Abstract Plants are best understood as evolutionary holobionts, in which the host and its associated microbiomes operate as an integrated unit to influence growth, health, and stress resilience. This comprehensive review synthesizes the most current knowledge of plant‐associated microbiomes across key ecological compartments, including the rhizosphere, endosphere, phyllosphere, and seeds, highlighting their assembly drivers, functional mechanisms, and translational potential. We dissect the molecular foundations of rhizobial and arbuscular mycorrhizal (AM) symbioses, the plant‐AM fungus‐bacterium continuum, alongside emerging concepts including the aerial root mucilagesphere, phyllosphere homeostasis, and the pathobiome. We further explore host genetic, metabolic, and environmental determinants of microbiome assembly, and present cutting‐edge methodologies ranging from quantitative profiling to artificial intelligence‐driven synthetic community design. Finally, we outline a strategic blueprint for harnessing standardized synthetic microbiomes and precision microbiome engineering to advance sustainable agriculture. This integrative framework bridges fundamental ecology with practical applications, delineating a path toward climate‐resilient crop production.

Interactions between phosphate (Pi) and zinc (Zn) or iron (Fe) nutrition in plants have been widely studied; however, the underlying mechanisms of their cross-talks in arbuscular mycorrhizal (AM) plants remain obscure. Here, we examine the physiological and molecular responses of tomato (Solanum lycopersicum L.) to the combination of Pi, Zn and/or Fe nutrient stresses during symbiosis with Rhizophagus irregularis, revealing the existence of a tripartite Pi-Zn-Fe cross-talk in AM symbiosis. Interestingly, the mycorrhiza-activated SlPT3, a member of the PHOSPHATE TRANSPORTER 1 (PHT1) gene family in tomato, is remarkably induced upon the simultaneous Pi and Zn deficiencies. Reverse genetics analysis revealed that SlPT3 not only contributes to Pi transport but is also essential for arbuscule development during Zn deficiency. Moreover, knockdown of SlPT3 leads to reduced Fe accumulation and arbuscule degeneration in mycorrhizal roots by integration of Pi and Zn deficiencies. In silico analysis indicated that the SlPT3 and its homologs contain the IRT (Iron-regulated transporter) domain across dicot and monocot species. Further heterogeneous expression of SlPT3 in yeasts can restore the Δpho84 defects in high-affinity Pi uptake and regulate iron (Fe+2) homeostasis in the fet3fet4 mutant. Collectively, SlPT3 serves as a context-dependent transporter that reshapes nutrient transport priorities in response to combined stresses, revealing a sophisticated mechanism for maintaining symbiosis under fluctuating soil nutrient conditions. These findings provide new avenues for exploring how arbuscular mycorrhizas integrate multiple nutrient stress signals into intricate plant development.

This article is a Commentary on Silvestri et al. (2025), 246: 924–935.

About 70% of land plants form symbioses with arbuscular mycorrhizal (AM) fungi. Some plant genes important for accommodating AM fungi within roots have been characterized, but AM fungal genes involved in asymbiotic growth and hyphopodium formation remain elusive due to a lack of methods for genetic manipulation. Here, we introduce an innovative gene silencing technology based on spraying double-stranded RNA (dsRNA) to characterize the functions of three genes encoding G-protein signaling proteins, including the regulator of G-protein signaling RiRgs3, the Gα subunit RiGpa3, and the Gβ subunit RiGpb1 from the AM fungus Rhizophagus irregularis at the asymbiotic and initial symbiotic stages. RiRgs3, RiGpa3, and RiGpb1 expression is induced in the early stages of AM symbiosis. Using spray-induced gene silencing (SIGS), we discovered that R. irregularis can take up dsRNA. Moreover, SIGS of RiRgs3, RiGpa3, or RiGpb1 led to defects in spore germination and hyphopodium formation. In conclusion, our results reveal that SIGS is a suitable technique for the analysis of gene function in AM fungi and that G-protein signaling is required for spore germination and hyphopodium formation.

It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus Rhizophagus irregularis. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi. Key features • The chitosan can carry the dsRNA derived from the AM fungus Rhizophagus irregularis. • CS/dsRNA polyplex nanoparticles (PNs) can successfully silence the target gene in the AM fungus R. irregularis. • CS/dsRNA PNs can be applied to the characterization of AM fungal genes via the spray-induced gene silencing (SIGS) approach. • This protocol can be applied in asymbiotic and symbiotic cultures of AM fungi. Graphical overview Overview of the chitosan/dsRNA gene silencing procedures.

Arbuscular mycorrhizal (AM) fungi are crucial components of the plant microbiota and can form symbioses with 72% of land plants. Researchers have long known that AM symbioses have dramatic effects on plant performance and also provide multiple ecological services in terrestrial environments. The successful establishment of AM symbioses relies on the host plant recognition of the diffusible mycorrhizal (Myc) factors, lipo-chitooligosaccharides (LCOs) and chitooligosaccharides (COs). Among them, the short-chain COs such as CO4/5 secreted by AM fungi are the major Myc factors in COs. In this review, we summarize current advances, develop the concept of mycorrhizal biceptor complex (double receptor complexes for Myc-LCOs and CO4/5 in the same plant), and provide a perspective on the future development of mycorrhizal receptors. First, we focus on the distinct perception of two Myc factors by different host plant species, highlighting the essential role of Lysin-Motif (LysM)-type mycorrhizal receptors in perceiving them. Second, we propose the underlying molecular mechanisms by which LysM-type mycorrhizal receptors in various plants recognize both the Myc-LCOs and -COs. Finally, we explore future prospects for studies on the biceptor complex (Myc-LCO and -CO receptors) in dicots to facilitate the utilization of them in cereal crops (particularly in modern cultivated rice). In conclusion, our understanding of the precise perception processes during host plant interacting with AM fungi, where LysM-type mycorrhizal receptors act as recruiters, provides the tools to design biotechnological applications addressing agricultural challenges.

Summary In terrestrial ecosystems, most plant species can form beneficial associations with arbuscular mycorrhizal (AM) fungi. Arbuscular mycorrhizal fungi benefit plant nutrient acquisition and enhance plant tolerance to drought. The high osmolarity glycerol 1 mitogen‐activated protein kinase (HOG1‐MAPK) cascade genes have been characterized in Rhizophagus irregularis. However, the upstream receptor of the HOG1‐MAPK cascade remains to be investigated. We identify the receptor kinase RiSho1 from R. irregularis, containing four transmembrane domains and one Src homology 3 (SH3) domain, corresponding to the homologue of Saccharomyces cerevisiae. Higher expression levels of RiSho1 were detected during the in planta phase in response to drought. RiSho1 protein was localized in the plasma membrane of yeast, and interacted with the HOG1‐MAPK module RiPbs2 directly by protein–protein interaction. RiSho1 complemented the growth defect of the yeast mutant ∆sho1 under sorbitol conditions. Knock‐down of RiSho1 led to the decreased expression of downstream HOG1‐MAPK cascade (RiSte11, RiPbs2, RiHog1) and drought‐resistant genes (RiAQPs, RiTPSs, RiNTH1 and Ri14‐3‐3), hampered arbuscule development and decreased plants antioxidation ability under drought stress. Our study reveals the role of RiSho1 in regulating arbuscule development and drought‐resistant genes via the HOG1‐MAPK cascade. These findings provide new perspectives on the mechanisms by which AM fungi respond to drought.

Microbiota living in the rhizosphere influences plant growth and fitness, from the opposite perspective; whether host genotypes control its root microbiota is of great interest to forest breeders and microbiologists. To improve low-yield plantations and promote sustainable management of Camellia oleifera, high-throughput sequencing was used to study the chemical properties and microbiome in rhizosphere soil of Camellia forests under three genotypes (common C. oleifera, local C. gauchowensis, and C. chekiangoleosa) and three growth stages (sapling stage at 4-year-old, primary fruit stage at 7-year-old, and full fruiting stage at 11-year-old). The results showed that the rhizosphere soil organic matter (OM), nutrient concentrations, diversity, and community composition of the microbiome were significantly varied among different Camellia genotypes. The relative abundance of symbiotic and pathotrophic fungi in the rhizosphere soil of C. chekiangoleosa was significantly higher than that of C. gauchowensis. Concentrations of OM, available phosphorus (AP), and bacterial alpha diversity increased with tree age. Fungi of Saitozyma, Mortierella, and Glomeromycota and bacteria of Burkholderia-Caballeronia-Paraburkholderia and Vicinamibacterales had potential for fertilizer development for Camellia plantation. Camellia genotypes and growth stages were significantly correlated with the rhizosphere soil pH, OM, and available potassium (AK). Soil pH and OM were key factors that affected the microbiome in the Camellia rhizosphere soils. In conclusion, tree genotypes and growth stages shaped microbial communities in Camellia rhizosphere soils, and some plant growth-promoting rhizobacteria were identified as preliminary candidates for improving Camellia plantation growth.

The association between plants and arbuscular mycorrhizal fungi (AMF) affects plant performance and ecosystem functioning. Recent studies have identified AMF-associated bacteria as cooperative partners that participate in AMF-plant symbiosis: specific endobacteria live inside AMF, and hyphospheric bacteria colonize the soil that surrounds the extraradical hyphae. In this Review, we describe the concept of a plant-AMF-bacterium continuum, summarize current advances and provide perspectives on soil microbiology. First, we review the top-down carbon flow and the bottom-up mineral flow (especially phosphorus and nitrogen) in this continuum, as well as how AMF-bacteria interactions influence the biogeochemical cycling of nutrients (for example, carbon, phosphorus and nitrogen). Second, we discuss how AMF interact with hyphospheric bacteria or endobacteria to regulate nutrient exchange between plants and AMF, and the possible molecular mechanisms that underpin this continuum. Finally, we explore future prospects for studies on the hyphosphere to facilitate the utilization of AMF and hyphospheric bacteria in sustainable agriculture.

During the evolution of higher plants, many microorganisms evolve alongside them, creating a synergistic plant–microbe world [...]

Irrigation and fertilization are essential management practices for increasing forest productivity. They also impact the soil ecosystem and the microbial population. In order to examine the soil bacterial community composition and structure in response to irrigation and fertilization in a Eucalyptus plantations, a total of 20 soil samples collected from Eucalyptus plantations were analyzed using high-throughput sequencing. Experimental treatments consisting of control (CK, no irrigation or fertilization), fertilization only (F), irrigation only (W), and irrigation and fertilization (WF). The results showed a positive correlation between soil enzyme activities (urease, cellulase, and chitinase) and fertilization treatments. These enzyme activities were also significantly correlated with the diversity of soil bacterial communities in Eucalyptus plantations.. Bacteria diversity was considerably increased under irrigation and fertilization (W, F, and WF) treatments when compared with the CK treatment. Additionally, the soil bacterial richness was increased in the Eucalyptus plantations soil under irrigation (W and WF) treatments. The Acidobacteria (38.92–47.9%), Proteobacteria (20.50–28.30%), and Chloroflexi (13.88–15.55%) were the predominant phyla found in the Eucalyptus plantations soil. Specifically, compared to the CK treatment, the relative abundance of Proteobacteria was considerably higher under the W, F, and WF treatments, while the relative abundance of Acidobacteria was considerably lower. The contents of total phosphorus, accessible potassium, and organic carbon in the soil were all positively associated with fertilization and irrigation treatments. Under the WF treatment, the abundance of bacteria associated with nitrogen and carbon metabolisms, enzyme activity, and soil nutrient contents showed an increase, indicating the positive impact of irrigation and fertilization on Eucalyptus plantations production. Collectively, these findings provide the scientific and managerial bases for improving the productivity of Eucalyptus plantations.

Drought stress has a negative impact on plant growth and production. Arbuscular mycorrhizal (AM) fungi, which establish symbioses with most terrestrial vascular plant species, play important roles in improving host plant mineral nutrient acquisition and resistance to drought. However, the physiological and molecular regulation mechanisms occurring in mycorrhizal Eucalyptus grandis coping with drought stress remain unclear. Here, we studied the physiological changes and mitogen-activated protein kinase (MAPK) cascade gene expression profiles of E. grandis associated with AM fungi under drought stress. The results showed that colonization by AM fungi significantly enhanced plant growth, with higher plant biomass, shoot height, root length, and relative water content (RWC) under drought conditions. Mycorrhizal plants had lower levels of accumulation of proline, malondialdehyde (MDA), H2O2, and O2·- than seedlings not colonized with AM fungi. In addition, mycorrhizal E. grandis also had higher peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities under drought conditions, improving the antioxidant system response. Eighteen MAPK cascade genes were isolated from E. grandis, and the expression levels of the MAPK cascade genes were positively induced by symbiosis with AM fungi, which was correlated with changes in the proline, MDA, H2O2, and O2·- contents and POD, SOD, and CAT activities. In summary, our results showed that AM symbiosis enhances E. grandis drought tolerance by regulating plant antioxidation abilities and MAPK cascade gene expression. IMPORTANCE Arbuscular mycorrhizal (AM) fungi play an important role in improving plant growth and development under drought stress. The MAPK cascade may regulate many physiological and biochemical processes in plants in response to drought stress. Previous studies have shown that there is a complex regulatory network between the plant MAPK cascade and drought stress. However, the relationship between the E. grandis MAPK cascade and AM symbiosis in coping with drought remains to be investigated. Our results suggest that AM fungi could improve plant drought tolerance mainly by improving the antioxidant ability to protect plants from reactive oxygen species (ROS) and alleviate oxidative stress damage. The expression of the MAPK cascade genes was induced in mycorrhizal E. grandis seedlings under drought stress. This study revealed that MAPK cascade regulation is of special significance for improving the drought tolerance of E. grandis. This study provides a reference for improving mycorrhizal seedling cultivation under stress.

Introduction Phosphorus (P) is one of the most important nutrient elements for plant growth and development. Under P starvation, arbuscular mycorrhizal (AM) fungi can promote phosphate (Pi) uptake and homeostasis within host plants. However, the underlying mechanisms by which AM fungal symbiont regulates the AM symbiotic Pi acquisition from soil under P starvation are largely unknown. Here, we identify a HLH domain containing transcription factor RiPho4 from Rhizophagus irregularis. Methods To investigate the biological functions of the RiPho4, we combined the subcellular localization and Yeast One-Hybrid (Y1H) experiments in yeasts with gene expression and virus-induced gene silencing approach during AM symbiosis. Results The approach during AM symbiosis. The results indicated that RiPho4 encodes a conserved transcription factor among different fungi and is induced during the in planta phase. The transcription of RiPho4 is significantly up-regulated by P starvation. The subcellular localization analysis revealed that RiPho4 is located in the nuclei of yeast cells during P starvation. Moreover, knock-down of RiPho4 inhibits the arbuscule development and mycorrhizal Pi uptake under low Pi conditions. Importantly, RiPho4 can positively regulate the downstream components of the phosphate (PHO) pathway in R. irregularis. Discussion In summary, these new findings reveal that RiPho4 acts as a transcriptional activator in AM fungus to maintain arbuscule development and regulate Pi uptake and homeostasis in the AM symbiosis during Pi starvation.

Arbuscular mycorrhizal (AM) fungi can form beneficial associations with the most terrestrial vascular plant species. AM fungi not only facilitate plant nutrient acquisition but also enhance plant tolerance to various environmental stresses such as drought stress. However, the molecular mechanisms by which AM fungal mitogen-activated protein kinase (MAPK) cascades mediate the host adaptation to drought stimulus remains to be investigated. Recently, many studies have shown that virus-induced gene silencing (VIGS) and host-induced gene silencing (HIGS) strategies are used for functional studies of AM fungi. Here, we identify the three HOG1 (High Osmolarity Glycerol 1)-MAPK cascade genes RiSte11, RiPbs2 and RiHog1 from Rhizophagus irregularis. The expression levels of the three HOG1-MAPK genes are significantly increased in mycorrhizal roots of the plant Astragalus sinicus under severe drought stress. RiHog1 protein was predominantly localized in the nucleus of yeast in response to 1 M sorbitol treatment, and RiPbs2 interacts with RiSte11 or RiHog1 directly by pull-down assay. Importantly, VIGS or HIGS of RiSte11, RiPbs2 or RiHog1 hampers arbuscule development and decreases relative water content in plants during AM symbiosis. Moreover, silencing of HOG1-MAPK cascade genes led to the decreased expression of drought-resistant genes (RiAQPs, RiTPSs, RiNTH1 and Ri14-3-3) in the AM fungal symbiont in response to drought stress. Taken together, this study demonstrates that VIGS or HIGS of AM fungal HOG1-MAPK cascade inhibits arbuscule development and expression of AM fungal drought-resistant genes under drought stress.

Arbuscular mycorrhizal (AM) fungi can form mutualistic endosymbiosis with > 70% of land plants for obtaining fatty acids and sugars, in return, AM fungi promote plant nutrients and water acquisition to enhance plant fitness. However, how AM fungi orchestrate its own signaling components in response to drought stress remains elusive. Here, we identify a transcription factor containing C2H2 zinc finger domains, RiMsn2 from Rhizophagus irregularis. To characterize the RiMsn2, we combined heterologous expression, subcellular localization in yeasts, and biochemical and molecular studies with reverse genetics approaches during the in planta phase. The results indicate that RiMsn2 is highly conserved across AM fungal species and induced during the early stages of symbiosis. It is significantly upregulated in mycorrhizal roots under severe drought conditions. The nucleus-localized RiMsn2 regulates osmotic homeostasis and trehalose contents of yeasts. Importantly, gene silencing analyses indicate that RiMsn2 is essential for arbuscule formation and enhances plant tolerance to drought stress. Results from yeasts and biochemical experiments suggest that the RiHog1-RiMsn2-STREs module controls the drought stress-responsive genes in AM fungal symbiont. In conclusion, our findings reveal that a module centered on the transcriptional activator RiMsn2 from AM fungus regulates drought stress tolerance in host plant.

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.

Rhizobium lipopolysaccharide (LPS) is an important component of the cell wall of gram-negative bacteria and serves as a signal molecule on the surface of rhizobia, participating in the symbiosis during rhizobia–legume interaction. In this study, we constructed a deletion mutant of ADP-L-glycerol-D-mannoheptosyl-6-exoisomerase (rfaD) of Mesorhizobium huakuii 7653R and a functional complementary strain. The results showed that the deletion of rfaD did not affect the free-living growth rate of 7653R, but that it did affect the LPS synthesis and that it increased sensitivity to abiotic stresses. The rfaD promoter-GUS reporter assay showed that the gene was mainly expressed in the infection zone of the mature nodules. The root nodules formation of the rfaD mutant was delayed during symbiosis with the host plant of Astragalus sinicus. The symbiotic phenotype analyses showed that the nodules of A. sinicus lost symbiotic nitrogen fixation ability, when inoculated with the rfaD mutant strain. In conclusion, our results reveal that the 7653R rfaD gene plays a crucial role in the LPS synthesis involved in the symbiotic interaction between rhizobia and A. sinicus. This study also provides new insights into the molecular mechanisms by which the rhizobia regulate their own gene expression and cell wall components enabling nodulation in legumes.

Plantation forests productivity is severely limited by the seasonal drought and fertilization practices in South China. Soil nutrient and water availability influence soil fungal community, functional group diversity and the variation of plant productivity; however, the effects of irrigation and fertilization on fungal responses have rarely been studied. Here, we investigate the responses of fungal community structure and functional groups in Eucalyptus plantation soils to short-term fertilization (F), dry-season irrigation (W), short-term fertilization combined with dry-season irrigation (FW), and control (CK) treatments for ten months. A higher proportion of Basidiomycota was observed in the irrigation and/or fertilization treatments; conversely, lower proportions of Ascomycota and Mucoromycotina were observed in the only irrigation and fertilization treatments. Higher soil carbon contents and symbiotroph fungi (mainly Ectomycorrhizas) proportion were detected in the FW treatment, while low proportions of saprophytic and pathogenic fungi were observed in the FW treatment when compared with those in other treatments. These results may indicate that Eucalyptus tree growth under irrigation and fertilization condition was better than under fertilization only, irrigation only, or neither management. The results highlight that short-term fertilization and dry-season irrigation can shift fungal community structure and functional groups by regulating available soil moisture and nutrients. They also provide a theoretical basis for the development of more appropriate management approaches in the early stages of forest plantation.

[目的]筛选巨桉金属耐受蛋白基因EgMTP6,分析其生物信息学特征、亚细胞定位、锌转运功能和表达模式,揭示丛枝菌根真菌、锌、磷与EgMTP6的相互关系,探究其在缓解锌胁迫中的作用.[方法]通过生物信息学分析,得到EgMTP6的核苷酸和氨基酸序列特征.利用农杆菌注射烟草叶表皮细胞分析EgMTP6的亚细胞定位,酵母突变体互补分析EgMTP6转运锌的功能.利用荧光定量PCR技术,分析锌(ZnSO4)、磷(NaH2PO4)和异形根孢囊霉对EgMTP6表达模式的影响.[结果]基因预测显示:EgMTP6包含一个长度为1 524bp的开放阅读框,编码507个氨基酸,包含12个外显子和11个内含子.结构预测显示:EgMTP6蛋白氨基端位于膜内侧,羧基端位于膜外侧,包含5个跨膜螺旋和HD-HD基序.系统进化分析表明:EgMTP6属于Group6,归为Fe/Zn-CDF类群.多序列比对发现,MTP6含有亚铁离子外排泵结构(Ferrous-iron efflux pump,FieF).亚细胞定位显示EgMTP6定位在内质网,酵母突变体互补表明EgMTP6是锌转运蛋白.荧光定量结果说明:EgMTP6的表达不受锌浓度影响,但受高浓度磷和异形根孢囊霉诱导.[结论]EgMTP6属于Fe/Zn-CDF类群,参与锌的转运,表达量不受锌浓度影响,高浓度磷处理和接种异形根孢囊霉时表达量显著提高.EgMTP6在分子特征和功能方面的研究有助于进一步探讨该基因在巨桉菌根耐受锌胁迫时的功能.