Vascular plants require boron to cross-link the rhamnogalacturonan-II (RG-II) domain of pectin to form functional cell walls. Boronic acids, which form reversible esters with cis-diols like borate, have been proposed to influence RG-II cross-linking, though the mechanism remains unclear. We used suspension-cultured rose cells adapted to grow without boron to investigate the effect of boronic acids on RG-II dimerization. When grown with phenylboronic acid (PBA) as the sole boron source, nearly all RG-II was crosslinked, whereas methylboronic acid (MBA) only partially restored cross-linking. In contrast, in vitro assays showed that homogeneous RG-II monomers did not dimerize with alkyl or aryl boronic acids unless supplemented with hydrogen peroxide (H₂O₂), which oxidatively converts boronic acids to boric acid. Real-time NMR spectroscopy and density functional theory calculations provided insight into the reaction mechanism and energetics of oxidation respectively. Together, our data show that exogenous boronic acids are a source of boric acid for plants, and that the deboronation reaction generates aryl or alkyl alcohol byproducts that can undergo further chemical modification in planta. The fate and potential roles of these byproducts in planta remain to be determined.
A model plant, Nicotiana benthamiana, was examined as a host for persistent fungal viruses capable of crossing organismal kingdoms. Protoplasts of N. benthamiana were transfected with a mixture of virions of a betapartitivirus, Rosellinia necatrix partitivirus 18 (RnPV18), and an alphapartitivirus, RnPV19, and were then subjected to plantlet regeneration. Primary RT-PCR-based screening showed that nearly 100% of the resulting calli tested positive for RnPV18, whereas approximately 90% were positive for RnPV19. However, secondary screening performed at a later stage of tissue culture revealed that only 6% of the calli retained RnPV19, whereas approximately 33% retained RnPV18. These results suggest that the calli were chimeric, consisting of virus-infected and virus-free sectors, and that the partitiviruses were progressively lost during callus maintenance. It is also possible that these fungal partitiviruses were unable to fully adapt to, or counteract, host defense responses sufficiently to establish stable infection. We succeeded in obtaining RnPV18-positive calli and suspension cultures that maintained the virus at detectable levels, as shown by northern blotting, after prolonged subculture for at least 9 months. High-throughput small RNA analyses revealed both similarities and differences in virus-derived small RNA profiles among protoplasts, calli, and suspension cultures. Viral genome analyses further revealed developmental stage-specific and stage-independent substitutions compared with the RnPV18 genomic sequence maintained in the original fungal host. Interestingly, a C-to-U mutation in the RNA-dependent RNA polymerase-encoding region of RnPV18 was detected much more frequently in a particular line, designated B21, than in another stably RnPV18-infected line, P8, irrespective of whether the virus was maintained in suspension cultures or calli. This may explain why virus accumulation in B21 calli and suspension cultures was much lower than that in P8 cell cultures, as RNA-seq analyses showed 159 K counts per million for P8 versus 44 K counts per million for B21. Taken together, this study provides a platform for investigating partitiviruses and other ubiquitous persistent viruses, which are generally difficult to inoculate experimentally.
Synthetic biology enables efficient production of valuable compounds in biological systems, including plants that capture atmospheric CO2 to synthesize and accumulate abundant and diverse specialized metabolites. Most plant synthetic biology studies to produce specialized metabolites have primarily used Nicotiana benthamiana as an underpinning metabolic chassis, due to its rapid agroinfiltration method, leaving much of the metabolic potential of other plant species underexplored. Here we engineered three distinct plant chassis-Arabidopsis, tobacco and soybean-by stably introducing an optimized betalain biosynthetic pathway and analyzed their metabolic impacts. Betalains are tyrosine-derived pigments, which are used as natural red and yellow food dyes with rapidly growing demand due to a recent regulatory shift. We fine-tuned metabolic balances by redesigning the RUBY betalain construct (RUBYv2), adding an extra DODA enzyme ("pull") and modulating tyrosine precursor supply ("push") using two different promoters. The "push-and-pull (push+pull)" lines produced higher betalain levels than the "pull" lines in all three species, even exceeding those of beet roots. While Arabidopsis and tobacco "push+pull" lines driven by a strong promoter showed dwarfism, corresponding soybean lines did not show severe growth defects, suggesting greater tolerance of soybean to the engineered pathway. This study demonstrates that careful plant chassis selection, coupled with precise control of pathway expression, is essential for maximizing the yield of target specialized metabolites, such as betalain pigments, without impairing overall plant growth.
Cancer is a globally devastating disease that severely threatens human health and impedes social development. Camptothecin and its derivatives serve as crucial chemotherapeutic agents in clinical cancer treatment, owing to their unique inhibitory activity against DNA topoisomerase I (TOP1). Although the post-modifications of camptothecin (e.g., hydroxylation and subsequent methoxylation) have been well elucidated, the core biosynthetic pathway of camptothecin remains largely unclear. In the present study, we report an unexpected and significant finding: strictosamide can be directly converted to pumiloside both in several plant species (Nicotiana benthamiana, Salvia miltiorrhiza, and Atractylodes macrocephala) and in vitro. We term this process the "Direct Express Train" of the camptothecin biosynthetic pathway. We further demonstrate that this direct conversion proceeds more efficiently under alkaline conditions in vitro. Intriguingly, light was found to effectively facilitate this conversion. Under light exposure, exogenous flavin adenine dinucleotide (FAD) supplementation markedly promoted the reaction, with the final conversion rate reaching 38.8%. These findings not only deepen our understanding of the camptothecin biosynthetic pathway but also provide a novel strategy for the efficient biosynthesis of pumiloside (7) using plant chassis such as N. benthamiana.
Specialized or secondary metabolites mediate biotic interactions, including virulence and defense. In plant-pathogenic Pseudomonas, certain specialized metabolites can enhance colonization of plant hosts, yet their broader contribution to plant-microbe interactions and the relative importance of different metabolites remain unclear. Specialized metabolites are products of enzymes encoded in biosynthetic gene clusters (BGCs), whose prediction from genome sequences has become routine but whose functional roles are rarely tested experimentally. Here, we characterize the BGC repertoire of 225 P. viridiflava isolates from Arabidopsis thaliana and assess BGC contributions to fitness and disease severity in planta. The BGC landscape of P. viridiflava was dominated by non-ribosomal peptide synthetase (NRPS) and NRPS-like BGCs, which accounted for 50% of the predicted BGCs. One-third of the BGC families were restricted to a single isolate. Transposon mutagenesis coupled with random barcode transposon sequencing (RB-TnSeq) revealed that the majority of BGCs reduce rather than increase fitness during A. thaliana infection, with the magnitude of the fitness cost varying across host genotypes. This cost could be due to exploitation of public goods by cheater mutant strains. In single-isolate plant infections, where public goods are not available, we found 11/34 BGC families correlated with disease severity. Yet, only two of these (an N-acetylglutaminylglutamine amide [NAGGN] and an NRPS) were negatively associated with disease severity, which is positively correlated with bacterial growth in this pathosystem, further indicating that BGCs are generally not beneficial in planta. Our findings reveal extensive and largely uncharacterized biosynthetic potential in populations of P. viridiflava and indicate that candidate metabolites are likely not adaptive for direct interactions with the plant, but perhaps for microbe-microbe interactions either in planta or in other ecological niches. Bacteria, including plant-associated bacteria such as Pseudomonas viridiflava, produce a vast array of chemical compounds, called secondary or specialized metabolites, that can mediate their interaction with the plant host or other microorganisms. Some of these compounds are known to directly influence how bacteria interact with plants, but it has been unclear whether this is a general rule. We studied a large collection of closely related leaf-dwelling P. viridiflava-a plant pathogen-that varied in their ability to cause disease. We found that very few of the gene clusters responsible for making specialized metabolites improved the ability of the bacteria to colonize its natural host Arabidopsis thaliana. On the contrary, carrying these gene clusters often reduced bacterial growth and disease severity in plants. Specialized metabolites may instead primarily be important for interacting with other microbes, different host species, or under environmental conditions we did not test. These are questions that remain for future research.
Two endophytic actinobacteria of the genus Streptomyces-EKL1.1T and EKS8.28T-were isolated from the surface-sterilized leaf and twig of a red gum tree (Eucalyptus camaldulensis Dehn.) grown in highly saline soil, as reported in a previous study. These strains were aerobic and feature well-developed substrate mycelia and aerial mycelia with spiral spore chains. Strains EKL1.1T and EKS8.28T shared the highest 16S rRNA gene sequence similarity with Streptomyces mexicanus NRRL B-24196T (99.2%) and Streptomyces glomeratus LMG 19903T (99.0%), respectively. The comparative genome study of strain EKL1.1T and its closest type strain, S. mexicanus NRRL B-24196T, showed the highest dDDH, ANIb, and ANIm values of 31.8, 85.3, and 88.5%, respectively. The comparative genome study of strain EKS8.28T and its closest type strain, Streptomyces cynarae HUAS 13-4T, had the highest dDDH, ANIb, and ANIm values of 41.8, 89.6, and 91.4%, respectively. The genotypic and phenotypic properties of strains EKL1.1T and EKS8.28T distinguished these two strains from the closely related species with validly published names. The name proposed for the new species of strain EKL1.1T is Streptomyces kalasinensis (= NRRL B-65753T = TBRC 19936T). The name proposed for the novel species of strain EKS8.28T is Streptomyces phytorum (= NRRL B-65754T = TBRC 19937T). Strain EKL1.1T could only inhibit one fungus, Cladosporium sp. LB1, moderately (35%). Strain EKS8.28T could inhibit Fusarium sp. RE1 (71.1%), Curvularia sp. LB12 (62.2%), and Cladosporium sp. LB1 (50%). Strains EKL1.1T and EKS8.28T could produce indole acetic acid (IAA) and solubilize phosphate. The optimum spore inoculum of strains EKL1.1T and EKS8.28T to promote seedling growth parameters of eucalyptus was 107 and 108 spores/mL, respectively. Strains EKL1.1T and EKS8.28T facilitated the development of eucalyptus seedlings under saline conditions. They also enhanced the growth of eucalyptus seedlings in planta by augmenting shoot length and fresh weight. Genome mining of these strains reveals their in vitro and in planta characteristics, including biosynthetic gene clusters of bioactive compounds and several genes associated with plant growth enhancement under stressful conditions. They can be formulated as inoculants to improve eucalyptus plantations for sustainable agriculture in the future.
Transcriptional Gene Silencing (TGS) is an essential process in plants for development, gene regulation, defense against viruses, and genome integrity. TGS is predominantly established by RNA-directed DNA methylation (RdDM), a self-reinforcing mechanism in which sRNAs guide transcriptional suppressors to target genomic loci by sequence complementarity, and methylated DNA in turn facilitates sRNA genesis. Recently, exogenous application of promoter targeting long dsRNAs was associated with promoter methylation without any detectable gene silencing. A plethora of sRNAs of different sizes and types form as cleavage products of precursor dsRNAs such as pre-miRNAs, inverted-repeats, viral replication intermediates, or exogenous dsRNAs. Due to the complexity of sRNA products, the features of the sRNAs, which trigger de novo RdDM, remain enigmatic. Here, we demonstrated that in planta delivery of chemically synthesized 24-nucleotide(nt) small interfering RNAs (siRNAs), targeting the 35S promoter of GFP-expressing Nicotiana benthamiana (Nb) 16c line, was sufficient to induce RdDM, H3K9me2 deposition, and also TGS. Using CRISPR/Cas-mediated gene editing, we showed that exogenous 24nt siRNA-triggered TGS is dependent on ARGONAUTE 4A (NbAGO4A) but not on NbAGO4B. Exogenously administered 24nt siRNAs could provide the means to investigate such initiation events, while allowing functional dissection of siRNA classes and their modifications in planta.
Numerous fungicides are essential for controlling phytopathogens, enabling modern industrial agriculture to maximize productivity and profitability. However, the extensive use of widely employed fungicides, such as tebuconazole and carbendazim, has led to reduced efficacy due to the emergence of resistant plant-pathogenic fungi. Accordingly, in this study, we synthesized a 2‑pyrone chemotype bearing a ynone substituent, designated as ynone 6 and evaluated its potential as an antifungal scaffold. This representative lead compound exhibited broad-spectrum antifungal activity against five major phytopathogens, including Fusarium graminearum and Botrytis cinerea. Ynone 6 demonstrated potent in planta control efficacy across various host tissues, including wheat spikes, fruits, and leaves, with inhibitory effects comparable to those of tebuconazole. Sensitivity screening using six knockout mutants targeting well-established antifungal pathways revealed that the sensitivity profiles of the mutants differed significantly between conidial germination and hyphal growth, suggesting that the mode of action of ynone 6 is stage-dependent and may involve multiple targets. Collectively, the potent in vitro activity of ynone 6 and its consistent in planta efficacy across multiple host-pathogen systems highlight the ynone-tethered 2-pyrone scaffold as a promising lead scaffold for the development of effective antifungal agents.
Ziziphus lotus (L.) Desf. is a resilient shrub native to Morocco's arid and semi-arid regions. This study aimed to isolate and characterize plant growth-promoting bacteria (PGPB) from the root endosphere of Z. lotus, and to evaluate the most effective strain in planta, assessing its impact on plant germination, growth, and modification of the soil microbiota associated to wheat under greenhouse conditions. Our results showed that among the isolated endophytes, Bacillus subtilis ED24 demonstrated strong plant growth-promoting properties, including solubilization of insoluble phosphorus and zinc, nitrogen fixation, and siderophores production. Findings from the pot experiment demonstrated that B. subtilis ED24 significantly improved germination (96% vs. 83% in control), shoot height (52.76 cm vs. 44.98 cm), leaf area (46.2 cm² vs. 30.82 cm²), and shoot biomass (4.28 g vs. 3.45 g). Inoculation also enhanced shoot potassium content (106.6 mg/pot vs. 63.92 mg/pot), grain yield and quality along with a slight increase in grain protein content (19.50% vs. 18.39%) and grain crude fiber content (2.53% vs. 2.48%). Furthermore, inoculation with B. subtilis ED24 enriched beneficial rhizospheric bacteria (e.g., Cicerobacter, Streptomyces, Ferrovibrio, Rhizobium) and reduced pathogenic fungi, such as Fusarium. These findings highlight the potential of B. subtilis ED24 to promote wheat growth and beneficially shape the wheat-associated microbial community.
Burkholderia glumae is a major causal agent of bacterial panicle blight (BPB) in rice, which is characterized by seedling and grain rot. Therefore, the aim of this study was to isolate potential biocontrol bacteria from rice and investigate their efficacies against B. glumae. Potential biocontrol bacteria were isolated from rice seedlings asymptomatic to B. glumae infection using the in planta evaluation screening (IPES) method. In vitro experiments were performed to examine the biocontrol activities of the bacterial isolates. Cultivation of rice seeds in soil (Bonsol No. 2) inoculated with these bacteria and B. glumae suppressed the pathogenicity of B. glumae. Isolates L5 and L6 showed 99.78% and 99.60% identity with Paenibacillus hunanensis, respectively, and isolate R2 was identified as Sphingomonas aquatilis. In vitro evaluation showed that R2 did not exhibit any biocontrol activity. Although the isolates showed inhibitory effects against B. glumae in rice seedlings grown in Bonsol No. 2, they did not show biocontrol activities in Naemidori soil. Notably, inoculation with L5, L6, and R2 bacterial mixture inhibited B. glumae pathogenicity in Naemidori. Conclusively, this study provides a strategy for the effective screening of biocontrol organisms for the sustainable prevention of B. glumae infection in rice.
Dihydrochalcones (DHCs) are highly accumulated in tender leaves of Lithocarpus litseifolius but their biosynthetic pathway and accumulation mechanism remain unclear. In this study, candidate genes including one cinnamoyl-CoA reductase (LlCCR), two double bond reductases (LlDBR1 ~ 2), three aldehyde hydrogenases (LlALDH1 ~ 3), two 4-coumaroyl:CoA ligases (Ll4CL1 ~ 2) and four phloretin glycosyltransferases (LlP4'GT, LlP2'GT1 ~ 3) were comprehensively investigated. The substrate specificities and catalytic kinetics of these gene-encoded enzymes were achieved. Through successive catalysis of LlALDH1, Ll4CL2, and chalcone synthase 1 (LlCHS1) or combined action of LlCCR and LlCHS1, phloretin was biosynthesized from direct precursor dihydro-p-coumaraldehyde, which had been converted from initial precursor p-coumaroyl-CoA by LlCCR-mediated carboxylic acid reduction and LlDBR1-catalyzed α,β-double bond saturation. High accumulation of the DHCs in tender leaves of L. litseifolius was mainly driven by efficient catalysis of LlCCR toward p-coumaroyl-CoA and highly expressed genes in the pathway, especially the LlP4'GT and LlP2'GT1 which contributed to biosynthesis of trilobatin and phlorizin, respectively. Antisense oligodeoxyribonucleotide treatments against the LlCCR, LlDBR1, LlALDH1, Ll4CL2, LlP4'GT, and LlP2'GT1 significantly reduced transcripts of the target genes and content of DHCs, confirming these genes might be involved in the pathway. This finding provides insight into the biosynthesis and accumulation mechanism of DHCs in planta.
Gibberellin (GA) promotes plant growth primarily by triggering degradation of DELLA transcription regulators, yet how DELLA activity is fine-tuned dynamically by phosphorylation independently of proteolysis remains poorly understood. Here we show that the CDK8 kinase module of the Mediator complex attenuates activity of the Arabidopsis DELLA protein RGA by modulating coactivator recruitment. Using TurboID-based proximity labeling, biochemical and genetic analyses, we identify CDK8 as an in-planta kinase that phosphorylates RGA at Ser170 within its disordered PolyS/T region. This phosphorylation does not affect RGA stability, localization or interactions with transcription factors or histone H2A, but selectively weakens RGA association with the Mediator subunit MED15, thereby reducing DELLA-dependent transcription activation. Consistently, cdk8 mutants show impaired GA-responses and delayed developmental phase transitions that are partially rescued by loss of DELLA function. Our findings uncover a phosphorylation-dependent mechanism by which the Mediator kinase module fine-tunes hormone-responsive transcription through selective control of DELLA-coactivator interactions.
Volatile terpenes constitute a predominant class of floral scent emitted by Paeonia lactiflora. Despite their ecological and economical significance, the genetic blueprint of the underlying biosynthetic pathway remains poorly elucidated. Although a few terpene synthase (TPS) genes have been reported, the broader network of genes orchestrating terpene production in P. lactiflora is still largely unresolved. In this study, we attempted to address this gap by exploring the terpene biosynthetic pathway genes in P. lactiflora 'Zifengyu'. β-caryophyllene, geraniol, citronellol, and 1, 8-cineole were identified as the dominant floral terpenes, and catalytic functions of key proteins-terpene synthase (PlTPS), Nudix hydrolase (PlNUDX), and prenyltransferase (PlPT) were comprehensively characterized. Briefly, biochemical analyses revealed that six of the nine identified PlTPS proteins utilized diverse prenyl diphosphates to generate both monoterpenes and sesquiterpenes, while their products specificity were determined by plastidic or cytosolic localizations in planta. In particular, PlTPS4, PlTPS5, and PlTPS9 catalyzed the production of β-caryophyllene, 1, 8-cineole, and geraniol, respectively. Besides, two amino acid residues were found to drive catalytic activity and product profiles in PlTPS4 and PlTPS5. Markedly, PlNUDX hydrolyzed GPP and NPP to yield geraniol and nerol thereby providing a plastid-independent pathway for monoterpene biosynthesis, and prenyltransferases were further functionally characterized to clarify the supply of prenyl diphosphates feeding into volatile terpenes. Collectively, these findings not only provide a mechanistic framework for understanding floral terpene biosynthesis in P. lactiflora but also reveal alternative metabolic routes that enrich its volatile profiles that could be utilized in scent improvement of ornamental plants.
RNA 5-methylcytidine (m5C) has been identified as a key epitranscriptomic modification of mRNAs involved in regulating multiple post-transcriptional processes. Here, we found that knockout of the RNA m5C demethylase, OsNOP2, results in elevated m5C levels and positively influences numerous agronomic traits in rice. After verifying OsNOP2 RNA m5C demethylase function in vitro and in planta, we found that enhanced m5C levels arising from OsNOP2 knockout results in increased translation, particularly for transcripts involved in carbon assimilation and nitrogen metabolism. OsNOP2-KO boosts grain yield by ∼28% per plot in the Nipponbare genetic background in normal conditions. Furthermore, these increased yield traits are maintained under both heat treatment and saline soil conditions. More importantly, knockout of OsNOP2 in the rice varieties Longgeng31 and Xiushui134, as well as its orthologs in wheat and tomato, also increases the RNA m5C level to enhance yield, supporting functional conservation of OsNOP2's regulatory impacts. Together, our findings unveil an RNA m5C-elevating mechanism by OsNOP2 that epigenetically governs carbon assimilation and nitrogen utilization efficiency in plants, providing a potential strategy for genetic improvement in multiple crops.
Bacteria-phage coevolution often results in correlated fitness effects on partner species. Whether coevolutionary changes impact the ecology of the surrounding communities is unclear. Here we link coevolution between the phytopathogenic bacterium Ralstonia pseudosolanacearum and its phage parasites to bacterial wilt disease patterns across four geographically disconnected tomato fields. We find that bacteria and phages are locally adapted between and within fields. Phage infectivity was highest on sympatric bacteria, and bacteria showed greater phage resistance when isolated from healthy than diseased plants. The modularity of phage-bacteria coevolution was associated with field-specific anti-phage defence system patterns and locally adapted phage populations. Moreover, phages selected for field-specific mutations in different phage receptor genes, which were negatively associated with virulence measured in planta, suggesting why phage-resistant but weakly virulent pathogen isolates are associated with healthy tomato plants within fields. Our findings show that bacteria-phage coevolution results in patchy plant disease distribution through phage resistance-virulence trade-offs.
Global food security requires innovative strategies for sustainable crop improvement. Gene editing offers a precise and rapid approach to plant modification, but its success depends on efficient delivery and robust expression systems. Geminivirus-derived replicons (GVRs) enhance transient expression by amplifying introduced DNA within plant cells. In this study, we evaluated three previously deconstructed geminiviral backbones -Bean yellow dwarf virus (BeYDV), Tomato leaf curl virus (ToLCV), and Wheat dwarf virus (WDV)- against a non-replicating T-DNA control for their ability to sustain GFP expression in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). Constructs were delivered via Agrobacterium tumefaciens, and in planta GVR circularization was verified, with accumulation levels dependent on the specific replicon and host species. GFP RNA and protein accumulation was assessed by RT-qPCR, fluorescence imaging, and ELISA; all GVRs prolonged GFP fluorescence relative to control. In tobacco, transcript levels increased significantly by 3 days post infiltration (dpi), reaching up to 221- fold by 6 dpi with BeYDV, while BeYDV and ToLCV produced approximately fivefold higher protein levels. In tomato, ToLCV and WDV showed the strongest enhancement, with transcript and protein levels increasing up to 6.3-fold and 2.4-fold, respectively. These results demonstrate that GVRs markedly enhance and extend transient gene expression in solanaceous hosts, with performance dependent on the replicon and plant species. ToLCV and BeYDV were most effective in tobacco, whereas ToLCV and WDV performed best in tomato. Overall, GVRs represent versatile tools for transient protein production and for improving the delivery and efficiency of genome-editing reagents in plants.
Highbush blueberry (Vaccinium corymbosum) is an important horticultural crop of significant nutritional, therapeutic, and economic value. However, the development of elite cultivars via genetic transformation has been severely restricted by labor-intensive tissue culture requirements and low transformation efficiency. Here, a simple, efficient, and tissue culture-independent Agrobacterium rhizogenes-mediated hairy root transformation system was established for the highbush blueberry cultivar "Bluerain." By eliminating the need for aseptic manipulation, new shoots generated from semilignified stem segments were subjected to vacuum-assisted A. rhizogenes strain K599 infiltration. Approximately 3 months post-infiltration, high biomass hairy roots was successfully induced, and the transformation efficiency reached 74.4% following stepwise optimization. This in planta protocol was further successfully applied to the cultivars "O'Neal," "Legacy," and "Emerald," yielding transformation efficiencies range from 26.7% to 40.0%. This optimized genetic transformation approach provides a valuable tool for the functional characterization of root-specific gene and for accelerating clonal propagation and genetic improvement in highbush blueberry.
Leaves are the plant's main photosynthetic organs and drive Earth's primary production. Grasses form longitudinal leaves with parallel venation and graminoid stomata. Yet, how distinct leaf tissues coordinately develop to build functional grass leaf anatomy is not well understood. Here, we decoded the developing grass leaf from vegetative meristems to mature tissues using single-cell RNA-sequencing in the wild grass Brachypodium distachyon. In-depth analysis of epidermal clusters and multiplexed whole-mount RNA-fluorescence in situ hybridization resolved most epidermal lineages and confirmed them in planta. Gene regulatory network analysis distinguished the targetomes of the two co-expressed, yet functionally divergent stomatal transcription factors BdMUTE and BdFAMA. Finally, we used our dataset to identify and functionally describe the role of the transcription factor gene BdGRAS32 in inhibiting cell division and a stomata-specific role for the putative cell wall-modifying enzyme BdPME53-like. Our single-cell grass leaf atlas enables the dissection of developmental processes that make the leaf sustain global food production.
Plant morphology is a critical determinant of crop productivity in Brassica napus, influencing both lodging resistance and optimal planting density. Here, we investigated the genetic regulation of plant architecture by focusing on the BAK gene family. Using a multi-target sgRNA CRISPR/Cas9 strategy, we generated knockout mutants of several BAK homologs. Phenotypic characterization showed that specific higher-order mutants (Bnbak_sp and Bnbak_n) displayed significantly reduced plant height and altered silique arrangement, while maintaining normal yield-related traits. Expression and localization analyses revealed that BnBAK genes are primarily expressed in leaves and stem internodes, with proteins localizing to multiple membrane systems, including the nuclear envelope. Transcriptomic analysis of internodes indicated extensive reprogramming of photosynthesis- and defense-related pathways. Additionally, we identified TCP transcription factors as key downstream regulators, with motif enrichment confirming TCP-binding sites in promoters of differentially expressed genes. Furthermore, BnBAK proteins are required for the accumulation of BnTCP transcription factors in planta. Functional complementation in Arabidopsis further demonstrated that overexpression of TCP21 substantially rescued the dwarf phenotype of bak1-3 bkk1-1 mutants. Together, these findings elucidate the molecular mechanisms through which BnBAK genes modulate plant architecture in B. napus, providing critical insights and practical targets for breeding compact, high-yielding rapeseed varieties.
Fungal diseases seriously threaten global crop production, highlighting the need for fungicides with novel modes of action. Here, we show that shikonin exerts antifungal activity by targeting dihydroorotase (DHOase). Shikonin significantly inhibited mycelial growth, spore germination, and appressorium formation in representative phytopathogenic fungi in vitro. Biophysical analyses showed that shikonin directly binds to the DHOase of Magnaporthe oryzae (MoPyr4), with dissociation constants of 3.084, 1.22, and 1.26 μM determined by MST, SPR, and ITC, respectively. Molecular docking and mutagenesis identified R90, N178, and H320 as key binding residues. Consistently, deletion of MoPYR4 in M. oryzae reduced sensitivity to shikonin. In planta, shikonin restricted invasive hyphal growth in rice cells and showed both protective and curative activities against rice blast disease. Shikonin also bound to and inhibited the DHOases of Fusarium graminearum and Fusarium oxysporum, indicating broad-spectrum antifungal potential. These findings identify fungal DHOase as a promising target for fungicide development.