Plant-Microbe Symbiosis

Les sols sont le support de la vie terrestre et le substrat de la végétation. Ce sont des écosystèmes complexes et fragiles qui contribuent à la qualité de notre environnement. Leur étude est, par essence, pluridisciplinaire et se situe au carrefour de la géologie, de la physique, de la chimie, de la biologie, de l'agriculture et de la climatologie. Leurs caractéristiques physicochimiques et biologiques conditionnent la nature de la végétation, la qualité et le rendement des cultures. Les pratiques de l'agriculture intensive et industrielle les appauvrissent considérablement dans nombre de régions du globe, y compris dans notre pays et il convient d'en prendre conscience et de tenter d'y remédier. Les sols contribuent aussi au stockage et au piégeage du gaz carbonique, au travers de la minéralisation de la matière organique, et sont donc un puits de carbone, mais ils peuvent aussi, dans certaines conditions, en libérer et devenir une source supplémentaire de ce gaz à effet de serre. Leur gestion est donc un facteur important à maitriser dans les efforts pour atténuer le changement climatique .

L'objectif de cette séance, commune avec l'Académie d'agriculture de France est de faire un point des connaissances sur quelques aspects de la science des sols et sur les enjeux qui s'y rattachent.

Nodulation is the most efficient nitrate assimilation system in the ecosystem, while excessive fertilization has an increased nitrate inhibition effect; deciphering the nitrate signal transduction mechanism in the process is of the utmost importance. In this study, genome-wide analyses of the GmCEP genes were applied to identify nodulation-related CEP genes; 22 GmCEP family members were identified, while GmCEP6 was mainly expressed in nodules and significantly responded to nitrate treatment and rhizobium infection, especially in later stages. Overexpression and CRISPR-Cas9 were used to validate its role in nodulation. We found that GmCEP6 overexpression significantly increased the nodule number, while GmCEP6 knock-out significantly decreased the nodule number, which suggests that GmCEP6 functions as a positive regulator in soybean nodulation. qRT-PCR showed that alterations in the expression of GmCEP6 affected the expression of marker genes in the Nod factor signaling pathway. Lastly, the function of GmCEP6 in nitrate inhibition of nodulation was analyzed; nodule numbers in the GmCEP6-overexpressed roots significantly increased under nitrogen treatments, which suggests that GmCEP6 functions in the resistance to nitrate inhibition. The study helps us understand that GmCEP6 promotes nodulation and participates in the regulation of nitrate inhibition of nodulation, which is of great significance for high efficiency utilization of nitrogen in soybeans.

Nitrogen (N) is a crucial nutrient for the growth and activity of rhizosphere microorganisms, particularly during drought conditions. Plant root-secreted mucilage contains N that could potentially nourish rhizosphere microbial communities. However, there remains a significant gap in understanding mucilage N content, its source, and its utilization by microorganisms under drought stress. In this study, we investigated the impact of four maize varieties (DH02 and DH04 from Kenya, and Kentos and Keops from Germany) on the secretion rates of mucilage from aerial roots and explored the origin of mucilage N supporting microbial life in the rhizosphere. We found that DH02 exhibited a 96% higher mucilage secretion rate compared to Kentos, while Keops showed 114% and 89% higher secretion rates compared to Kentos and DH04, respectively. On average, the four maize varieties released 4 μg N per root tip per day, representing 2% of total mucilage secretion. Notably, the natural abundance of 15N isotopes increased (higher δ15N signature) with mucilage N release. This indicates a potential dilution of the isotopic signal from biological fixation of atmospheric N by mucilage-inhabiting bacteria as mucilage secretion rates increase. We proposed a model linking mucilage secretion to a mixture of isotopic signatures and estimated that biological N fixation may contribute to 45 - 75% of mucilage N per root tip. The N content of mucilage from a single maize root tip can support a bacterial population ranging from 107 to 1010 cells per day. In conclusion, mucilage serves as a significant N-rich resource for microbial communities in the rhizosphere during drought conditions.

Here, we used in vitro and pot cultivation systems of AM fungi to investigate whether certain keystone bacteria were able to shape the microbial communities growing in the hyphosphere and potentially improved the fitness of the AM fungal host. Based on various AM fungi, soil leachates, and synthetic microbial communities, we found that under organic phosphorus (P) conditions, AM fungi could selectively recruit bacteria that enhanced their P nutrition and competed with less P-mobilizing bacteria. Specifically, we observed a privileged interaction between the isolate Streptomyces sp. D1 and AM fungi of the genus Rhizophagus, where (1) the carbon compounds exuded by the fungus were acquired by the bacterium which could mineralize organic P and (2) the in vitro culturable bacterial community residing on the surface of hyphae was in part regulated by Streptomyces sp. D1, primarily by inhibiting the bacteria with weak P-mineralizing ability, thereby enhancing AM fungi to acquire P.

In the relentless pursuit of sustainable agricultural practices, society has pivoted its gaze towards alternatives to synthetic chemical fertilizers, recognizing the significant environmental impact they impose. Among the myriad of alternatives, the use of plant growth-promoting bacteria (PGPB) has emerged as a promising solution, encouraging potential to revolutionize plant nutrition in a manner that is both effective and environmentally sustainable. The interaction between plants and PGPB is a wonder of nature, encompassing a wide array of interactions that extend far beyond simple nutrient provision. These remarkable microorganisms, through their ability to harness unavailable nutrients and synthesize essential phytohormones, exert a profound influence on plant metabolism, enhancing growth and resilience even in challenging conditions.

Although plants are sessile, their ubiquitous distribution, ability to harness energy from the sun, and ability to sense above and belowground signals make them ideal candidates for biosensor development. Synthetic biology has allowed scientists to reimagine biosensors as engineered devices that are focused on accomplishing novel tasks. As such, a new wave of plant-based sensors, phytosensors, are being engineered as multi-component sense-and-report devices that can alert human operators to a variety of hazards. While phytosensors are intrinsically tied to agriculture, a new generation of phytosensors has been envisioned to function in the built environment and even in austere environments, such as space. In this review, we will explore the current state of the art with regard to phytosensor engineering.

•The quantity and activity of nutrient acquisition agents, e.g., roots, rhizobium and mycorrhizal fungi, are important for plant growth. How the invasive plants and the native legume adjust the quantity and activity of these agents in competing for soil resources remains unclear.

•The invasive plant, Xanthium strumarium, did not directly scavenge biologically fixed nitrogen (N) of a common native legume, Glycine max, but rather depleted rhizosphere soil N of the legume.

•The native legume increased its dependence on N-fixation in competition with the invasive plant, which was due to promotion of nodule activity in N-fixing by the invasive plant as well as inhibition of legume root quantity and activity.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH or Gap) is a ubiquitous enzyme essential for carbon and energy metabolism in most organisms. Despite its primary role in sugar metabolism, GAPDH is recognized for its involvement in diverse cellular processes, being considered a paradigm among multifunctional/moonlighting proteins. Besides its canonical cytoplasmic location, GAPDH has been detected on cell surfaces or as a secreted protein in prokaryotes, yet little is known about its possible roles in plant symbiotic bacteria. Here we report that Rhizobium etli, a nitrogen-fixing symbiont of common beans, carries a single gap gene responsible for both GAPDH glycolytic and gluconeogenic activities. An active Gap protein is required throughout all stages of the symbiosis between R. etli and its host plant Phaseolus vulgaris. Both glycolytic and gluconeogenic Gap metabolic activities likely contribute to bacterial fitness during early and intermediate stages of the interaction, whereas GAPDH gluconeogenic activity seems critical for nodule invasion and nitrogen fixation. Although the R. etli Gap protein is secreted in a c-di-GMP related manner, no involvement of the R. etli gap gene in c-di-GMP related phenotypes, such as flocculation, biofilm formation or EPS production, was observed. Notably, the R. etli gap gene fully complemented a double gap1/gap2 mutant of Pseudomonas syringae for free life growth, albeit only partially in planta, suggesting potential specific roles for each type of Gap protein. Nevertheless, further research is required to unravel additional functions of the R. etli Gap protein beyond its essential metabolic roles.

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonia, they have great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have sufficient genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants, or symbiotic association with nonlegumes.

Although the molecular mechanism underlying IT development remains obscure, several host players have been characterized, including VAPYRIN (VPY). The vpy mutant shows delayed initiation of ITs, often resulting in oversized MCs (Murray et al., 2011; Liu et al., 2019). VPY is also required for arbuscule development in arbuscular mycorrhiza (AM) symbiosis (Feddermann et al., 2010; Pumplin et al., 2010; Murray et al., 2011; Zhang et al., 2015; Bapaume et al., 2019; Chen et al., 2021). VPY encodes a plant-specific protein with a major sperm protein (MSP) domain and several ankyrin repeats, both of which are protein–protein interaction domains (Tarr & Scott, 2005; Li et al., 2006). In the context of nodulation, VPY interacts with LIN, a protein also essential for IT development, via its ankyrin repeats region, and is positively regulated by LIN, likely by stabilizing VPY protein by so far unknown mechanisms, in both M. truncatula and L. japonicus (Kuppusamy et al., 2004; Kiss et al., 2009; Yano et al., 2009; Liu et al., 2019; Liu et al., 2021). VPY and LIN form an ‘infectosome’ protein complex with RHIZOBIUM-DIRECTED POLAR GROWTH (RPG) and an exocyst subunit EXO70H4, regulating IT development, likely via polarized exocytosis (Arrighi et al., 2008; Liu et al., 2019; Roy et al., 2020; Wang et al., 2022; Jhu & Oldroyd, 2023; Lace et al., 2023; Li et al., 2023). The core infectosome components, VPY, LIN and RPG accumulate at the extending IT tip and form one or a few cytoplasmic puncta near the nucleus (Liu et al., 2019; Lace et al., 2023; Li et al., 2023). The functional significance and mechanism underlying the interaction of these infectosome components remain to be elucidated.

Here, we identify a single amino acid in VPY, which is essential for its role in rhizobial infection and its interaction with LIN, and determines its subcellular localization pattern.

Root-associated microbiota profoundly affect crop health and productivity. Plants can selectively recruit beneficial microbes from the soil and actively balance microbe-triggered plant-growth promotion and stress tolerance enhancement. The cost associated with this is the root-mediated support of a certain number of specific microbes under nutrient limitation. Thus, it is important to consider the dynamic changes in microbial quantity when it comes to nutrient condition-induced root microbiome reassembly. Quantitative microbiome profiling (QMP) has recently emerged as a means to estimate the specific microbial load variation of a root microbiome (instead of the traditional approach quantifying relative microbial abundances) and data from the QMP approach can be more closely correlated with plant development and/or function. However, due to a lack of detailed-QMP data, how soil nutrient conditions affect quantitative changes in microbial assembly of the root-associated microbiome remains poorly understood. A recent study quantified the dynamics of the soybean root microbiome, under unbalanced fertilization, using QMP and provided data on the use of specific synthetic communities (SynComs) for sustaining crop productivity. In this editorial, we explore potential opportunities for utilizing QMP to decode the microbiome for sustainable agriculture.

Taxonomic identification of arbuscular mycorrhizal (AM) fungal spores extracted directly from the field is sometimes difficult because spores are often degraded or parasitized by other organisms. Single-spore inoculation of a suitable host plant allows for establishing monosporic cultures of AM fungi. This study aimed to propagate AM fungal spores isolated from maize soil using single spores for morphological characterization. First, trap cultures were established to trigger the sporulation of AM fungal species. Second, trap cultures were established with individual morphotypes by picking up only one spore under a dissecting microscope and transferring it to a small triangle of sterilized filter paper, which was then carefully inoculated below a root from germinated sorghum seeds in each pot and covered with a sterile substrate. All pots were placed in sunbags and maintained in a plant growth room for 120 days. Spores obtained from single spore trap cultures from each treatment, maize after oats (MO), maize after maize (MM), maize after peas (MP), and maize after soybean (MS), were extracted using the sieving method. Healthy spores were selected for morphological analysis. Direct PCR was conducted by crushing spores in RNAlater and applying three sets of primer pairs: ITS1 × ITS4, NS31 × AML2, and SSUmcf and LSUmBr. Nucleotide sequences obtained from Sanger sequencing were aligned on MEGA X. The phylogenetic tree showed that the closest neighbors of the propagated AM fungal species belonged to the genera Claroideoglomus, Funneliformis, Gigaspora, Paraglomus, and Rhizophagus. The morphological characteristics were compared to the descriptive features of described species posted on the INVAM website, and they included Acaulospora cavernata, Diversispora spurca, Funneliformis geosporus, Funneliformis mosseae, Gigaspora clarus, Gigaspora margarita, Glomus macrosporum, Paraglomus occultum, and Rhizophagus intraradices. These findings can provide a great contribution to crop productivity and sustainable management of the agricultural ecosystem. Also, the isolate analyzed could be grouped into efficient promoters of growth and mycorrhization of maize independent of their geographical location.

Symbiotic nitrogen fixation in legume nodules requires substantial energy investment from host plants, and soybean (Glycine max (L.) supernodulation mutants show stunting and yield penalties due to overconsumption of carbon sources. We obtained soybean mutants differing in their nodulation ability, among which rhizobially induced cle1a/2a (ric1a/2a) has a moderate increase in nodule number, balanced carbon allocation, and enhanced carbon and nitrogen acquisition. In multi-year and multi-site field trials in China, two ric1a/2a lines had improved grain yield, protein content and sustained oil content, demonstrating that gene editing towards optimal nodulation improves soybean yield and quality.

The fight against biopiracy -- plundering genetic resources and the traditional knowledge surrounding them -- could soon be based on an international treaty which is being finalised at negotiations that began on Monday.

Background
After two decades of extensive microbiome research, the current forefront of scientific exploration involves moving beyond description and classification to uncovering the intricate mechanisms underlying the coalescence of microbial communities. Deciphering microbiome assembly has been technically challenging due to their vast microbial diversity but establishing a synthetic community (SynCom) serves as a key strategy in unravelling this process. Achieving absolute quantification is crucial for establishing causality in assembly dynamics. However, existing approaches are primarily designed to differentiate a specific group of microorganisms within a particular SynCom.

Results
To address this issue, we have developed the differential fluorescent marking (DFM) strategy, employing three distinguishable fluorescent proteins in single and double combinations. Building on the mini-Tn7 transposon, DFM capitalises on enhanced stability and broad applicability across diverse Proteobacteria species. The various DFM constructions are built using the pTn7-SCOUT plasmid family, enabling modular assembly, and facilitating the interchangeability of expression and antibiotic cassettes in a single reaction. DFM has no detrimental effects on fitness or community assembly dynamics, and through the application of flow cytometry, we successfully differentiated, quantified, and tracked a diverse six-member SynCom under various complex conditions like root rhizosphere showing a different colonisation assembly dynamic between pea and barley roots.

Conclusions
DFM represents a powerful resource that eliminates dependence on sequencing and/or culturing, thereby opening new avenues for studying microbiome assembly.

A trait is an attribute influencing an organism’s performance within its environment, encompassing morphological, genetic and physiological characteristics measured at the individual or population levels (Salguero-Gómez et al. , 2018; Zhang et al. , 2023a). Understanding the ecology of species using a trait-based approach can contribute to a mechanistic explanation of processes mediated by microbes, including those that affect ecosystem functioning (Romero-Olivares et al. , 2021). This approach holds particular significance for arbuscular mycorrhizal (AM) fungi - Phylum Glomeromycota. As obligate symbionts of plants, where multiple species colonize both roots and soils in a network, predicting the functional outcomes (e.g., host growth, plant community diversity, soil characteristics) of individual AM fungal genotypes and communities within ecosystems remains challenging, despite major developments in molecular methods in the last two decades (Tisserant et al. , 2013; Montoliu-Nerin et al. , 2021). Indeed, establishing connections between AM fungal taxa and/or genotypes (e.g., within species) and their functional roles is a laborious process, which is expected to continue in the foreseeable future (Serghi et al. , 2021; Manley et al. , 2023). This is needed due to the complex links between AM fungi and functional outcomes for both hosts (e.g. , plant growth and fitness, nutrient uptake and stress tolerance) and soil functions/properties (e.g. , carbon storage, aggregate stability, biotic diversity), which appear to be highly context dependent and relatively poorly predicted by taxonomy alone (Munkvold et al. , 2004; Koch et al. , 2017; Yang et al. , 2017; Qiu et al. , 2021). However, this effort is also required because AM fungal traits have not been systematically assessed alongside with hypotheses of adaptation or with specific mechanisms in mind. For example, small-spored AM fungi may be dispersed longer distances by wind than large-spored AM fungi. It is then a reasonable hypothesis that small spore size is an adaptation for wind dispersal. One could empirically observe that small-spored AM fungi are geographically more widespread than large-spored fungi and this potential result could be viewed as evidence in support of this hypothesis. However, this finding would not necessarily prove that such dispersal difference has “functional” or “adaptive” value. Alternatively, producing small spores is a correlated response to producing many spores quickly, which itself could be an adaptive response to the likelihood of unpredictable soil disturbance such as from tillage. In this scenario, the adaptation and/or function is the production of many spores quickly to confer resistance to disturbance and then, after soil disturbance with wind erosion, small spores may also be blown farther (which may or may not improve fitness). Another example, variation in rooting depth among plants in a community may contribute to resource partitioning. But the mechanism (differential resource depletion with depth) still needs to be demonstrated separately from the trait evidence. AM fungi could contribute to equalize resource partitioning if plants with short roots associate with AM fungi that form more extensive extra-radical mycelium and vice-versa. Given these complexities, we consider the development of a robust, universally applicable trait-based framework for predicting key AM fungal functional outcomes a priority. To achieve this objective, first we must identify AM fungal traits that can be measured at morphological, physiological, and genetic levels. Second, considering the important roles of AM fungi in ecosystems, affecting host plants, soil, and the AM fungi themselves, we need to discern/hypothesize how measuring AM fungal traits impacts each of these components. For the host plant, it is crucial to consider nutrition, biomass, fitness, and survival in face of pathogens, heavy metals, salinity, drought, etc. (Delavaux et al. , 2017; Wehneret al. ). Within the soil environment, AM fungal effects on soil structure (Rillig & Mummey, 2006), nutrient cycling, carbon storage, and other members of the soil food-web are paramount (Antunes & Koyama, 2016; Frew et al. , 2021; Horsch et al. , 2023a). Regarding the fungal organism, we should focus on key aspects of their life-history strategies; reproduction and fitness, survival, dispersal, competitive ability, infectivity and abundance both within the host and soil environments (Aguilar-Trigueros et al. , 2019; Chaudharyet al. , 2020; Deveautour et al. , 2020). This requires identifying relevant proxies (sometimes termed “soft traits” in the plant ecophysiology literature) to provide easy-to-measure quantitative metrics for such complex facets of fungal life-history that can be measured across several species. Third, we need to evaluate existing standardized methods and experimental designs, or develop new ones, to measure such relevant (soft) traits, as has been done in plant ecophysiology (Pérez-Harguindeguy et al. , 2013). Measurement standardization and relevant metadata for hypothesis-driven analysis and interpretation is essential if we are to aggregate trait information from different studies into a public database, facilitating their incorporation into earth system models (e.g., (Fry et al. , 2019) and enhancing the predictability of functional processes and/or adaptations associated with AM fungi. Analogous libraries on plant traits (Kattge et al. , 2020) have proved useful to better understand trait variation along global climatic gradients (Butleret al. , 2017). Here, we aim to:

Molecular innovations within key metabolisms can have profound impacts on element cycling and ecological distribution. Yet, much of the molecular foundations of early evolved enzymes and metabolisms are unknown. Here, we bring one such mystery to relief by probing the birth and evolution of the G-subunit protein, an integral component of certain members of the nitrogenase family, the only enzymes capable of biological nitrogen fixation. The G-subunit is a Paleoproterozoic-age orphan protein that appears more than 1 billion years after the origin of nitrogenases. We show that the G-subunit arose with novel nitrogenase metal dependence and the ecological expansion of nitrogen-fixing microbes following the transition in environmental metal availabilities and atmospheric oxygenation that began ∼2.5 billion years ago. We identify molecular features that suggest early G-subunit proteins mediated cofactor or protein interactions required for novel metal dependency, priming ancient nitrogenases and their hosts to exploit these newly diversified geochemical environments. We further examined the degree of functional specialization in G-subunit evolution with extant and ancestral homologs using laboratory reconstruction experiments. Our results indicate that permanent recruitment of the orphan protein depended on the prior establishment of conserved molecular features and showcase how contingent evolutionary novelties might shape ecologically important microbial innovations.