Virus-induced Gene Silencing System Research Articles

Virus-Induced Gene Silencing (VIGS) in Hydrangea macrophylla and Functional Analysis of HmF3'5'H.

Hydrangea macrophylla, renowned for its large inflorescences and a diverse range of colors, highlights a significant limitation in current gene function research, which is the lack of effective molecular genetic tools. This study utilized a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate gene function through posttranscriptional gene silencing in H. macrophylla for the first time. The ortholog of phytoene desaturase (PDS) in H. macrophylla, termed HmPDS, was identified. Infection of tissue-cultured seedlings with TRV-HmPDS led to photobleaching of the leaves. Additionally, infection with TRV containing the HmCHS1 fragment in the flowers resulted in decreased anthocyanin production in sepals and a lightening of sepal coloration in the infected flowers. The phenomena and RT-qPCR results proved that the PDS and CHS genes of hydrangea were successfully silenced via the vacuum infiltration method. Furthermore, the introduction of TRV-HmF3'5'H revealed a decrease in delphinidin-3-glucoside content in sepals and caused a color change in the sepals from blue to pink. This study demonstrated that the TRV-VIGS system was successfully established in H. macrophylla and effectively applied to the function analysis of HmF3'5'H.

Efficient virus-induced gene silencing in Primulina using two virus vector systems

Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing method and an effective technique for analyzing plant gene function. However, to date there have been no reports on the establishment of a VIGS system in Primulina, which includes over 200 species of plants, most of which have high ornamental value. In this study, we successfully established a VIGS system in P. 'Alfonso' and P. carinata using TRV vector system with phytoene desaturase (PDS) as marker gene. After being treated with pTRV2-PDS vector, both P. 'Alfonso' and P. carinata plants showed a pronounced photobleaching phenotype. The system was further optimized by exploring different infection conditions. The optimized TRV-based VIGS system was as follows: Agrobacterium OD600 = 0.5, leaf vacuum infiltration, and the silencing efficiency of the system can reach 75 %. Chlorata42 was used as another marker gene and further verified the establishment of TRV VIGS system in P. 'Alfonso'. In addition, the CaLCuV-based VIGS system was also established with PDS as marker gene in P. 'Alfonso'. The establishment of the TRV-based and CaLCuV-based VIGS systems provided a breakthrough in the validation methods for identifying gene function in Primulina genus.

Rapid and Visual Screening of Virus Infection in Sugar Beets Through Polerovirus-Induced Gene Silencing.

Since the ban of neonicotinoid insecticides in the European Union, sugar beet production is threatened by outbreaks of virus yellows (VY) disease, caused by several aphid-transmitted viruses, including the polerovirus beet mild yellowing virus (BMYV). As the symptoms induced may vary depending on multiple infections and other stresses, there is an urgent need for fast screening tests to evaluate resistance/tolerance traits in sugar beet accessions. To address this issue, we exploited the virus-induced gene silencing (VIGS) system, by introducing a fragment of a Beta vulgaris gene involved in chlorophyll synthesis in the BMYV genome. This recombinant virus was able to generate early clear vein chlorosis symptoms in infected sugar beets, allowing easy and rapid visual discernment of infected plants across five sugar beet lines. The recombinant virus displayed similar infectivity as the wild-type, and the insert remained stable within the viral progeny. We demonstrated that the percentage of VIGS-symptomatic plants was representative of the infection rate of each evaluated line, and depending on the susceptibility of the line to BMYV infection, VIGS symptoms may last over months. Our work provides a polerovirus-based VIGS system adapted to sugar beet crop allowing visual and rapid large-scale screens for resistance or functional genomic studies.

Developing and applying a virus-induced gene silencing system for functional genomics in walnut (Juglans regia L.) mediated by tobacco rattle virus

Walnut (Juglans regia L.) is a high-value tree species planted worldwide, but the incomplete less developed genetic transformation system limits its gene function analysis. In this study, virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) technology was applied to walnut seedlings to degrade the transcript of target gene. Different infiltration methods were used to explore the effects of infection mode, Agrobacterium cell density, silencing fragment length, and walnut cultivars. The results showed that spray infiltration of seedlings resulted in a photobleaching phenotype of the whole plant. Leaf injection was a more effective way of infiltration. The optimal combination was the Agrobacterium cell density at OD600 = 1.1 with target fragment = 255 bp for the treatment of walnut early-fruiting cultivar ’Xiangling.’ This combination can reach up to 48 % of gene silencing efficiency. Based on this optimized VIGS system, silencing a walnut chlorophyll synthesis-related gene, JrPOR (Protochlorophyllide reductase), to further validate the system’s effect. The results showed that the expression of JrPOR was significantly repressed, and the chlorophyll level of the silenced plants was significantly decreased compared with the control. The above results indicate that the walnut TRV-VIGS system has been successfully established and can be used for reverse genetic studies, providing an option for verifying gene function in walnut.

An Engineered Citrus Tristeza Virus (T36CA)-Based Vector Induces Gene-Specific RNA Silencing and Is Graft Transmissible to Commercial Citrus Varieties.

A protein-expressing citrus tristeza virus-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus-induced gene silencing system intended for use with studies of California citrus. Virus-induced gene silencing constructs engineered with a truncated Citrus macrophylla PHYTOENE DESATURASE (CmPDS) gene sequence in the sense or antisense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two nonsynonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3, were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 weeks postinoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8- to 12-month postinoculation window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 months postinoculation, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.

Abscisic acid-, stress-, ripening-induced 2 like protein, TaASR2L, promotes wheat resistance to stripe rust.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive wheat diseases. The plant hormone abscisic acid (ABA) plays a key regulatory role in plant response to stress. ABA-, stress-, ripening-induced proteins (ASR) have been shown to be abundantly induced in response to biotic and abiotic stresses to protect plants from damage. However, the function of wheat ASR2-like protein (TaASR2L) in plants under biotic stress remains unclear. In this study, transient silencing of TaASR2L using a virus-induced gene silencing system substantially reduced wheat resistance to Pst. TaASR2L interaction with serine/arginine-rich splicing factor SR30-like (TaSR30) was validated mainly in the nucleus. Knockdown of TaSR30 expression substantially reduced wheat resistance to Pst. Overexpression of TaASR2L and TaSR30 demonstrated that they can promote the expression of ABA- and resistance-related genes to enhance wheat resistance to Pst. In addition, the expression levels of TaSR30 and TaASR2L were substantially increased by exogenous ABA, and the resistance of wheat to Pst was increased, and the expression of PR genes was induced. Therefore, these results suggest that TaASR2L interacts with TaSR30 by promoting PR genes expression and enhancing wheat resistance to Pst.

CsNAC17 enhances resistance to Colletotrichum gloeosporioides by interacting with CsbHLH62 in Camellia sinensis

Abstract The pathogen Colletotrichum gloeosporioides causes anthracnose, a serious threat to tea trees around the world, particularly in warm and humid regions. RNA-Seq data have previously indicated NAC transcription factors are involved in anthracnose resistance, but underlying mechanisms remain unclear. The BiFC, Split-LUC, and Co-IP assays validated the interaction between CsbHLH62 and CsNAC17 identified through yeast two-hybrid (Y2H) screening. CsNAC17 or CsbHLH62 overexpression enhanced anthracnose resistance, as well as enhanced levels of H2O2, hypersensitivity, and cell death in Nicotiana benthamiana. The NBS-LRR gene CsRPM1 is regulated by CsNAC17 by binding directly to its promoter (i.e., CACG, CATGTG), while CsbHLH62 facilitates CsNAC17’s binding and increases trascriptional activity of CsRPM1. Additionally, transient silencing of CsNAC17 and CsbHLH62 in tea plant leaves using the virus-induced gene silencing (VIGS) system resulted in decreased resistance to anthracnose. Conversely, transient overexpression of CsNAC17 and CsbHLH62 in tea leaves significantly enhanced the resistance against anthracnose. Based on these results, it appears that CsbHLH62 facilitates the activity of CsNAC17 on CsRPM1, contributing to increased anthracnose resistance.

Overexpression of GmSRC2 confers resistance to Phytophthora sojae in soybean

Phytophthora root and stem rot caused by Phytophthora sojae (P. sojae) is one of the most destructive diseases to affect soybean (Glycine max (L.) Merr) production. GmSRC2 that encodes a C2 domain-containing protein can respond to various stresses, however, the molecular mechanism of GmSRC2 in resistance of soybean to P. sojae is yet to be fully elucidated. In this study, GmSRC2 was found to be significantly up-regulated under P. sojae treatment; GmSRC2-overexpression (OE) transgenic lines and GmSRC2-silencing transient plants were generated via Agrobacterium tumefaciens mediated transformation and virus-induced gene silencing (VIGS) system, respectively. Infected leaves and cotyledons of OE-GmSRC2–1 and OE-GmSRC2–2 lines showed significant decreases in the disease symptoms and P. sojae biomass than those of wild type (WT); the activities of superoxide dismutase (SOD) and peroxidase (POD) confirmed the accumulation of reactive oxygen species (ROS) in overexpressed transgenic lines. Whereas, silencing of GmSRC2 severely increased the disease symptoms and the biomass of P. sojae. Further, we confirmed that GmSRC2 interacted with the effector PsAvh23 of P. sojae, and the C2 domain was crucial for the interaction. Overexpression of GmSRC2 upregulated the ADA2/GCN5 module upon P. sojae. The aforementioned results demonstrated that GmSRC2 played vital roles in regulating soybean resistance to oomycetes.

Construction and optimization of the TRV-mediated VIGS system in Areca catechu embryoids

Areca catechu, a member of the palm family Arecaceae native to tropical regions, holds significant economic importance as a tropical crop. This study explores the application of virus-induced gene silencing (VIGS) in A. catechu embryogenic callus tissues, utilizing the phytoene desaturase gene (AcPDS) of A. catechu as a reporter gene. Results demonstrate that employing EHA105 strain, an Agrobacterium infection liquid concentration of OD600 = 0.5, an infection duration of 5 min, acetosyringone (AS) concentration of 21.5 mg/L, co-cultivation at 19 °C for 2 days followed by 28 °C for 3 days, and a subsequent 14-day post-co-cultivation period, the experimental group (pTRV1+pTRV2-AcPDS) exhibited significantly reduced gene expression compared to the control group CK (pTRV1+pTRV2), manifesting the anticipated photobleaching phenotype by the 21st day. RT-PCR analysis revealed that AcPDS gene expression in TRV-mediated photobleached A. catechu embryos decreased to 0.227 times that of the control group CK. This established VIGS system in A. catechu embryoids establishes an effective experimental platform for studying functional genes in A. catechu.

Enhancing virus-induced gene silencing efficiency in tea plants (Camellia sinensis L.) and the functional analysis of CsPDS

Virus-induced gene silencing (VIGS) is an effective technology for gene functional analysis in tea plants, yet its efficiency needs enhancement, particularly in the Qiancha 1 (QC1) cultivar of Camellia sinensis. Employing pTRV1/pTRV2-CsPDS as a vector and leaf albinism rate as a silencing index, this study optimized the VIGS system by examining the effects of vacuum treatment duration, pressure, injection method, and cutting size on silencing efficiency. The results indicate that both injection and vacuum infiltration methods are viable in QC1, with the vacuum method exhibiting superior efficiency. The optimal vacuum infiltration conditions were determined to be 0.8 kPa for approximately 5 min. Phenotypic analysis showed that silencing the CsPDS gene resulted in leaves yellowing to varying degrees. Transcriptome analysis revealed disruptions in various pathways in these tea cuttings with differing levels of coloration, particularly in photosynthetic pigment biosynthesis and nitrogen metabolism. Interestingly, the content of theanine was higher in the cuttings with lower chlorophyll content. Analysis of differential gene expression profile and quantitative real-time PCR confirms that theanine accumulation is primarily due to increased synthesis and reduced degradation. These findings deepen our understanding of the metabolic and molecular mechanisms behind leaf coloration in tea plants.

A G6P1E isomerase of sugar metabolism is involved in the flower colors of Dianthus chinensis

Dianthus chinensis L. is indigenous to northern China, Korea, Mongolia, Kazakhstan, and southeastern Russia. It is widely cultivated in urban landscapes. Its flower has a great variety of colors and color schemes. Sugars control and induce anthocyanin synthesis and accumulation in plants. In sugar metabolism, many enzymes are specific for their substrate’s α or β anomer. Gaining and characterizing genes involved in sugar metabolism and flower color will be beneficial in clarifying the role of sugar in the flower colors of D. chinensis. Glucose-6-phosphate-1-epimerase (G6P1E, EC 5.1.3.15) catalyzes the α or β change of glucose-6-phosphate at the branch point of glucose metabolism. DchG6P1E1 (MZ292712) was isolated in D. chinensis and characterized using the tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system. Its cDNA full length is 1401 bp, including an open reading frame of 918 bp. In the DchG6P1E1-silenced flowers, the reducing purple was observed, as well as the anthocyanin content, reducing sugar content, G6P1E activity, and DchG6P1E1 expression were significantly decreased. During the development of floral buds and among the three flower colors, the anthocyanin content, reduced sugar content, G6P1E activity, and DchG6P1E1 expression rose dramatically, with pigments increasing in the petals. Among the organs, the flowers had the highest anthocyanin contents and reducing sugar. The highest levels of G6P1E activity and DchG6P1E1 expression were in the roots. The anthocyanin content was positively related to the reducing sugar content at 0.05 levels by correlation analysis. In conclusion, DchG6P1E1 is a root-enriched gene associated with flower colors in D. chinensis.

Characterisation and Expression Analysis of LdSERK1, a Somatic Embryogenesis Gene in Lilium davidii var. unicolor.

The Lanzhou lily (Lilium davidii var. unicolor) is a variant of the Sichuan lily of the lily family and is a unique Chinese 'medicinal and food' sweet lily. Somatic cell embryogenesis of Lilium has played an important role in providing technical support for germplasm conservation, bulb propagation and improvement of genetic traits. Somatic embryogenesis receptor-like kinases (SERKs) are widely distributed in plants and have been shown to play multiple roles in plant life, including growth and development, somatic embryogenesis and hormone induction. Integrating the results of KEGG enrichment, GO annotation and gene expression analysis, a lily LdSERK1 gene was cloned. The full-length open reading frame of LdSERK1 was 1875 bp, encoding 624 amino acids. The results of the phylogenetic tree analysis showed that LdSERK1 was highly similar to rice, maize and other plant SERKs. The results of the subcellular localisation in the onion epidermis suggested that the LdSERK1 protein was localised at the cell membrane. Secondly, we established the virus-induced gene-silencing (VIGS) system in lily scales, and the results of LdSERK1 silencing by Tobacco rattle virus (TRV) showed that, with the down-regulation of LdSERK1 expression, the occurrence of somatic embryogenesis and callus tissue induction in scales was significantly reduced. Finally, molecular assays from overexpression of the LdSERK1 gene in Arabidopsis showed that LdSERK1 expression was significantly enhanced in the three transgenic lines compared to the wild type, and that the probability of inducing callus tissue in seed was significantly higher than that of the wild type at a concentration of 2 mg/L 2,4-D, which was manifested by an increase in the granularity of the callus tissue.

Establishment of a Virus-Induced Gene-Silencing (VIGS) System in Passiflora edulis Sims and Its Use in the Functional Analysis of PechlH

Passion fruit is a very important tropical and subtropical fruit that not only has a good edible flavour, but also has high ornamental value. However, the functions of important trait genes in passion fruit have been difficult to elucidate due to the difficulty of stable genetic transmission in passion fruit. The VIGS system for the tobacco rattle virus (TRV) has been widely used in a number of crops, but no success has been reported in passion fruit. In this study, we used the PePDS marker gene to unravel the passion fruit VIGS system. The use of albino phenotype observation and quantitative PCR to detect gene expression showed that the PDS albino phenotype was successfully induced in the young leaves of passion fruit seedlings after 7 days, while a significant down-regulation of expression occurred in the phenotypic plants. The albino phenotype can be maintained for 14–16 d. In addition, we chose the chlorophyll-synthesis-related gene PechlH for the VIGS silencing of passion fruit leaves. When the PechlH gene was knocked out, the leaves of the passion fruit lost their green colour and showed yellowing symptoms. This revealed that the PechlH gene was involved in the chloroplast synthesis pathway. Finally, using different OD600 gradients in PePDS, we demonstrated that the silencing efficiency reached 46.70% at an OD600 of 0.8. Thus, an effective and stable VIGS system was achieved in passion fruit. This will help to accelerate the study of gene function in passion fruit in the future.

Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish

Virus-induced gene silencing (VIGS) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology. However, the investigation of gene silencing and editing in radish remains limited. In this study, a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus (TRV)- and turnip yellow mosaic virus (TYMV)-mediated gene silencing vectors. The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish. The expression level of RsPDS was significantly inhibited using VIGS in ‘NAU-067’ radish leaves. The rootless seedlings of ‘NAU-067’ were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences. Nine adventitious roots were blue with GUS staining, and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing. Furthermore, albino lines were generated with A. tumefaciens-mediated transformation of radish cotyledons. Five base substitutions and three base deletions occurred at target sequence 2 in Line 1, and three base insertions and three base substitutions occurred at target sequence 1 in Line 2. This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish, which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

Establishment of virus-induced gene-silencing system in Juglans sigillata Dode and functional analysis of JsFLS2 and JsFLS4

Juglans sigillata Dode is one of the important tree species in southwest China, and it has significant economic and ecological value. However, there is still a lack of effective methods to identify the functional genes of J. sigillata. By verifying the model plant tobacco, the pTRV2::JsPDS vector was able to cause photobleaching. This study showed that photobleaching occurred 24 and 30 d after the silencing vector was infected with aseptic seedlings and fruits of J. sigillata, respectively. When the OD600 was 0.6, and the injection dose was 500 μL, the gene silencing efficiency of aseptic seedlings was the highest at 16.7 %, significantly better than other treatments. Moreover, when the OD600 was 0.8, and the injection dose was 500 μL, the gene silencing efficiency in the walnut fruit was the highest (20 %). In addition, the VIGS system was successfully used to silence JsFLS2 and JsFLS4 genes in J. sigillata. This study also showed that the flavonol content and gene expression in the treatment group were decreased compared to the control group. In addition, the proteins transcribed and translated from the JsFLS4 gene may have higher catalytic activity for dihydroquercetin. The above results indicate that the TRV-mediated VIGS system can be an ideal tool for studying J. sigillata gene function.

A simple and effective VIGS system facilitates the control of citrus canker by silencing CsLOB1

Virus-induced gene silencing (VIGS) is a powerful technology for molecular characterization of gene functions in plants and has the potential to prevent and control plant diseases. Unfortunately, VIGS in many woody perennials such as citrus are severely hindered because they have a long juvenile period and are recalcitrant to infection compared to herbaceous plants. Here, we report the development of a simple and effective VIGS system based on citrus leaf blotch virus (CLBV), a virus endemic in most citrus-producing countries but showing no symptoms in most citrus varieties. By Agrobacterium-mediated vacuum-infiltration (AVI), pCLBV201-su245 and pCLBV201-pds391 triggered efficient gene silencing (85%) and silencing phenotypes began to appear at 30 days post infiltration/inoculation (dpi), a similar period for achieving that on herbaceous plants. Moreover, the silencing phenotypes could still be observed four years post inoculation. Further, pCLBV201-lob369 was constructed to silence the gene lateral organ boundaries1 (lob1), encoding a key factor for susceptibility to citrus canker that caused by Xanthomonas citri ssp. citri (Xcc). The resistance evaluation results showed that the lesion area, disease index, and bacterial content in the pCLBV201-lob369 treatment group were decreased by 64%, 14%, and 67%, respectively. This work provides a simple and effective VIGS system for citrus which has the potential to be used for diseases control.

Applications of Virus-Induced Gene Silencing in Cotton.

Virus-induced gene silencing (VIGS) is an RNA-mediated reverse genetics technique that has become an effective tool to investigate gene function in plants. Cotton is one of the most important economic crops globally. In the past decade, VIGS has been successfully applied in cotton functional genomic studies, including those examining abiotic and biotic stress responses and vegetative and reproductive development. This article summarizes the traditional vectors used in the cotton VIGS system, the visible markers used for endogenous gene silencing, the applications of VIGS in cotton functional genomics, and the limitations of VIGS and how they can be addressed in cotton.

Construction and application of a virus-induced gene silencing system in taro

Construction and application of a virus-induced gene silencing system in taro

Phenotype and signaling pathway analysis to explore the interaction between tomato plants and TYLCV in different organs

Tomato yellow leaf curl disease (TYLCD), caused by Tomato yellow leaf curl virus (TYLCV), is one of the most destructive diseases in tomato cultivation. By comparing the phenotypic characteristics and virus quantities in the susceptible variety 'Cooperation 909 Red Tomatoes' and the resistant variety 'Huamei 204' after inoculation with TYLCV infectious clones, our study discovered that the root, stem and leaf growth of the susceptible variety 'Cooperation 909 Red Tomatoes' were severely hindered and the resistant variety 'Huamei 204' showed growth inhibition only in roots. TYLCV accumulation in roots were significantly higher than in leaves. Further, we examined the expression of key genes in the SA and JA signalling pathways in leaves, stems and roots and found the up-regulation of SA-signalling genes in all organs of the susceptible variety after inoculation with TYLCV clones. Interestingly, SlJAZ2 in roots of the resistant variety was significantly down-regulated upon TYLCV infection. Further, we silenced the SlNPR1 and SlCOI1 genes individually using virus induced gene silencing system in tomato plants. We found that viruses accumulated to a higher level in SlNPR1 silenced plants than wild type plants, and the virus quantity in roots was significantly increased in SlCOI1 silenced plants. These results provide new insights for advancing research in understanding tomato-TYLCV interaction.

Analysis of the emitted pattern of floral volatiles and cloning and functional analysis of the PsuLIS gene in tree peony cultivar ‘High Noon’

The floral volatiles, many of which have fragrances, are one of the important quality characteristics of ornamental plants. Tree peony (Paeonia suffruticosa) is a famous traditional Chinese flower that is well-known in many countries around the world. However, the composition and formation mechanism of volatiles in tree peony flowers is unclear. Here, we analyzed the pattern of floral volatiles emitted and further cloned and analyzed the function of the PsuLIS (linalool synthetase) gene in cultivated tree peony cultivar ‘High Noon’ cultivated in the field. 49 compounds of floral volatiles were detected by using GC–MS in cultivated tree peony cultivar ‘High Noon’, with linalool having the highest content among all substances, which is much higher than that of other substances. Their relative content was separately highest at full blooming during the flowering process and at petals in various floral organ. The diurnal change of floral volatiles is highest during 10:00–12:00 and lowest during 16:00–18:00. Furthermore, the PsuLIS gene was cloned with an open reading frame of 1698 bp and encodes 565 amino acids. The phylogenetic analysis showed that the protein of PsuLIS has the closest evolutionary relationship with LIS in Actinidia arguta. The expression pattern of the PsuLIS gene, which exhibits its highest expression during the full blooming stage, in petals and from 10:00–12:00, demonstrated consistency with the emission pattern of linalool and displayed a significant positive correlation. Additionally, tree peony cultivar ‘High Noon’ plants were obtained through knockdown using the virus-induced gene silencing (VIGS) system. The relative expression of the PsuLIS gene in transient overexpression plants and knockdown plants were separately increasing by 3.8 times and decreasing by 74.7% compared to wild type plants, and the expression pattern of the PsuLIS gene was consistent with the variation trend observed in linalool content across different genotype cultivated tree peony cultivar ‘High Noon’ plants. The recombinant PsuLIS protein can convert geranyl diphosphate (GPP) to linalool in vitro. The aforementioned findings indicate that the PsuLIS gene plays a crucial role in regulating linalool synthesis in cultivated tree peony cultivar ‘High Noon’, and laying the foundation for cultivating different fragrance types of peonies.