Single-cell RNA sequencing facilitates the elucidation of the complete biosynthesis of the antidepressant hyperforin in St. John's wort.

Hyperforin is the compound responsible for the effectiveness of St. John's wort (Hypericum perforatum) as an antidepressant, but its complete biosynthetic pathway remains unknown. Gene discovery based on co-expression analysis of bulk RNA-sequencing data or genome mining failed to discover the missing steps in hyperforin biosynthesis. In this study, we sequenced the 1.54-Gb tetraploid H. perforatum genome assembled into 32 chromosomes with the scaffold N50 value of 42.44 Mb. By single-cell RNA sequencing, we identified a type of cell, "Hyper cells", wherein hyperforin biosynthesis de novo takes place in both the leaves and flowers. Through pathway reconstitution in yeast and tobacco, we identified and characterized four transmembrane prenyltransferases (HpPT1-4) that are localized at the plastid envelope and complete the hyperforin biosynthetic pathway. The hyperforin polycyclic scaffold is created by a reaction cascade involving an irregular isoprenoid coupling and a tandem cyclization. Our findings reveal how and where hyperforin is biosynthesized, enabling synthetic-biology reconstitution of the complete pathway. Thus, this study not only deepens our comprehension of specialized metabolism at the cellular level but also provides strategic guidance for elucidation of the biosynthetic pathways of other specializied metabolites in plants.

Mapping the molecular landscape of Lotus japonicus nodule organogenesis through spatiotemporal transcriptomics.

Legumes acquire nitrogen-fixing ability by forming root nodules. Transferring this capability to more crops could reduce our reliance on nitrogen fertilizers, thereby decreasing environmental pollution and agricultural production costs. Nodule organogenesis is complex, and a comprehensive transcriptomic atlas is crucial for understanding the underlying molecular events. Here, we utilized spatial transcriptomics to investigate the development of nodules in the model legume, Lotus japonicus. Our investigation has identified the developmental trajectories of two critical regions within the nodule: the infection zone and peripheral tissues. We reveal the underlying biological processes and provide gene sets to achieve symbiosis and material exchange, two essential aspects of nodulation. Among the candidate regulatory genes, we illustrate that LjNLP3, a transcription factor belonging to the NIN-LIKE PROTEIN family, orchestrates the transition of nodules from the differentiation to maturation. In summary, our research advances our understanding of nodule organogenesis and provides valuable data for developing symbiotic nitrogen-fixing crops.

Release of a ubiquitin brake activates OsCERK1-triggered immunity in rice.

Plant pattern-recognition receptors perceive microorganism-associated molecular patterns to activate immune signalling1,2. Activation of the pattern-recognition receptor kinase CERK1 is essential for immunity, but tight inhibition of receptor kinases in the absence of pathogen is crucial to prevent autoimmunity3,4. Here we find that the U-box ubiquitin E3 ligase OsCIE1 acts as a molecular brake to inhibit OsCERK1 in rice. During homeostasis, OsCIE1 ubiquitinates OsCERK1, reducing its kinase activity. In the presence of the microorganism-associated molecular pattern chitin, active OsCERK1 phosphorylates OsCIE1 and blocks its E3 ligase activity, thus releasing the brake and promoting immunity. Phosphorylation of a serine within the U-box of OsCIE1 prevents its interaction with E2 ubiquitin-conjugating enzymes and serves as a phosphorylation switch. This phosphorylation site is conserved in E3 ligases from plants to animals. Our work identifies a ligand-released brake that enables dynamic immune regulation.

Genetically optimizing soybean nodulation improves yield and protein content.

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.

Spatially distributed metabolites SWEETen the root for microbes.

Limited understanding exists on the spatial configuration of underground plant-microbe interactions. In this issue of Cell Host & Microbe, Loo et al. illustrate the sugar transporter-involved interdependent interaction between root metabolites and microbial spatial colonization, providing insights into metabolic-associated organization of plant-microbe interactions.

Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification.

Biological nitrogen fixation by free-living bacteria and rhizobial symbiosis with legumes plays a key role in sustainable crop production. Here, we study how different crop combinations influence the interaction between peanut plants and their rhizosphere microbiota via metabolite deposition and functional responses of free-living and symbiotic nitrogen-fixing bacteria. Based on a long-term (8 year) diversified cropping field experiment, we find that peanut co-cultured with maize and oilseed rape lead to specific changes in peanut rhizosphere metabolite profiles and bacterial functions and nodulation. Flavonoids and coumarins accumulate due to the activation of phenylpropanoid biosynthesis pathways in peanuts. These changes enhance the growth and nitrogen fixation activity of free-living bacterial isolates, and root nodulation by symbiotic Bradyrhizobium isolates. Peanut plant root metabolites interact with Bradyrhizobium isolates contributing to initiate nodulation. Our findings demonstrate that tailored intercropping could be used to improve soil nitrogen availability through changes in the rhizosphere microbiome and its functions.

Dynamic root microbiome sustains soybean productivity under unbalanced fertilization.

Root-associated microbiomes contribute to plant growth and health, and are dynamically affected by plant development and changes in the soil environment. However, how different fertilizer regimes affect quantitative changes in microbial assembly to effect plant growth remains obscure. Here, we explore the temporal dynamics of the root-associated bacteria of soybean using quantitative microbiome profiling (QMP) to examine its response to unbalanced fertilizer treatments (i.e., lacking either N, P or K) and its role in sustaining plant growth after four decades of unbalanced fertilization. We show that the root-associated bacteria exhibit strong succession during plant development, and bacterial loads largely increase at later stages, particularly for Bacteroidetes. Unbalanced fertilization has a significant effect on the assembly of the soybean rhizosphere bacteria, and in the absence of N fertilizer the bacterial community diverges from that of fertilized plants, while lacking P fertilizer impedes the total load and turnover of rhizosphere bacteria. Importantly, a SynCom derived from the low-nitrogen-enriched cluster is capable of stimulating plant growth, corresponding with the stabilized soybean productivity in the absence of N fertilizer. These findings provide new insights in the quantitative dynamics of the root-associated microbiome and highlight a key ecological cluster with prospects for sustainable agricultural management.

Phosphorus/nitrogen sensing and signaling in diverse root-fungus symbioses.

Establishing mutualistic relationships between plants and fungi is crucial for overcoming nutrient deficiencies in plants. This review highlights the intricate nutrient sensing and uptake mechanisms used by plants in response to phosphate and nitrogen starvation, as well as their interactions with plant immunity. The coordination of transport systems in both host plants and fungal partners ensures efficient nutrient uptake and assimilation, contributing to the long-term maintenance of these mutualistic associations. It is also essential to understand the distinct responses of fungal partners to external nutrient levels and forms, as they significantly impact the outcomes of symbiotic interactions. Our review also highlights the importance of evolutionarily younger and newly discovered root-fungus associations, such as endophytic associations, which offer potential benefits for improving plant nutrition. Mechanistic insights into the complex dynamics of phosphorus and nitrogen sensing within diverse root-fungus associations can facilitate the identification of molecular targets for engineering symbiotic systems and developing plant phenotypes with enhanced nutrient use efficiency. Ultimately, this knowledge can inform tailored fertilizer management practices to optimize plant nutrition.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Root microbiota confers rice resistance to aluminium toxicity and phosphorus deficiency in acidic soils.

Aluminium (Al) toxicity impedes crop growth in acidic soils and is considered the second largest abiotic stress after drought for crops worldwide. Despite remarkable progress in understanding Al resistance in plants, it is still unknown whether and how the soil microbiota confers Al resistance to crops. Here we found that a synthetic community composed of highly Al-resistant bacterial strains isolated from the rice rhizosphere increased rice yield by 26.36% in acidic fields. The synthetic community harvested rhizodeposited carbon for successful proliferation and mitigated soil acidification and Al toxicity through extracellular protonation. The functional coordination between plants and microbes offers a promising way to increase the usage of legacy phosphorus in topsoil. These findings highlight the potential of microbial tools for advancing sustainable agriculture in acidic soils.

Low phosphorus promotes NSP1-NSP2 heterodimerization to enhance strigolactone biosynthesis and regulate shoot and root architecture in rice.

Phosphorus is an essential macronutrient for plant development and metabolism, and plants have evolved ingenious mechanisms to overcome phosphate (Pi) starvation. However, the molecular mechanisms underlying the regulation of shoot and root architecture by low phosphorus conditions and the coordinated utilization of Pi and nitrogen remain largely unclear. Here, we show that Nodulation Signaling Pathway 1 (NSP1) and NSP2 regulate rice tiller number by promoting the biosynthesis of strigolactones (SLs), a class of phytohormones with fundamental effects on plant architecture and environmental responses. We found that NSP1 and NSP2 are induced by Oryza sativa PHOSPHATE STARVATION RESPONSE2 (OsPHR2) in response to low-Pi stress and form a complex to directly bind the promoters of SL biosynthesis genes, thus markedly increasing SL biosynthesis in rice. Interestingly, the NSP1/2-SL signaling module represses the expression of CROWN ROOTLESS 1 (CRL1), a newly identified early SL-responsive gene in roots, to restrain lateral root density under Pi deficiency. We also demonstrated that GR244DO treatment under normal conditions inhibits the expression of OsNRTs and OsAMTs to suppress nitrogen absorption but enhances the expression of OsPTs to promote Pi absorption, thus facilitating the balance between nitrogen and phosphorus uptake in rice. Importantly, we found that NSP1p:NSP1 and NSP2p:NSP2 transgenic plants show improved agronomic traits and grain yield under low- and medium-phosphorus conditions. Taken together, these results revealed a novel regulatory mechanism of SL biosynthesis and signaling in response to Pi starvation, providing genetic resources for improving plant architecture and nutrient-use efficiency in low-Pi environments.

Single-nucleus transcriptomes reveal spatiotemporal symbiotic perception and early response in Medicago.

Establishing legume-rhizobial symbiosis requires precise coordination of complex responses in a time- and cell type-specific manner. Encountering Rhizobium, rapid changes of gene expression levels in host plants occur in the first few hours, which prepare the plants to turn off defence and form a symbiotic relationship with the microbes. Here, we applied single-nucleus RNA sequencing to characterize the roots of Medicago truncatula at 30 min, 6 h and 24 h after nod factor treatment. We found drastic global gene expression reprogramming at 30 min in the epidermis and cortex and most of these changes were restored at 6 h. Moreover, plant defence response genes are activated at 30 min and subsequently suppressed at 6 h in non-meristem cells. Only in the cortical cells but not in other cell types, we found the flavonoid synthase genes required to recruit rhizobia are highly expressed 30 min after inoculation with nod factors. A gene module enriched for symbiotic nitrogen fixation genes showed that MtFER (MtFERONIA) and LYK3 (LysM domain receptor-like kinase 3) share similar responses to symbiotic signals. We further found that MtFER can be phosphorylated by LYK3 and it participates in rhizobial symbiosis. Our results expand our understanding of dynamic spatiotemporal symbiotic responses at the single-cell level.

Microbe-dependent and independent nitrogen and phosphate acquisition and regulation in plants.

Nitrogen (N) and phosphorus (P) are the most important macronutrients required for plant growth and development. To cope with the limited and uneven distribution of N and P in complicated soil environments, plants have evolved intricate molecular strategies to improve nutrient acquisition that involve adaptive root development, production of root exudates, and the assistance of microbes. Recently, great advances have been made in understanding the regulation of N and P uptake and utilization and how plants balance the direct uptake of nutrients from the soil with the nutrient acquisition from beneficial microbes such as arbuscular mycorrhiza. Here, we summarize the major advances in these areas and highlight plant responses to changes in nutrient availability in the external environment through local and systemic signals.

Control of arbuscule development by a transcriptional negative feedback loop in Medicago.

Most terrestrial plants establish a symbiosis with arbuscular mycorrhizal fungi (AMF), which provide them with lipids and sugars in exchange for phosphorus and nitrogen. Nutrient exchange must be dynamically controlled to maintain a mutually beneficial relationship between the two symbiotic partners. The WRI5a and its homologues play a conserved role in lipid supply to AMF. Here, we demonstrate that the AP2/ERF transcription factor MtERM1 binds directly to AW-box and AW-box-like cis-elements in the promoters of MtSTR2 and MtSTR, which are required for host lipid efflux and arbuscule development. The EAR domain-containing transcription factor MtERF12 is also directly activated by MtERM1/MtWRI5a to negatively regulate arbuscule development, and the TOPLESS co-repressor is further recruited by MtERF12 through EAR motif to oppose MtERM1/MtWRI5a function, thereby suppressing arbuscule development. We therefore reveal an ERM1/WRI5a-ERF12-TOPLESS negative feedback loop that enables plants to flexibly control nutrient exchange and ensure a mutually beneficial symbiosis.

Plant immunity in soybean: progress, strategies, and perspectives.

Soybean (Glycine max) is one of the most important commercial crops worldwide. Soybean hosts diverse microbes, including pathogens that may cause diseases and symbionts that contribute to nitrogen fixation. Study on soybean-microbe interactions to understand pathogenesis, immunity, and symbiosis represents an important research direction toward plant protection in soybean. In terms of immune mechanisms, current research in soybean lags far behind that in the model plants Arabidopsis and rice. In this review, we summarized the shared and unique mechanisms involved in the two-tiered plant immunity and the virulence function of pathogen effectors between soybean and Arabidopsis, providing a molecular roadmap for future research on soybean immunity. We also discussed disease resistance engineering and future perspectives in soybean.

Phytophthora sojae boosts host trehalose accumulation to acquire carbon and initiate infection.

Successful infection by pathogenic microbes requires effective acquisition of nutrients from their hosts. Root and stem rot caused by Phytophthora sojae is one of the most important diseases of soybean (Glycine max). However, the specific form and regulatory mechanisms of carbon acquired by P. sojae during infection remain unknown. In the present study, we show that P. sojae boosts trehalose biosynthesis in soybean through the virulence activity of an effector PsAvh413. PsAvh413 interacts with soybean trehalose-6-phosphate synthase 6 (GmTPS6) and increases its enzymatic activity to promote trehalose accumulation. P. sojae directly acquires trehalose from the host and exploits it as a carbon source to support primary infection and development in plant tissue. Importantly, GmTPS6 overexpression promoted P. sojae infection, whereas its knockdown inhibited the disease, suggesting that trehalose biosynthesis is a susceptibility factor that can be engineered to manage root and stem rot in soybean.

Diversity and regulation of symbiotic nitrogen fixation in plants.

Plants associate with nitrogen-fixing bacteria to secure nitrogen, which is generally the most limiting nutrient for plant growth. Endosymbiotic nitrogen-fixing associations are widespread among diverse plant lineages, ranging from microalgae to angiosperms, and are primarily one of three types: cyanobacterial, actinorhizal or rhizobial. The large overlap in the signaling pathways and infection components of arbuscular mycorrhizal, actinorhizal and rhizobial symbioses reflects their evolutionary relatedness. These beneficial associations are influenced by environmental factors and other microorganisms in the rhizosphere. In this review, we summarize the diversity of nitrogen-fixing symbioses, key signal transduction pathways and colonization mechanisms relevant to such interactions, and compare and contrast these interactions with arbuscular mycorrhizal associations from an evolutionary standpoint. Additionally, we highlight recent studies on environmental factors regulating nitrogen-fixing symbioses to provide insights into the adaptation of symbiotic plants to complex environments.