Virus-induced Gene Silencing System Research Articles

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.

Soybean 40S Ribosomal Protein S8 (GmRPS8) Interacts with 6K1 Protein and Contributes to Soybean Susceptibility to Soybean Mosaic Virus.

Soybean mosaic virus (SMV), a member of Potyvirus, is the most destructive and widespread viral disease in soybean production. Our earlier studies identified a soybean 40S ribosomal protein S8 (GmRPS8) using the 6K1 protein of SMV as the bait to screen a soybean cDNA library. The present study aims to identify the interactions between GmRPS8 and SMV and characterize the role of GmRPS8 in SMV infection in soybean. Expression analysis showed higher SMV-induced GmRPS8 expression levels in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting that GmRPS8 was involved in the response to SMV in soybean. Subcellular localization showed that GmRPS8 was localized in the nucleus. Moreover, the yeast two-hybrid (Y2H) experiments showed that GmRPS8 only interacted with 6K1 among the eleven proteins encoded by SMV. The interaction between GmRPS8 and 6K1 was further verified by a bimolecular fluorescence complementation (BiFC) assay, and the interaction was localized in the nucleus. Furthermore, knockdown of GmRPS8 by a virus-induced gene silencing (VIGS) system retarded the growth and development of soybeans and inhibited the accumulation of SMV in soybeans. Together, these results showed that GmRPS8 interacts with 6K1 and contributes to soybean susceptibility to SMV. Our findings provide new insights for understanding the role of GmRPS8 in the SMV infection cycle, which could help reveal potyviral replication mechanisms.

Development and application of virus-induced gene silencing (VIGS) for studying ApTT8 gene function in Agapanthus praecox ssp. orientalis

Agapanthus praecox ssp. orientalis is a famous summer-blooming perennial that produces hundreds of florets with blue or white tepal. In comparison, other colours are infrequent in the genus Agapanthus. Previous analysis revealed that delphinidin was the main but not the only anthocyanidin, petunidin and cyanidin also existed in the tepal. This indicated that the colouration and regulation mechanism in A. praecox was unique and worthy of study. However, the stable genetic transformation was low efficiency and time-consuming. A rapid and effective gene functional identification method was urgently needed. In this study, the first use of the VIGS system in A. praecox was described in detail. Tobacco rattle virus (TRV) caused systemic infection with mild symptoms in A. praecox, which was also confirmed by qRT-PCR. The reporter gene ApPDS (phytoene desaturase) was silenced using the injection and soaking method in basal plates of inflorescence, and the photo-bleaching symptoms were observed in tepal. The optimal Agrobacterium concentration was OD600 = 1.0. Then the VIGS system was used to silence a bHLH-like transcriptional factor, ApTT8, to identify its function in anthocyanin biosynthesis. The results showed that the total anthocyanin content was reduced to 12.21-fold compared with the empty control. Additionally, tepals were taken as recipients of VIGS using vacuum infiltration method. Total anthocyanin content was reduced to 3.44-fold in ApTT8-silenced tepal. The silencing efficiency of tepal was as high as 85.09 %. The structure genes, ApF3’H, ApF3’5’H, ApF3H, ApDFR, ApANS and ApUFGT were also significantly down-regulated. The successful silencing of the ApPDS and ApTT8 in this study indicated that the optimized TRV-mediated VIGS system could be used to target gene silencing in florets and tepal of A. praecox. This method will significantly shorten the time required for studying molecular mechanism of flower colouration and is of great significance for the research of A. praecox functional genomics.

Targeting wheat fusarium head blight with mycovirus-mediated VIGS

Fusarium head blight (FHB) caused by Fusarium graminearum occurs in wheat (Triticum aestivum) and threatens food production worldwide. Wheat lacks broad, durable FHB resistance. However, Zhang et al. developed a mycovirus-based virus-induced gene-silencing system in F. graminearum, providing efficient biocontrol of this devastating fungal disease.

Transcriptomic analysis of wheat reveals possible resistance mechanism mediated by Yr10 to stripe rust

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a catastrophic disease that threatens global wheat yield. Yr10 is a race-specific all-stage disease resistance gene in wheat. However, the resistance mechanism of Yr10 is poorly characterized. Therefore, to elucidate the potential molecular mechanism mediated by Yr10, transcriptomic sequencing was performed at 0, 18, and 48 h post-inoculation (hpi) of compatible wheat Avocet S (AvS) and incompatible near-isogenic line (NIL) AvS + Yr10 inoculated with Pst race CYR32. Respectively, 227, 208, and 4050 differentially expressed genes (DEGs) were identified at 0, 18, and 48 hpi between incompatible and compatible interaction. The response of Yr10 to stripe rust involved various processes and activities, as indicated by the results of Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Specifically, the response included photosynthesis, defense response to fungus, metabolic processes related to salicylic acid (SA) and jasmonic acid (JA), and activities related to reactive oxygen species (ROS). Ten candidate genes were selected for qRT-PCR verification and the results showed that the transcriptomic data was reliable. Through the functional analysis of candidate genes by the virus-induced gene silencing (VIGS) system, it was found that the gene TaHPPD (4-hydroxyphenylpyruvate dioxygenase) negatively regulated the resistance of wheat to stripe rust by affecting SA signaling, pathogenesis-related (PR) gene expression, and ROS clearance. Our study provides insight into Yr10-mediated resistance in wheat.

Small Heat Shock Protein (sHsp22.98) from Trialeurodes vaporariorum Plays Important Role in Apple Scar Skin Viroid Transmission.

Trialeurodes vaporariorum, commonly known as the greenhouse whitefly, severely infests important crops and serves as a vector for apple scar skin viroid (ASSVd). This vector-mediated transmission may cause the spread of infection to other herbaceous crops. For effective management of ASSVd, it is important to explore the whitefly's proteins, which interact with ASSVd RNA and are thereby involved in its transmission. In this study, it was found that a small heat shock protein (sHsp) from T. vaporariorum, which is expressed under stress, binds to ASSVd RNA. The sHsp gene is 606 bp in length and encodes for 202 amino acids, with a molecular weight of 22.98 kDa and an isoelectric point of 8.95. Intermolecular interaction was confirmed through in silico analysis, using electrophoretic mobility shift assays (EMSAs) and northwestern assays. The sHsp22.98 protein was found to exist in both monomeric and dimeric forms, and both forms showed strong binding to ASSVd RNA. To investigate the role of sHsp22.98 during ASSVd infection, transient silencing of sHsp22.98 was conducted, using a tobacco rattle virus (TRV)-based virus-induced gene silencing system. The sHsp22.98-silenced whiteflies showed an approximate 50% decrease in ASSVd transmission. These results suggest that sHsp22.98 from T. vaporariorum is associated with viroid RNA and plays a significant role in transmission.

Function verification of a chlorophyll a/b binding protein gene through a newly established tobacco rattle virus-induced gene silencing system in Kandelia obovata.

As an important mangrove species, Kandelia obovata plays an irreplaceable role in the coastal ecosystem. However, due to a lack of genetic technology, there is limited research on its functional genes. As such, establishing an efficient and rapid functional verification system is particularly important. In this study,tobacco rattle virus (TRV) and the phytoene desaturase gene KoPDS were used as the vector and target gene, respectively, to establish a virus-induced gene silencing system (VIGS) in K. obovata. Besides, the system was also used to verify the role of a Chlorophyll a/b binding protein (Cab) gene KoCAB in leaf carbon sequestration of K. obovata. RNA-Seq and qRT-PCR showed that the highest gene-silencing efficiency could reach 90% after 10 days of inoculation and maintain above 80% after 15 days, which was achieved with resuspension buffer at pH 5.8 and Agrobacterium culture at OD600 of 0.4-0.6. Taken together, the TRV-mediated VIGS system established herein is the first genetic analysis tool for mangroves, which may greatly impel functional genomics studies in mangrove plants.