Browning in tobacco leaves during the flue-curing process severely reduces the quality and industrial usability, and the reasons and mechanisms are still unclear. Here, the reactive oxygen species (ROS) levels, antioxidant capacity, and membrane lipid oxidation in upper, middle, and lower leaves during the early flue-curing process were comprehensively compared with the severe-browning cultivar Dabaijin and the low-browning cultivar Yunyan87. During the later yellowing stage and early color-fixing stage, higher H₂O₂ content and greater membrane lipid oxidation, but lower O₂•− content were exhibited in the leaves of Dabaijin, while significantly lower SOD activity and higher POD and CAT activities were shown in Yunyan87. The antioxidant-related pathways (glutathione metabolism, carotenoid biosynthesis) were enriched in the shared differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) from integrated transcriptomic and metabolomic analyses. We identified 62 ROS and antioxidant-related DEGs. Among these, 9 genes associated with ROS generation were upregulated in Dabaijin, while most antioxidant enzyme genes were downregulated. Meanwhile, 100 non-enzymatic antioxidant-related DAMs were screened, and most of the DAMs were significantly accumulated in Yunyan87. Additionally, exogenous application of H₂O₂ to Yunyan87 significantly promoted browning, while the antioxidants (200 µM glutathione and 0.1

Plant molecular marker technologies have reshaped crop genetics and breeding by making it possible to analyse genome-wide variation with a precision that phenotype-based selection, even in experienced programmes, cannot reach in routine practice. This review summarises recent progress in marker platforms from classical RFLP and SSR systems to high-throughput SNP genotyping, with emphasis on KASP, multiple nucleotide polymorphism and multi-gene panel technologies, and on sequencing-based methods such as GBS, GBTS and Hyper-seq that often serve as an upstream discovery layer for targeted assays and databases. These platforms are increasingly integrated into practical workflows for marker-assisted and genomic selection, DNA fingerprinting, germplasm characterisation and plant variety protection, and multi-locus markers have become a central tool for high-resolution DUS testing and EDV determination that adds an independent layer of evidence to morphology-based assessments. Key challenges now include cross-platform standardisation, design of marker panels that balance cost with information content, interoperability of databases across institutions and countries, and the definition of molecular distance thresholds that are acceptable both biologically and in legal and regulatory settings. The review also considers the rapid integration of molecular marker data with artificial intelligence, including AI-driven marker discovery and panel optimisation, genomic prediction in multi-environment trials and the concept of an intelligent seed-industry operating system that links genotypic, phenotypic and environmental information in a coherent data framework. These developments collectively point to a shift from isolated marker assays towards platform-level, AI-supported infrastructures that can accelerate variety innovation and contribute to the modernisation and quality improvement of the seed industry.

Bacterial wilt is a devastating vascular disease caused by the soil-borne bacterium Ralstonia solanacearum, which invades and blocks the xylem of susceptible plants, especially economically important solanaceous crops. In addition to classical plant defense hormones, recent research has focused on understanding the role of auxin as a key and continuous regulator of infection and development. Auxin connects changes during infection to root development and the progression of bacterial wilt disease. The auxin related shift in plant hormone patterns associated with root development has been observed in both infected and non-infected host plant species. This shift is accompanied by the upregulation of several auxin regulating transcriptional programs, the accumulation of indole-3-acetic acid, and the enhancement of auxin signaling in specific plant tissues. The auxin-associated reprogramming of roots infected with R. solanacearum is also linked to the reorganization of root developmental programs, including reduced primary root elongation, pronounced induction of root hair formation, and increased lateral root development at later stages of infection. Root hair formation in response to infection requires an intact and functional auxin signaling pathway. There is also support for a positive relationship between the level of auxin responsiveness and a plant’s susceptibility to infection. Susceptible genotypes activate their auxin pathway more than resistant genotypes, while reducing the ability to transport or perceive auxin increases plant resistance and decreases the severity of key root-associated phenotypes during infections. At the molecular level, R. solanacearum is reported to influence the auxin homeostasis of the host plant indirectly through effectors, which induce changes in the plant immune response, alter metabolism and energy status, and modify the interactions within plant hormone pathways. These changes create conditions that facilitate the colonization and spread of R. solanacearum. Therefore, auxin is best understood as a dynamically regulated “balancer” that mediates the trade-off between plant growth and defense, integrating the plant developmental flexibility with immune response. Furthermore, leveraging these findings to develop precision resistance offers strategies for enhancing plant resistance. These include improving auxin modulation within the growth environment and employing spatiotemporal transcriptomics to identify spatially and temporally correlated losses of auxin sensitivity or transport in root zones susceptible to infection.

Flower color in tobacco is largely determined by flavonoid and anthocyanin accumulation. Flavonol synthase (FLS) is a key branch-point enzyme in the flavonoid biosynthetic pathway that competes with dihydroflavonol 4-reductase for common substrates, thereby influencing the biosynthesis of flavonols and anthocyanins. However, the role of NtFLS1 in tobacco flower pigmentation remains unclear. To investigate the function of NtFLS1, CRISPR/Cas9-mediated mutagenesis was performed in Nicotiana tabacum. Compared with wild-type K326 plants, ntfls1 mutants exhibited markedly darker flowers at the full-bloom stage, with floral pigmentation changing from light pink to deep purple. Transcriptome analysis identified 7,052 differentially expressed genes between mutant and wild-type flowers, with significant enrichment in phenylpropanoid and flavonoid biosynthesis pathways. Expression analysis showed that CHS and CHI were downregulated, whereas COMT and F3’H were upregulated in ntfls1 flowers. Metabolomic analysis further revealed substantial accumulation of ( +)-trans-Taxifolin, Scopoletin, and multiple anthocyanins, including Cyanidin 3-O-glucoside and Pelargonidin-derived compounds. Integrated transcriptomic and metabolomic analyses suggested that NtFLS1 disruption altered flavonoid pathway partitioning and promoted anthocyanin accumulation in tobacco flowers. NtFLS1 is involved in the regulation of flavonoid biosynthesis and flower pigmentation in tobacco. Disruption of NtFLS1 was associated with increased anthocyanin accumulation and enhanced floral pigmentation, likely through redistribution of flavonoid pathway intermediates. These findings provide new insights into the molecular basis of flower color formation in tobacco and may facilitate molecular breeding of ornamental traits in Solanaceous crops.

Crops are the primary source of food for humans, providing essential nutrients such as carbohydrates and proteins. With economic development and rising living standards, demand for high-quality crops has increased, encompassing not only high and stable yields but also enhanced nutritional profiles, flavor, taste, and functional components. These quality traits are typically controlled by complex polygenic networks and often involve trade-offs among yield, nutrition, and processing performance. Traditional breeding methods such as crossbreeding and phenotypic selection are limited by low efficiency, long breeding cycles, and difficulties in the coordinated improvement of multiple traits. The CRISPR/Cas9 system, characterized by its programmability, simplicity, and high editing efficiency, offers a novel strategy for precise trait improvement and the breeding of high-quality crop varieties. Through targeted modification of key genes, CRISPR/Cas9 can effectively regulate the synthesis and accumulation of nutrients such as starch, proteins, lipids, and vitamins, thereby enabling the targeted optimization of crop quality traits. This review systematically summarizes recent applications of CRISPR/Cas9 in enhancing nutritional components, improving eating quality, and reducing harmful substances in crops. In addition, current challenges and future prospects are discussed to provide theoretical guidance and practical support for the precise and efficient application of gene-editing technologies in crop quality improvement.

Tobacco is an important economic crop and a model plant for molecular biology research. It exists in various cultivars and is processed using different curing methods. Fatty acids play a crucial role in the quality and flavor of tobacco leaves. However, there is limited information on the fatty acid composition across different cultivars, developmental stages, and curing methods. This study employed targeted metabolomics and transcriptomics to investigate the fatty acids and related pathway genes in tobacco leaves from different cultivars, developmental stages, and curing methods. This study focused on four tobacco cultivars: K326, Basma, Samsun, and Cuba1, and investigated fatty acid differences in the leaves at four developmental stages (seedling, transplanting, budding, and topping) under two curing methods (air-curing and flue-curing). K326 was used as the main cultivar for comparison with the other three. The analysis included short-chain fatty acids (C2-C6), free fatty acids (C8-C24), and gene expression differences. The fatty acid metabolic profile of different tissue types in K326 at the budding stage was also examined. The results showed significant differences in fatty acid content among the different tissues of K326 at the budding stage, with the highest levels of short-chain fatty acids found in flower buds and upper leaves. At the seedling stage, there were marked variations in short-chain fatty acid content across different periods. Three key genes Nta01g31980, Nta08g22780, and Nta23g11140 were identified as major differential genes in fatty acid-related pathways in K326 compared to the other three cultivars during this stage. Regarding the four cultivars, the total short-chain fatty acid content at the budding stage was ranked as Basma > Samsun > Cuba1 > K326 before topping, but the order was reversed after topping. At the budding stage, 35 fatty acid pathway-related genes showed similar expression levels in Basma and Samsun, differing from K326 and Cuba1. Among the two curing methods, air-curing resulted in higher short-chain fatty acid content than flue-curing. Under air-curing, Samsun and Basma showed more downregulation of differential fatty acids compared to K326, while the opposite was observed under flue-curing. This study expands our understanding of fatty acids in tobacco across different cultivars and developmental stages, providing a molecular basis for the study of fatty acids and genes related to their biosynthesis and metabolism.

Pathogenic bacteria utilize a type III secretion system to translocate effector proteins into plant cells, where they inhibit plant immunity or interfere with normal cellular functions to facilitate infection. Whether and how pathogen effectors manipulate plant adenosine 5'-triphosphate (ATP) to facilitate infection remains largely unknown. In this work, we show that an effector protein, RipAF1, from the plant pathogen Ralstonia solanacearum suppresses flg22-induced immune activation and contributes to virulence. RipAF1 physically interacts with plant ferredoxin-NADP+ reductase (FNR), which is involved in NADPH and ATP production, in chloroplast. Transient expression of FNR leads to increased ATP accumulation and resistance against R. solanacearum, while co-expression of FNR with RipAF1 significantly reduced ATP levels. We further show that exogenous application of ATP enhances plant resistance to R. solanacearum infection. Our findings indicate a key role of ATP in plant resistance against R. solanacearum, and elucidate a bacterial virulence strategy wherein pathogenicity is enhanced through targeted modification of host ATP homeostasis via bacterial effector proteins.

Chromosome segment substitution lines (CSSLs) represent a powerful genetic resource for quantitative trait loci (QTL) mapping, gene cloning and breeding. Here, we developed two sets of CSSLs consisting of 245 and 128 unique lines, which derived from OX2028 x K326 and Samsun x K326 crosses. On average, each CSSL carried 1.8 and 2.9 introgressed segments in the two sets, with an average physical segment length of approximately 34.3 Mb and 27.6 Mb, respectively. These CSSLs covered similar to 97 % and similar to 77 % of the genomes of OX2028 and Samsun, respectively. By performing QTL mapping based on best linear unbiased prediction (BLUP) of phenotypic traits, we identified a total of 64 QTLs associated with six agronomic traits and three disease resistance traits. These QTLs explained phenotypic variation ranging from 1.5 % to 50.8 %. Among them, 22 QTLs detected in OX2028 derived population and 42 detected in Samsun derived CSSLs. Notably, a new QTL for tobacco leaf width, qLW1-1 was narrowed down to an 8-Mb interval on chromosome 1, and NtZY01G00114, encoding an auxin-response factor protein, was considered as the candidate gene. Our study provides valuable genetic resources for tobacco breeding and enhances our understanding of the genetic basis of complex traits in tobacco.

Tobacco is a significant industrial crop, serving as a model for plant science and a promising specie for the production of proteins and small molecules. However, system biology studies of tobacco under natural field cultivation conditions remain scarce. Here, we construct a genome-scale metabolic regulatory network through integration of dynamic transcriptomic and metabolomic profiles from field-grown tobacco leaves across two ecologically distinct regions. We map 25,984 genes and 633 metabolites into 3.17 million regulatory pairs using multi-algorithm integration. This network reveals three pivotal transcriptional hubs, including NtMYB28 (promoting hydroxycinnamic acids synthesis by modifying Nt4CL2 and NtPAL2 expression), NtERF167 (amplifying lipid synthesis via NtLACS2 activation) and NtCYC (driving aroma production through NtLOX2 induction). These transcriptional hubs achieve substantial yield improvements of target metabolites by rewiring metabolic flux. The present work provides a systems-level atlas of tobacco metabolic regulation and may help to guide metabolic engineering.

The biochemical and transcriptional regulatory mechanisms of chlorophyll metabolism have been extensively studied, but the translational regulatory mechanisms remain poorly understood. In this study, we found that Nt DHS1 deficiency in N. tabacum resulted in smaller leaves and increased leaf chlorophyll content. Protein content determination experiments revealed that the global protein synthesis of the Ntdhs1 mutant was decreased. A ribosome profiling sequence (Ribo-seq) assay showed that the translation level of genes related to cell growth was significantly reduced, while the translation level of chlorophyll metabolism related genes was significantly increased in Ntdhs1 mutant. Biochemical analysis further demonstrated that Nt DHS interacts with the translation initiation factor Nt eIF5A. Moreover, the Nteif5a1 mutant exhibited phenotypes similar to the Ntdhs1 mutant, including a reduced translation level of cell growth related genes and increased translation level of chlorophyll metabolism related genes. Our studies suggest that the Nt DHS-Nt eIF5A complex regulates leaf senescence by modulating the translation of specific genes.

The epigenomic landscape regulates gene expression and chromatin dynamics, with histone and RNA modifications playing crucial roles. Although studies have elucidated the interactions among chromatin modifications, DNA methylation, and mRNA modifications, the relationships among RNA modifications and their collective influence on RNA metabolism remain poorly understood. Grasping these epigenetic mechanisms is essential for improving crop resilience and productivity. In this study, we explored the co-occurrence and functional interactions of three significant mRNA modifications in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa): N-4-acetylcytidine (ac(4)C), N-6-methyladenosine (m(6)A), and 5-methylcytosine (m(5)C). Our results indicate that these modifications frequently coexist in the same transcripts, exhibiting distinct spatial distributions across species. Notably, the m(6)A modification enhances the ac(4)C-mediated destabilization of RNA secondary structures, especially when modifications are clustered, thereby promoting RNA stability. In Arabidopsis, the ac(4)C modification improved translational efficiency and the m(6)A modification amplified this effect in a distance-dependent manner; by contrast, in rice the influence of m(6)A is independent of distance. The m(5)C modification has minimal impact on RNA structure or stability but modulates m(6)A-associated transcript stability in a context-dependent manner. Our findings shed light on the dynamic regulatory code of combinatorial RNA modifications, highlighting species-specific mechanisms of post-transcriptional regulation. This research offers valuable insights into the intricate interplay of RNA modifications, with implications for advancing agricultural biotechnology through a deeper understanding of plant RNA functionality.

Some pathogens colonize plant leaves, but others invade the roots, including the vasculature, causing severe disease symptoms. Plant innate immunity has been extensively studied in leaf pathosystems; however, the precise regulation of immunity against vascular pathogens remains largely unexplored. We previously demonstrated that loss of function of the receptor kinase FERONIA (FER) increases plant resistance to the typical vascular bacterial pathogen Ralstonia solanacearum. Here, we show that upon infection with R. solanacearum, root xylem cell walls in Arabidopsis thaliana become highly lignified. FER is specifically upregulated in the root xylem in response to R. solanacearum infection, and inhibits lignin biosynthesis and resistance to this pathogen. We determined that FER interacts with and phosphorylates the transcription factor RESPONSIVE TO DESICCATION 26 (RD26), leading to its degradation. Overexpression and knockout of RD26 demonstrated that it positively regulates plant resistance to R. solanacearum by directly activating the expression of lignin-related genes. Tissue-specific expression of RD26 in the root xylem confirmed its role in vascular immunity. We confirmed that the FER-RD26 module regulates lignin biosynthesis and resistance against R. solanacearum in tomato ( Solanum lycopersicum). Taken together, our findings unveil that the FER-RD26 cascade governs plant immunity against R. solanacearum in vascular tissues by regulating lignin deposition. This cascade may represent a key defense mechanism against vascular pathogens in plants.

Being as a useful model plant for biological study and significant industrial-crop that is sensitive to low temperature, tobacco is frequently adopted to evaluate functions of interested genes from other species by heterologous expression. Dehydration-responsive element binding (DREB) proteins are a type of transcription factors involved in plant responses to abiotic/biotic stimuli. However, genetic information and molecular-physiological roles of tobacco DREBs are little described. Here, we identified 20 NtDREBs containing a typical AP2-domain and phylogenetically classified them into five subgroups (A2-A6). Inspecting promoter sequences of NtDREBs revealed a different enrichment of certain abiotic stress-related cis-elements including 'MeJA- and ABAresponsive and A/T-rich binding' components. Analysis of publicly available RNA-seq data showed that many NtDREBs were transcriptionally regulated by cold-treatment. Furthermore, we assessed physiological roles in tobacco of NtDREB_A2.1, because of its rapid and/or strong expression responsive to low temperature and plant P- and N-nutritional status. Overexpression of NtDREB_A2.1 in tobacco cultivated under 15 degrees C improved the growth with increased plant-height and leaf-area compared to WT. Interestingly, NtDREB_A2.1 overexpression negatively influenced plant growth under conditions of low/high N-supply and 15 degrees C, whereas NtDREB_A2.1transgenic lines could effectively utilize external low- and/or high P for better growth with increased N- and Paccumulation under 15-25 degrees C. Our work added not only new members to the DREB-subfamily but a valuable molecular-target for engineering of crops, aiming at improvement of plant cold-tolerance and utilization of soil phosphorus occurring at low available levels for plants in most soils.

Transfer RNA (tRNA) can produce smaller RNA fragments called tRNA-derived fragments (tRFs). tRFs play critical roles in multiple cellular programs, although the functional mechanisms of tRFs remain largely unknown in plants. In this study, we examined the phenotype associated with 5' tRF-Ala (tRF-Ala, produced from tRNA-Ala) overexpression and knockdown lines (tDR-Ala-OE and tDR-Ala-kd, respectively) and the mechanisms by which tRF-Ala affects mRNA levels in Arabidopsis (Arabidopsis thaliana). We investigated the candidate proteins associated with tRF-Ala by quantitative proteomics and confirmed the direct interaction between tRF-Ala and the splicing factor SERINE-ARGININE RICH PROTEIN 34 (SR34). A transcriptome sequencing analysis showed that 318 genes among all the genes (786) with substantial alternative splicing (AS) variance in tDR-Ala-OE lines are targets of SR34. tRF-Ala diminished the binding affinity between SR34 and its targets by direct competition for interaction with SR34. These findings reveal the critical roles of tRF-Ala in regulating mRNA levels and splicing.

Nitrogen (N) is the key essential macronutrient for crop growth and yield. Over-application of inorganic N fertilizer in fields generated serious environmental pollution and had a negative impact to human health. Therefore, improving crop N use efficiency (NUE) is helpful for sustainable agriculture. The biological functions of nitrogen transporters and regulators have been intensively studied in many crop species. However, only a few nitrogen transporters have been identified in tobacco to date. We reported the identification and functional characterization of a nitrate transporter NtNPF2.11 from tobacco (Nicotiana tabacum). qRT-PCR assay revealed that NtNPF2.11 was mainly expressed in leaf and vein. Under middle N (MN, 1.57 kg N/100 m2) and high N (HN, 2.02 kg N/100 m2) conditions, overexpression of NtNPF2.11 in tobacco greatly improved N utilization and biomass. Moreover, under middle N and high N conditions, the expression of genes for nitrate assimilation, such as NtNR1, NtNiR, NtGS and NtGOGAT, were upregulated in NtNPF2.11 overexpression plants. Compared with WT, overexpression of NtNPF2.11 increased potassium (K) accumulation under high N conditions. These results indicated that overexpression of NtNPF2.11 could increase tobacco yield, N and K accumulation under higher N conditions. Overall, these findings improve our understanding the function of NtNPF2.11 and provide useful gene for sustainable agriculture.

为构建烟草全基因组模块库,开展烟草分子模块育种,以K326为轮回亲本,烤烟OX2028和香料烟Samsun为供体亲本,经过杂交、连续多代回交、自交,最终获得两套以K326为背景,覆盖供体亲本整个基因组的分子模块库.分子模块的基因组大部分回复到了轮回亲本K326,其田间农艺及抗病性等重要性状偏向亲本K326,不同材料间存在较广泛的遗传变异;群体的基本农艺性状呈现连续的正态或近似正态分布,符合数量性状的表型分布特性;鉴定了7个重要性状显著改变的分子模块,其中较K326中上部叶片开片明显改善的材料32份,叶数明显提高的材料25份,TMV、CMV、PVY病毒病抗性明显提高的材料分别为111、25和5份,黑胫病、青枯病抗性显著提高的材料44和52份.构建的烟草分子模块库可用于后续开展烟草重要性状的QTL定位和基因功能研究.

There are abundant polyphenols in tobacco leaves mainly including chlorogenic acid (CGA), rutin, and scopoletin, which not only influence plant growth, development, and environmental adaptation, but also have a great impact on the industrial utilization of tobacco leaves. Few transcription factors regulating the biosynthesis of polyphenols have been identified in tobacco so far. In this study, two NtWRKY33 genes were identified from N. tabacum genome. NtWRKY33a showed higher transcriptional activity than NtWRKY33b, and encoded a nuclear localized protein. Overexpression and knock-out of NtWRKY33a gene revealed that NtWRKY33a inhibited the accumulation of rutin, scopoletin, and total polyphenols, but meanwhile promoted the biosynthesis of CGA. Chromatin immunoprecipitation and Dual-Luc assays indicated that NtWRKY33a could directly bind to the promoters of NtMYB4 and NtHCT, and thus induced the transcription of these two genes. The contents of polyphenols in ntwrky33a, ntmy4, and ntwrky33a/ntmyb4 mutants further confirmed that the repression of NtWRKY33a on the biosynthesis of rutin, scopoletin, and total polyphenols depends on the activity of NtMYB4. Moreover, the promotion of NtHCT by NtWRKY33a modulates the distribution of metabolism flux into the synthesis of CGA. Ectopic expression of NtWRKY33a inhibit the expression of NtSAUR14, NtSAUR59, NtSAUR66, NtIAA4, NtIAA17, and NtIAA19 genes, indicating that NtWRKY33a might be involved in the regulation of plant auxin response. Our study revealed new functions of NtWRKY33a in regulating the synthesis of polyphenols, and provided a promising target for manipulating polyphenols contents in tobacco.

Being as a useful model plant for biological study and significant industrial-crop that is sensitive to low temperature, tobacco is frequently adopted to evaluate functions of interested genes from other species by heterologous expression. Dehydration-responsive element binding (DREB) proteins are a type of transcription factors involved in plant responses to abiotic/biotic stimuli. However, genetic information and molecular-physiological roles of tobacco DREBs are little described. Here, we identified 20 NtDREBs containing a typical AP2-domain and phylogenetically classified them into five subgroups (A2-A6). Inspecting promoter sequences of NtDREBs revealed a different enrichment of certain abiotic stress-related cis-elements including ‘MeJA- and ABA-responsive and A/T-rich binding’ components. Analysis of publicly available RNA-seq data showed that many NtDREBs were transcriptionally regulated by cold-treatment. Furthermore, we assessed physiological roles in tobacco of NtDREB_A2.1, because of its rapid and/or strong expression responsive to low temperature and plant P- and N-nutritional status. Overexpression of NtDREB2.1 in tobacco cultivated under 15 oC improved the growth with increased plant-height and leaf-area compared to WT. Interestingly, NtDREB_2A.1overexpression negatively influenced plant growth under conditions of low/high N-supply and 15oC, whereas NtDREB_2A.1-transgenic lines could effectively utilize external low- and/or high P for better growth with increased N- and P-accumulation under 15-25oC. Our work added not only new members to the DREB-subfamily but a valuable molecular-target for engineering of crops, aiming at improvement of plant cold-tolerance and utilization of soil phosphorus occurring at low available levels for plants in most soils.

Summary Receptor‐like kinases (RLKs) constitute the largest receptor family involved in the regulation of plant immunity and growth, but small‐molecule inhibitors that target RLKs to improve agronomic traits remain unexplored. The RLK member FERONIA (FER) negatively regulates plant resistance to certain soil‐borne diseases that are difficult to control and cause huge losses in crop yields and economy. Here, we identified 33 highly effective FER kinase inhibitors from 1494 small molecules by monitoring FER autophosphorylation in vitro . Four representative inhibitors (reversine, cenisertib, staurosporine and lavendustin A) inhibited the kinase activity of FER and its homologues in several crops by targeting the conserved ATP pocket in the kinase structure. FER contributes to the physiological impact of representative inhibitors in plants. The treatment of roots with reversine, staurosporine and lavendustin A enhanced innate immunity in plant roots and thus alleviated soil‐borne diseases in tobacco, tomato and rice without growth penalties. Consistently, RNA sequencing assays showed that lavendustin A and reversine exert profound impacts on immunity‐related gene expression. Our results will set a new milestone in the development of the plant RLK kinase regulation theory and provide a novel strategy for the prevention and control of plant soil‐borne diseases without growth penalties.

IntroductionNicotiana L. (Solanaceae) is of great scientific and economic importance, and polyploidization has been pivotal in shaping this genus. Despite many previous studies on the Nicotiana phylogenetic relationship and hybridization, evidence from whole genome data is still lacking.MethodsIn this study, we obtained 995 low-copy genes and plastid transcript fragments from the transcriptome datasets of 26 Nicotiana species, including all sections. We reconstructed the phylogenetic relationship and phylogenetic network of diploid species.ResultsThe incongruence among gene trees showed that the formation of N. sylvestris involved incomplete lineage sorting. The nuclear–plastid discordance and nuclear introgression absence indicated that organelle capture from section Trigonophyllae was involved in forming section Petunioides. Furthermore, we analyzed the evolutionary origin of polyploid species and dated the time of hybridization events based on the analysis of PhyloNet, sequence similarity search, and phylogeny of subgenome approaches. Our results highly evidenced the hybrid origins of five polyploid sections, including sections Nicotiana, Repandae, Rusticae, Polydicliae, and Suaveolentes. Notably, we provide novel insights into the hybridization event of section Polydicliae and Suaveolentes. The section Polydicliae formed from a single hybridization event between maternal progenitor N. attenuata and paternal progenitor N. undulata; the N. sylvestris (paternal progenitor) and the N. glauca (maternal progenitor) were involved in the formation of section Suaveolentes.DiscussionThis study represents the first exploration of Nicotiana polyploidization events and phylogenetic relationships using the high-throughput RNA-seq approach. It will provide guidance for further studies in molecular systematics, population genetics, and ecological adaption studies in Nicotiana and other related species.