burkholderia plantarii and Pseudomonas syringae pv. phaseolicola. These 6 LAGs encompassed a glycosyltransferase, extracellular binding proteins, homoserine dehydrogenases and hypothetical proteins. Collectively, our results highlight bacLIFE as an effective computational tool for prediction of LAGs and the generation of hypotheses for a better understanding of bacteria-host interactions.

Cyst nematodes are the most damaging species of plant-parasitic nematodes. They antagonize the colonization of beneficial microbial symbionts that are important for nutrient acquisition of plants. The molecular mechanism of the antagonism, however, remains elusive. Here, through biochemical combined with structural analysis, we reveal that Heterodera glycines, the most notorious soybean cyst nematode, suppresses symbiosis by secreting an enzyme named HgCht2 to hydrolyse the key symbiotic signalling molecules, lipochitooligosaccharides (LCOs). We solved the three-dimensional structures of apo HgCht2, as well as its chitooligosaccharide-bound and LCO-bound forms. These structures elucidated the substrate binding and hydrolysing mechanism of the enzyme. We designed an HgCht2 inhibitor, 1516b, which successfully suppresses the antagonism of cyst nematodes towards nitrogen-fixing rhizobia and phosphorus-absorbing arbuscular mycorrhizal symbioses. As HgCht2 is phylogenetically conserved across all cyst nematodes, our study revealed a molecular mechanism by which parasitic cyst nematodes antagonize the establishment of microbial symbiosis and provided a small-molecule solution.

Rates of molecular evolution vary greatly among even closely related species. Although theory predicts that antagonistic interactions between species increase rates of molecular evolution, predictions for how mutualism affects evolutionary rates are mixed. Here, we compared rates of molecular evolution between 1) mutualistic and non-mutualistic legumes, 2) an independent set of symbiotic rhizobia and their non-symbiotic close relatives, and 3) symbiotic and non-symbiotic clades within Ensifer, a diverse genus of bacteria with various lifestyles. We assembled transcriptomes de novo for 12 legume species and then calculated dN/dS ratios at orthologous genes in all species to determine if genes in mutualistic plants evolve faster or slower than in their non-mutualistic relatives. We also calculated dN/dS ratios in symbiosis genes known to be important for nodulation with rhizobia. We found that mutualists have higher rates of molecular evolution genome-wide compared to non-mutualist legumes. We next calculated dN/dS ratios in 14 bacteria species across the proteobacteria phylogeny that differ in whether they associate mutualistically with plants, using previously published data. We found that in most pairs, symbiotic rhizobia show higher dN/dS values compared to their non-symbiotic relatives. Finally, within a bacterial genus with many well-characterized mutualist species (Ensifer), we calculated dN/dS ratios in symbiotic and non-symbiotic clades and found that symbiotic lineages have higher rates of molecular evolution genome-wide, but not at genes on the symbiotic plasmid pSymB. Our results suggest that although mutualism between legumes and rhizobia is associated with elevated rates of molecular evolution genome-wide, symbiosis genes may be evolutionarily stagnant.

Plants have a remarkable ability to generate organs with a different identity to the parent organ, called ‘trans-organogenesis’. An example of trans-organogenesis is the formation of roots from stems (a type of adventitious root), which is the first type of root that arose during plant evolution. Despite being ancestral, stem-borne roots are often contextualised through lateral root research, implying that lateral roots precede adventitious roots. In this review we challenge that idea, highlight what is known about stem-borne root development across the plant kingdom, the remarkable diversity in form and function, and the many remaining evolutionary questions. Exploring stem-borne root evolutionary development can enhance our understanding of developmental decision making and the processes by which cells acquire their fates.

The transfer of nitrogen fixation (nif) genes from diazotrophs to non-diazotrophic hosts is of increasing interest for engineering biological nitrogen fixation. A recombinant Escherichia coli strain expressing Azotobacter vinelandii 18 nif genes (nifHDKBUSVQENXYWZMF, nifiscA, and nafU) were previously constructed and showed nitrogenase activity. In the present study, we constructed several E. coli strain derivatives in which all or some of the 18 nif genes were additionally integrated into the fliK locus of the chromosome in various combinations. E. coli derivatives with the chromosomal integration of nifiscA, nifU, and nifS, which are involved in the biosynthesis of the [4Fe–4S] cluster of dinitrogenase reductase, exhibited enhanced nitrogenase activity. We also revealed that overexpression of E. coli fldA and ydbK, which encode flavodoxin and flavodoxin-reducing enzyme, respectively, enhanced nitrogenase activity, likely by facilitating electron transfer to dinitrogenase reductase. The additional expression of nifM, putatively involved in maturation of dinitrogenase reductase, further enhanced nitrogenase activity and the amount of soluble NifH. By combining these factors, we successfully improved nitrogenase activity 10-fold.

Root development is controlled by local and systemic regulatory mechanisms that optimize mineral nutrient uptake and carbon allocation. The Autoregulation of Nodulation (AoN) pathway defines a negative regulation of nodule development in Legumes as a way to regulate the costly production of nitrogen-fixing organs. This pathway is defined as a response to symbiotic interaction and has been shown to also control root formation to some extent. However, it remains unclear if root and nodule development are under coordinated genetic regulation. Here, we identified mutants with altered root development in white lupin, constitutively producing specialized lateral roots called cluster roots. We showed that the CCR1 receptor-kinase negatively regulates cluster root and nodule development and targets common molecular modules such as NIN/LBD16-NFYA, defining a novel pathway that we named Autoregulation of Development (AoDev). AoDev defines a negative systemic pathway controlling several types of root organ development, independently of symbiotic partners and nutrient availability.

Plants rely on strigolactones (SLs) to regulate their development and form symbiotic relationships with microbes as part of the adaptive phosphorus (P) efficiency strategies. However, the impact of SLs on root-associated microbial communities in response to P availability remains unknown. Here, root microbiota of SL biosynthesis (max3-11) and perception (d14-1) were compared to wild-type Col-0 plants under different P concentrations. Using high-throughput sequencing, the relationship between SLs, P concentrations, and the root-associated microbiota was investigated to reveal the variation in microbial diversity, composition, and interaction. Plant genotypes and P availability played important but different roles in shaping the root-associated microbial community. Importantly, SLs were found to attract Acinetobacter in low P conditions, which included an isolated CP-2 (Acinetobacter soli) that could promote plant growth in cocultivation experiments. Moreover, SLs could change the topologic structure within co-occurrence networks and increase the number of keystone taxa (e.g., Rhizobiaceae and Acidobacteriaceae) to enhance microbial community stability. This study reveals the key role of SLs in mediating root-associated microbiota interactions.

Non-symbiotic N2-fixation would greatly increase the versatility of N-biofertilizers for sustainable agriculture. Genetic modification of diazotrophic bacteria has successfully enhanced NH4+ release. In this study, we compared the competitive fitness of A. vinelandii mutant strains, which allowed us to analyze the burden of NH4+ release under a broad dynamic range. Long-term competition assays under regular culture conditions confirmed a large burden for NH4+ release, exclusion by the wt strain, phenotypic instability, and loss of the ability to release NH4+. In contrast, co-inoculation in mild autoclaved soil showed a much longer co-existence with the wt strain and a stable NH4+ release phenotype. All genetically modified strains increased the N content and changed its chemical speciation in the soil. This study contributes one step forward towards bridging a knowledge gap between molecular biology laboratory research and the incorporation of N from the air into the soil in a molecular species suitable for plant nutrition, a crucial requirement for developing improved bacterial inoculants for economic and environmentally sustainable agriculture.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialised polyploid cells within root nodules. This results in a mutualistic relationship whereby the plant host receives fixed nitrogen from the bacteria in exchange for dicarboxylic acids. This plant-microbe interaction requires the regulation of multiple metabolic and physiological processes in both the host and symbiont in order to achieve highly efficient symbiosis. Recent studies have showed that the success of symbiosis is influenced by the circadian clock of the plant host. Medicago and soybean plants with altered clock mechanisms showed compromised nodulation and reduced plant growth. Furthermore, transcriptomic analyses revealed that multiple genes with key roles in recruitment of rhizobia to plant roots, infection and nodule development were under circadian control, suggesting that appropriate timing of expression of these genes may be important for nodulation. There is also evidence for rhythmic gene expression of key nitrogen fixation genes in the rhizobium symbiont, and temporal coordination between nitrogen fixation in the bacterial symbiont and nitrogen assimilation in the plant host may be important for successful symbiosis. Understanding of how circadian regulation impacts on nodule establishment and function will identify key plant-rhizobial connections and regulators that could be targeted to increase the efficiency of this relationship.

The introduction of Reduced height (Rht) dwarfing genes into elite wheat varieties has contributed to enhanced yield gain in high input agrosystems by preventing lodging. Yet, how modern selection for dwarfing has affected symbiosis remains poorly documented. In this study, we evaluated the response of both the plant and the arbuscular mycorrhizal fungus to plant genetic variation at a major Quantitative Trait Locus called QTL 4B2, known to harbor a Rht dwarfing gene, when forming the symbiosis. We used twelve inbred genotypes derived from a diversity base broadened durum wheat Evolutionary Pre-breeding Population and genotyped with a high-throughput Single Nucleotide Polymorphism (SNP) genotyping array. In a microcosm setup segregating roots and the extra-radical mycelium, each wheat genotype was grown with or without the presence of Rhizophagus irregularis. To characterize arbuscular mycorrhizal symbiosis, we assessed hyphal density, root colonization, spore production, and plant biomass. Additionally, we split the variation of these variables due either to genotypes or to the Rht dwarfing genes alone. The fungus exhibited greater development in the roots of Dwarf plants compared to non-Dwarf plants, showing increases of 27%, 37% and 51% in root colonization, arbuscules, and vesicles, respectively. In addition, the biomass of the extra-radical fungal structures increased by around 31% in Dwarf plants. The biomass of plant roots decreased by about 43% in mycorrhizal Dwarf plants. Interestingly, extraradical hyphal production was found to be partly genetically determined with no significant effect of Rht, as for plant biomasses. In contrast, variations in root colonization, arbuscules and extraradical spore production were explained by Rht dwarfing genes. Finally, when mycorrhizal, Dwarf plants had significantly lower total P content, pointing towards a less beneficial symbiosis for the plant and increased profit for the fungus. These results highlight the effect of Rht dwarfing genes on both root and fungal development. This calls for further research into the molecular mechanisms governing these effects, as well as changes in plant physiology, and their implications for fostering arbuscular mycorrhizal symbiosis in sustainable

Differences in functioning among various genotypes of arbuscular mycorrhizal (AM) fungi can determine their fitness under specific environmental conditions, although knowledge of the underlying mechanisms still is very fragmented. Here we compared seven homokaryotic isolates (genotypes) of Rhizophagus irregularis, aiming to characterize the range of intraspecific variability with respect to hyphal exploration of organic nitrogen (N) resources, and N supply to plants. To this end we established two experiments (one in vitro and one in open pots) and used 15N-chitin as the isotopically labeled organic N source. In Experiment 1 (in vitro), mycelium of all AM fungal genotypes transferred a higher amount of 15N to the plants than the passive transfer of 15N measured in the non-mycorrhizal (NM) controls. Noticeably, certain genotypes (e.g., LPA9) showed higher extraradical mycelium biomass production but not necessarily greater 15N acquisition than the others. Experiment 2 (in pots) highlighted that some of the AM fungal genotypes (e.g., MA2, STSI) exhibited higher rates of targeted hyphal exploration of chitin-enriched zones, indicative of distinct N exploration patterns from the other genotypes. Importantly, there was a high congruence of hyphal exploration patterns between the two experiments (isolate STSI always showing highest efficiency of hyphal exploration and isolate L23/1 being consistently the lowest), despite very different (micro) environmental conditions in the two experiments. This study suggests possible strategies that AM fungal genotypes employ for efficient N acquisition, and how to measure them. Implications of such traits for local mycorrhizal community assembly still need to be understood.

Life depends on a conserved set of chemical energy currencies that are relics of early biochemistry. One of these is ATP, a molecule that, when paired with a divalent metal ion such as Mg2+, can be hydrolyzed to support numerous cellular and molecular processes. Despite its centrality to extant biochemistry, it is unclear whether ATP supported the function of ancient enzymes. We investigate the evolutionary necessity of ATP by experimentally reconstructing an ancestral variant of the N2-reducing enzyme nitrogenase. The Proterozoic ancestor is predicted to be ~540–2,300 million years old, post-dating the Great Oxidation Event. Growth rates under nitrogen-fixing conditions are ~80% of those of wild type in Azotobacter vinelandii. In the extant enzyme, the hydrolysis of two MgATP is coupled to electron transfer to support substrate reduction. The ancestor has a strict requirement for ATP with no other nucleotide triphosphate analogs (GTP, ITP, and UTP) supporting activity. Alternative divalent metal ions (Fe2+, Co2+, and Mn2+) support activity with ATP but with diminished activities compared to Mg2+, similar to the extant enzyme. Additionally, it is shown that the ancestor has an identical efficiency in ATP hydrolyzed per electron transferred to the extant of two. Our results provide direct laboratory evidence of ATP usage by an ancient enzyme.