Abstract Soybean cyst nematode (SCN) is the most destructive pathogen of soybean, yet the cellular basis of host resistance remains poorly understood. Here, we present a high-quality, cell-type–resolved atlas of root responses during early SCN infection in the highly resistant genotype PI437654, capturing transcriptional states across all major tissues, including rare syncytial cells. Our analyses reveal that resistance is mediated not by a localized defense but by coordinated, multicell reprogramming spanning invasion layers, vascular tissues, and feeding site–associated cells. We identify the vascular cambium as the primary cellular origin of SCN-induced syncytia, resolving a long-standing question in nematology. Mechanistically, resistance arises from disruption of key processes required for feeding site establishment, secretory stress via imbalanced vesicle trafficking, suppression of endoreduplication to prevent hypertrophic syncytial growth, and activation of autophagy to maintain cellular homeostasis. Spatially organized hormone signaling networks, including jasmonic acid, salicylic acid, and ethylene pathways, further reinforce defense, with GmJAZ1 functioning as a central regulator of JA–SA crosstalk. Collectively, PI437654 enforces resistance by targeting host cell identity, nutrient sink formation, and sustained parasitism, deploying a multilayered, tissue-specific defense strategy. This study provides a mechanistic, systems-level framework for SCN resistance and establishes a single-cell resource capturing rare root cell states, offering actionable targets for engineering durable nematode resistance. Key points Soybean cyst nematode (SCN) is the most destructive pathogen of soybean worldwide, yet the cellular basis of early host responses and feeding site initiation remains poorly understood. Using single-nucleus RNA sequencing (snRNA-seq), we generated a cell-type–resolved atlas of early SCN infection in roots of a unique and highly resistant soybean genotype PI437654. Trajectory analysis integrated with syncytium marker genes revealed that cambium cells are selectively targeted as the cellular origin of syncytium formation. SCN infection triggers extensive cell-type–specific transcriptional reprogramming, particularly in vascular tissues (xylem, phloem, and cambium), involving pathways related to cell cycle and endoreduplication, vesicle trafficking, autophagy, and phytohormone signaling. Functional validation demonstrated enhanced autophagy activation in infected roots via increased GFP-GmATG8a–labeled autophagic puncta, while overexpression of the jasmonic acid regulator GmJAZ1 significantly enhanced SCN resistance in susceptible soybean. Together, these findings define the cellular origin of SCN-induced syncytia and reveal coordinated cell-type-specific defense programs, providing a mechanistic framework for engineering durable resistance to SCN.

Developing transgenic and/or gene-edited plants largely depends on tedious, lengthy, and costly in vitro regeneration protocols. While plants have remarkable regeneration ability, not all species, genotypes, or even explants exhibit the same transformation and regeneration potential under in vitro conditions. To tackle this bottleneck, we have developed a seamless and user-friendly system to induce transgenic and gene-edited de novo meristems via a synthetic cascade comprising a wound-induced regeneration pathway, plant developmental regulators (DRs), and gene-editing reagents. WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) was used as a transcriptional regulator to control the expression of various DR genes driven by ENHANCER OF SHOOT REGENERATION 1 (ESR1) promoter. This cascade was strategically applied in planta to the non-meristematic internode of Nicotiana benthamiana to induce meristematic activity and regenerate de novo shoots with knockout mutations of the phytoene desaturase (PDS) gene. Among the DR genes tested, the strategic expression of isopentenyl transferase (ipt) driven by the ESR1 promoter under the control of WIND1 proved most effective for efficient regeneration in tobacco. Subsequently, this synthetic toolkit was successfully applied to both tomato and soybean. WIND1 served as a key cellular reprogramming factor, initiating differentiation, while ipt complemented this process by promoting organogenesis through cytokinin biosynthesis. This methodology offers a transformative approach to overcome barriers in plant biotechnology, potentially accelerating the generation of transgenic and gene-edited plants without reliance, or with minimal reliance, on conventional tissue-culture intermediates.

CRISPR/Cas technologies have revolutionized plant genome editing, yet their inherent bias toward small insertions or deletions (indels) limits their utility for dissecting regulatory elements and generating impactful allelic variants. Here, we report the development and systematic evaluation of exonuclease-fused CRISPR/Cas systems in soybean to overcome this limitation. We engineered fusions of Cas9 and Cas12a with bacteriophage T5 exonuclease and human TREX2 and assessed their editing performance at the GmWOX5 locus using Agrobacterium rhizogenes-mediated transformation and deep amplicon sequencing. While native Cas9 and Cas12a predominantly generated micro-size deletions (1–10 bp), T5-Exo fusions shifted the mutation spectrum, producing a high frequency of moderate (26–50 bp) and large (> 50 bp) deletions. TREX2 fusions preferentially enhanced the generation of small (11–25 bp) to moderate deletions (26–50 bp). Fusion of exonucleases to Cas9 substantially reduced insertion frequencies and promoted more precise deletion patterns, as observed in T5-Exo-Cas9 and TREX2-Cas9. Deletions from both exonuclease fusions were biased toward the PAM-proximal region, reflecting altered repair outcomes likely driven by directional exonuclease activity and enhanced end resection. These results demonstrate that exonuclease fusions effectively expand the CRISPR toolkit by enabling efficient, targeted generation of larger deletions, which are often required for targeting cis-regulatory elements and microRNAs. We demonstrate that engineered exonuclease-fused CRISPR systems enable efficient, target-specific large deletions in soybean, providing a valuable tool to enhance functional genomics and accelerate trait discovery in crops.

Mineral nutrient uptake and deposition profoundly influence plant development, stress resilience, and productivity. Silicon (Si), though classified as a non-essential element, significantly influences a plant's physiology, particularly in fortifying defense responses and mitigating stress. While the genetic and molecular mechanisms of Si uptake and transport are well studied in monocots, particularly rice, their role in dicot species, such as soybean, remains unclear at the cellular and molecular levels. In this study, we utilized single-nucleus RNA sequencing (snRNA-seq) to dissect cellular responses to Si accumulation in soybean leaves. We identified distinct cellular populations, including a unique Si-induced or Si-associated cell cluster within vascular cells, suggesting a specialized mechanism of Si distribution. Si treatment notably induced the expression of defense-related genes, with a pronounced enrichment in vascular cells, underscoring their pivotal role in activating plant defense mechanisms. Moreover, Si modulated the expression of genes involved in phytoalexin biosynthesis, salicylic acid, and immune receptor signaling, suggesting transcriptional priming of genes involved in defense responses. Further investigation of Si transporters revealed precise expression of an Si efflux gene in epidermal cells in response to Si treatment. We also validated the role of efflux Si transporters using a Xenopus oocyte assay and CRISPR/Cas9 genome editing of composite soybean plant roots. This study provides critical insights into the biotic stress regulatory networks influenced by Si treatment in soybean leaves at the single-cell level, thus laying the foundation for enhancing stress tolerance through optimized mineral nutrient uptake.

Soybean cyst nematode (SCN, Heterodera glycine Ichinohe) is a major threat to global soybean yield. Resistance genes at the rhg1 locus from PI 88788 are majorly utilized in 95% of the U.S. breeding programs. Continuous use of this resistance source leads to a shift in the virulence of SCN populations and overcomes host resistance. Therefore, it is necessary to identify alternative SCN resistance sources to combat this ever-changing pest. Previously, we identified an exotic soybean line, PI 567516C, which carries a novel qSCN10 (O) locus for SCN resistance demonstrating different resistance responses compared to the known rhg1 and Rhg4 loci. Here, we narrowed the qSCN10 QTL region to 142-kb (containing 20 genes). Based on gene expression, gene ontology, in-silico analysis, and QTL-based haplotyping, two genes were identified for functional characterization. Overexpression of the transcription factor TGA1-related and Shugoshin C-terminus in the SCN-susceptible Williams 82 reduced the cyst number by 6.4-fold (84.6%) and 5.3-fold (81.2%), respectively. GmTGA1-10 and GmSCT-10 Tilling mutants showed high cyst numbers. The two genes associated with the qSCN10 QTL have significant potential to reduce the SCN population. They also offer an alternative source of durable SCN resistance that is independent of rhg1 and Rhg4.

Sorghum, a climate-resilient cereal, is crucial for meeting the growing demand for food and feed in arid and semi-arid regions, especially amid global population growth and climate change. Despite its natural drought tolerance and adaptability, sorghum faces challenges in increasing yield, enhancing resistance to abiotic and biotic stresses, and improving grain quality. Genetic engineering has emerged as a powerful tool to address these challenges by directly modifying genes associated with desirable traits. Recent advancements have utilized morphogenic regulators to improve transformation and regeneration efficiency in sorghum. This review explores the status of genomic resources and genetic diversity in sorghum, highlighting the advancements and challenges faced in its genetic engineering efforts. Genome editing technologies, particularly CRISPR/Cas systems, have improved key agronomic traits such as stress tolerance, nutrient use efficiency, and grain quality. However, significant obstacles still need to be addressed, including low regeneration rates, high genotype dependency, and labor-intensive transformation processes. We highlight potential strategies to overcome these barriers, such as optimizing transformation protocols, exploring alternative explants, using morphogenic regulators and advancing tissue culture techniques. Additionally, we discuss the biosafety considerations and potential applications of genetically engineered sorghum in global agriculture. This review underscores the need for ongoing innovation to unlock the potential of genetically engineered sorghum in addressing global food security challenges.

Mineral nutrients play an important role in plant growth, development, stress resilience, and overall yield. Among major and minor mineral nutrients, silicon (Si) is a non-essential but especially beneficial mineral nutrient that plays a critical role in cereal crops by enhancing defense response and mitigating abiotic stresses. The genetic and molecular mechanism of Si is well studied in rice; however, its role at the single-cell level remains elusive in sorghum and other cereal crops. Additionally, how Si treatment modulates the leaf transcriptome at the single-cell level remains unclear. Using single-nucleus RNA sequencing (snRNA-seq), we profiled the cellular and transcriptional responses of sorghum leaves to Si supplementation. Si-treated leaves showed substantial Si accumulation, primarily in specialized silica cells. Transcriptomic analysis identified 16 distinct cell clusters and revealed that Si treatment upregulated genes involved in stress-related metabolic pathways and transport processes. Importantly, we uncovered a cell-specific regulation of Si transporters, with differential expression in xylem and epidermal cells. These findings advance our understanding of nutrient regulation at the single-cell level and provide a foundation for enhancing nutrient efficiency and stress tolerance in sorghum.

Developing transgenic and/or gene-edited plants largely depends on tedious, lengthy, and costly in vitro regeneration protocols. While plants have remarkable regeneration ability, not all species, genotypes or even explants exhibit the same transformation and regeneration potential under in vitro conditions. To tackle this bottleneck, we have developed a seamless and user-friendly system to induce transgenic and gene-edited de novo meristems via a synthetic cascade comprising a wound-induced regeneration pathway, plant developmental regulators (DRs) and gene-editing reagents. WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) is used as a transcriptional regulator to control the expression of various DR genes through ENHANCER OF SHOOT REGENERATION 1 (ESR1) promoter. This cascade was strategically applied in planta to the non-meristematic internode of N. benthamiana to induce meristematic activity and regenerate de novo shoots with knock-out mutations of the phytoene desaturase (PDS) gene. This synthetic toolkit was further applied successfully to tomato and soybean. This methodology offers a transformative approach to overcome barriers in plant biotechnology, potentially accelerating the generation of transgenic and gene-edited plants without reliance on conventional tissue-culture intermediates. ### Competing Interest Statement GBP, AOK, and LHE have filed a patent application related to this work.

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most economically damaging soilborne pathogen affecting soybean, causing significant yield losses in the United States and worldwide. Current commercial cultivars rely heavily on a limited genetic resistance base, primarily from PI 88788 and Peking, which has led to the emergence of virulent SCN populations that threaten the durability of this resistance. To address this challenge, we performed a comprehensive allelic analysis of key resistance loci (rhg1, Rhg4, qSCN10 (O), and qSCN18 (G)) using whole-genome resequencing data from 1,110 diverse soybean accessions. Our study identified novel nonsynonymous single-nucleotide polymorphisms in 27 accessions, including PI 602492 (Glycine max) and two Glycine soja accessions (PI 522226, PI 522228), that display strong to moderate resistance across multiple SCN HG types (Heterodera glycines) or SCN races. Additionally, we identified two G. soja accessions, PI 507380B and PI 507752, that exhibited strong resistance to HG type 2.5.7 (race 5). Notably, accessions with genotypes similar to these five showed variable resistance phenotypes, suggesting the presence of additional, yet unidentified, genes contributing to broad-based SCN resistance. Among these, PI 602492 stands out as a valuable new resistance source with strong activity against multiple HG types (races), making it an excellent candidate for gene discovery and breeding efforts to enhance resistance independently of conventional germplasm. These findings provide important, underutilized genetic resources that can expand the resistance base and drive the development of more durable SCN-resistant soybean cultivars. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

In agriculture, mineral nutrient uptake and deposition profoundly influences plant development, stress resilience, and productivity. Despite its classification as a non-essential element, silicon (Si) uptake and deposition alters plant physiology and particularly improves defense response and stress mitigation. While genetic and molecular mechanisms of Si uptake and transport are well-studied in monocots, particularly rice, its role in dicot species, such as soybean, remains unclear at the cellular and molecular levels. Traditional bulk transcriptomics methods lack the resolution to uncover cellular heterogeneity. Here, we present a study utilizing single-nucleus RNA sequencing (snRNA-seq) to dissect cellular responses to Si accumulation in soybean leaves. Our analysis revealed distinct cellular populations, including a novel Si-induced cell cluster within vascular cells, suggesting a specific mechanism of Si distribution. Si treatment induced the expression of defense-related genes, particularly enriched in vascular cells, highlighting their specialized role in activating plant defense mechanisms. Moreover, Si modulated the expression of genes involved in RNA silencing, phytoalexin biosynthesis, and immune receptor signaling, suggesting transcriptional priming of genes involved in defense responses. We also investigated putative Si transporters, revealing differential expression patterns in response to Si treatment, suggesting presence of active and gradient-based transport mechanisms. Furthermore, by employing CRISPR/Cas9 genome editing we functionally validated the role of efflux Si transporters in composite soybean plants. Our findings shed light on the vital biotic stress regulatory networks governed by Si treatment in soybean leaves, paving potential strategies for enhancing stress tolerance and agronomic performance in crops.

In agriculture, mineral nutrients uptake and deposition profoundly influence plant development, stress resilience, and productivity. Despite its classification as a non-essential element, silicon (Si) is crucial in plant physiology, particularly in defense response and stress mitigation. While genetic and molecular mechanisms of Si uptake and transport are well-studied in monocots, particularly rice, its role in dicot species, such as soybean, remains unclear at the cellular and molecular levels. Traditional bulk transcriptomics methods lack the resolution to uncover cellular heterogeneity. Here, we present a study by utilizing single-nucleus RNA sequencing (snRNA-seq) to dissect cellular responses to Si accumulation in soybean leaves. Our analysis revealed distinct cellular populations, including a novel Si-induced cell cluster within vascular cells, suggesting a specific mechanism of Si distribution. Si treatment induced the expression of defense-related genes, particularly enriched in vascular cells, highlighting their specialized role in activating plant defense mechanisms. Moreover, Si modulated the expression of genes involved in RNA silencing, phytoalexin biosynthesis, and immune receptor signaling, suggesting a mechanism of transcriptional priming of genes involved in defense responses. We further investigated putative Si transporters, revealing differential expression patterns in response to Si treatment, suggesting presence of active and gradient-based transport mechanisms. Our findings shed light on the vital biotic stress regulatory networks governed by Si treatment in soybean leaves, paving potential strategies for enhancing stress tolerance and agronomic performance in crops. ### Competing Interest Statement The authors have declared no competing interest.

Bioavailability, uptake, and spatial distribution of essential and toxic mineral elements are pivotal factors that govern crop growth, development, and productivity. Soybean is a major leguminous crop globally, and yield losses are commonly attributed to various abiotic factors, including nutrient deficiencies or the influence of toxic minerals in the soil. Therefore, understanding the molecular basis of differential mineral element uptake, translocation, and accumulation in soybean is vital for developing improved cultivars. Here, we used portable X-ray fluorescence (p-XRF) for rapid and high-throughput mineral element profiling of a diverse set of soybean germplasm using leaves, stem, root, and seed tissues. Genome-wide association (GWAS) was performed on the element profiles of 219 soybean accessions, revealing lines with notable two to tenfold difference in various mineral nutrient uptake. These identified lines represent valuable genetic resources for germplasm development and gene discovery. The GWAS analysis pinpointed significant genomic loci and haplotypes associated with accumulation of aluminum (Al), silicon (Si), iron (Fe), and manganese (Mn). Remarkably, genes associated with the transport of solutes, metals, and ions have been pinpointed, indicating their potential involvement in the nutrient uptake and soybean improvement. To gain functional insights, Si deposition, allelic variants and expression of three Si transporter genes was studied in greater details. The high Si accumulating line PI548452 showed 12-fold increase in leaf Si content compared to low Si lines. Expression of HiSil2b and HiSil2c effluxer genes showed root and leaf specific expression, respectively, providing evidence for tissue specific Si transport and the basis for precise management of Si uptake to improve abiotic and biotic stresses. In summary, this study uncovered the novel accessions, haplotypes, allelic diversity, and the potential candidate genes underpinning the mineral nutrient accumulation in soybean.

The heavy metal contamination is creating devastating effects on ecosystem and environment and eventually hazardous to human health.Ujjani is one of the largest reservoir in Maharashtra state.It receives water from different metropolitan cities harboring numerous industries.Moreover, anthropogenic activities pollute water and fish fauna of the reservoir.In the present study, the concentrations of heavy metals such as Iron (Fe), Copper (Cu), Zinc (Zn), and Manganese (Mn) were analyzed from various tissues namely muscles, liver, gill, and fin of Chillapi fish.The variation in level of metals was found with respect to tissue types and body weight groups.The concentrations of Fe were ranged between 15.94 to 91.56µg/g, Cu concentration was between 1.88 to 48.88µg/g, Zn concentration was between 25.72 to 84.2µg/g and Mn was recorded between 2.12 to 28.48µg/ g in Chillapi fish.Fe and Cu have recorded highest in the liver, whereas peak of Zn and Mn was detected in fins.Furthermore, the minimal metal concentrations were observed in muscle samples of all different weight groups.It shows that Fe, Zn, Cu concentration in all four tissues was within the limit, whereas the Mn level was found to be exceeding the permissible limit as prescribed FAO/WHO, 1989.The presence of heavy metals higher than normal level in fish bodies is a clear indicator of biomagnifications.Furthermore, THQ and HI values were within limit for all studied metals in fish but if consumed in excess amount may cause toxicity in humans.Therefore, daily intake of fish should be strictly monitored to avoid excess intake of these heavy metals.Our study signifies the importance of addressing the heavy metal contamination issue to avoid health related problems.

Summary Salinity stress limits plant growth and has a major impact on agricultural productivity. Here, we identify NAC transcription factor SlTAF1 as a regulator of salt tolerance in cultivated tomato (Solanum lycopersicum). While overexpression of SlTAF1 improves salinity tolerance compared with wild‐type, lowering SlTAF1 expression causes stronger salinity‐induced damage. Under salt stress, shoots of SlTAF1 knockdown plants accumulate more toxic Na+ ions, while SlTAF1 overexpressors accumulate less ions, in accordance with an altered expression of the Na+ transporter genes SlHKT1;1 and SlHKT1;2. Furthermore, stomatal conductance and pore area are increased in SlTAF1 knockdown plants during salinity stress, but decreased in SlTAF1 overexpressors. We identified stress‐related transcription factor, abscisic acid metabolism and defence‐related genes as potential direct targets of SlTAF1, correlating it with reactive oxygen species scavenging capacity and changes in hormonal response. Salinity‐induced changes in tricarboxylic acid cycle intermediates and amino acids are more pronounced in SlTAF1 knockdown than wild‐type plants, but less so in SlTAF1 overexpressors. The osmoprotectant proline accumulates more in SlTAF1 overexpressors than knockdown plants. In summary, SlTAF1 controls the tomato’s response to salinity stress by combating both osmotic stress and ion toxicity, highlighting this gene as a promising candidate for the future breeding of stress‐tolerant crops.

SummaryWater deficit (drought stress) massively restricts plant growth and the yield of crops; reducing the deleterious effects of drought is therefore of high agricultural relevance. Drought triggers diverse cellular processes including the inhibition of photosynthesis, the accumulation of cell‐damaging reactive oxygen species and gene expression reprogramming, besides others. Transcription factors (TF) are central regulators of transcriptional reprogramming and expression of many TF genes is affected by drought, including members of the NAC family. Here, we identify the NAC factor JUNGBRUNNEN1 (JUB1) as a regulator of drought tolerance in tomato (Solanum lycopersicum). Expression of tomato JUB1 (SlJUB1) is enhanced by various abiotic stresses, including drought. Inhibiting SlJUB1 by virus‐induced gene silencing drastically lowers drought tolerance concomitant with an increase in ion leakage, an elevation of hydrogen peroxide (H2O2) levels and a decrease in the expression of various drought‐responsive genes. In contrast, overexpression of AtJUB1 from Arabidopsis thaliana increases drought tolerance in tomato, alongside with a higher relative leaf water content during drought and reduced H2O2 levels. AtJUB1 was previously shown to stimulate expression of DREB2A, a TF involved in drought responses, and of the DELLA genes GAI and RGL1. We show here that SlJUB1 similarly controls the expression of the tomato orthologs SlDREB1, SlDREB2 and SlDELLA. Furthermore, AtJUB1 directly binds to the promoters of SlDREB1, SlDREB2 and SlDELLA in tomato. Our study highlights JUB1 as a transcriptional regulator of drought tolerance and suggests considerable conservation of the abiotic stress‐related gene regulatory networks controlled by this NAC factor between Arabidopsis and tomato.