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Abstract Virus-Induced Gene Silencing (VIGS) is a versatile tool in plant science, yet its application to non-model species like sunflower demands extensive optimization due to transformation challenges. In this study, we aimed to elucidate the factors that significantly affect the efficiency of Agrobacterium-VIGS in sunflowers. After testing a number of approaches, we concluded that the seed vacuum technique followed by 6 h of co-cultivation produced the most efficient VIGS results. Genotype-dependency analysis revealed varying infection percentages (62–91%) and silencing symptom spreading in different sunflower genotypes. Additionally, we explored the mobility of tobacco rattle virus (TRV) and phenotypic silencing manifestation (photo-bleaching) across different tissues and regions of VIGS-infected sunflower plants. We showed the presence of TRV is not necessarily limited to tissues with observable silencing events. Finally, time-lapse observation demonstrated a more active spreading of the photo-bleached spots in young tissues compared to mature ones. This study not only offers a robust VIGS protocol for sunflowers but also provides valuable insights into genotype-dependent responses and the dynamic nature of silencing events, shedding light on TRV mobility across different plant tissues.

Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing (TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into siRNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.

Ralstonia pseudosolanacearum, a notorious phytopathogen, is responsible for causing bacterial wilt, leading to significant economic losses globally in many crops within the Solanaceae family. Despite various cultural and chemical control strategies, managing bacterial wilt remains a substantial challenge. This study demonstrates, for the first time, the effective use of plant-induced bacterial gene silencing against R. pseudosolanacearum, facilitated by Tobacco rattle virusmediated gene silencing, to control bacterial wilt symptoms in Nicotiana benthamiana. The methodology described in this study could be utilized to identify novel phytobacterial virulence factors through both forward and reverse genetic approaches. To validate plant-induced gene silencing, small RNA fractions extracted from plant exudates were employed to silence bacterial gene expression, as indicated by the reduction in the expression of GFP and virulence genes in R. pseudosolanacearum. Furthermore, treatment of human and plant pathogenic Gram-negative and Gram-positive bacteria with plantgenerated small RNAs resulted in the silencing of target genes within 48 hours. Taken together, the results suggest that this technology could be applied under field conditions, offering precise, gene-based control of target bacterial pathogens while preserving the indigenous microbiota. [ABSTRACT FROM AUTHOR]

Virus-Induced Gene Silencing (VIGS) is a versatile tool in plant science, yet its application to non-model species like sunflower demands extensive optimization due to transformation challenges. In this study, we aimed to elucidate the factors that significantly affect the efficiency of Agrobacterium-VIGS in sunflowers. After testing a number of approaches, we concluded that the seed vacuum technique followed by 6 h of co-cultivation produced the most efficient VIGS results. Genotype-dependency analysis revealed varying infection percentages (62–91%) and silencing symptom spreading in different sunflower genotypes. Additionally, we explored the mobility of tobacco rattle virus (TRV) and phenotypic silencing manifestation (photo-bleaching) across different tissues and regions of VIGS-infected sunflower plants. We showed the presence of TRV is not necessarily limited to tissues with observable silencing events. Finally, time-lapse observation demonstrated a more active spreading of the photo-bleached spots in young tissues compared to mature ones. This study not only offers a robust VIGS protocol for sunflowers but also provides valuable insights into genotype-dependent responses and the dynamic nature of silencing events, shedding light on TRV mobility across different plant tissues. [ABSTRACT FROM AUTHOR]

Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing (TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1 , were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into siRNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection. [ABSTRACT FROM AUTHOR]

In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used Agrobacterium-mediated VIGS inoculation methods include stem scratching, leaf infiltration, use of agrodrench, and air-brush spraying. In this study, we developed a root wounding-immersion method in which 1/3 of the plant root (length) was cut and immersed in a tobacco rattle virus (TRV)1:TRV2 mixed solution for 30 min. We optimized the procedure in Nicotiana benthamiana and successfully silenced N. benthamiana, tomato (Solanum lycopersicum), pepper (Capsicum annuum L.), eggplant (Solanum melongena), and Arabidopsis thaliana phytoene desaturase (PDS), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of PDS in N. benthamiana and tomato was 95-100%. In addition, we successfully silenced two disease-resistance genes, SITL5 and SITL6, to decrease disease resistance in tomatoes (CLN2037E). The root wounding-immersion method can be used to inoculate large batches of plants in a short time and with high efficiency, and fresh bacterial infusions can be reused several times. The most important aspect of the root wounding-immersion method is its application to plant species susceptible to root inoculation, as well as its ability to inoculate seedlings from early growth stages. This method offers a means to conduct large-scale functional genome screening in plants. [ABSTRACT FROM AUTHOR]

Summary: Plant‐derived volatiles mediate interactions among plants, pathogenic viruses, and viral vectors. These volatile‐dependent mechanisms have not been previously demonstrated belowground, despite their likely significant role in soil ecology and agricultural pest impacts. We investigated how the plant virus, tobacco rattle virus (TRV), attracts soil nematode vectors to infected plants.We infected Nicotiana benthamiana with TRV and compared root growth relative to that of uninfected plants. We tested whether TRV‐infected N. benthamiana was more attractive to nematodes 7 d post infection and identified a compound critical to attraction. We also infected N. benthamiana with mutated TRV strains to identify virus genes involved in vector nematode attraction.Virus titre and associated impacts on root morphology were greatest 7 d post infection. Tobacco rattle virus infection enhanced 2‐ethyl‐1‐hexanol production. Nematode chemotaxis and 2‐ethyl‐1‐hexanol production correlated strongly with viral load. Uninfected plants were more attractive to nematodes after the addition of 2‐ethyl‐1‐hexanol than were untreated plants. Mutation of TRV RNA2‐encoded genes reduced the production of 2‐ethyl‐1‐hexanol and nematode attraction.For the first time, this demonstrates that virus‐driven alterations in root volatile emissions lead to increased chemotaxis of the virus's nematode vector, a finding with implications for sustainable management of both nematodes and viral pathogens in agricultural systems. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Virus-induced gene silencing（VIGS）technology has been widely used in plant gene function research. The efficiency of gene silencing in soybean mediated by tobacco rattle virus（TRV）-based vector system remains to be clarified. The seamless cloning technique was used to construct a TRV-VIGS system to explore the silencing efficiency in soybean target genes in different tissues with different inoculation methods, providing a basis for gene function studies in soybean. Taken phytoene desaturase（GmPDS）and ubiquitin ligase（GmATL3）as target genes, pTRV1 and the recombinant vectors solution were inoculated into soybean（Glycine max）plants Zhonghuang13 by three methods: injection, agroinoculation and a combination of injection plus agroinoculation. The silencing phenotypes were observed at 28 d after inoculation, and the relative expressions of the genes in the roots and leaves were quantified by RT-qPCR to determine the silencing effectiveness of the different methods. Silencing of GmPDS resulted in yellowing and chlorosis on the edges and inside of the leaves, while chlorosis spots and fold chlorosis phenotypes in the leaves surface occurred. The results of RT-qPCR showed that silencing efficiency of GmPDS of all three inoculation methods was close to 100%. The silencing efficiency of GmATL3 by the injection was 80%-95% in the leaves and 40%-60% in the roots; the silencing efficiency of the root inoculation and injection plus root inoculation was 70%-90% in roots and 15%-50% in leaves. Different inoculation methods caused different levels of silencing phenotypes and silencing efficiencies for different endogenous genes. The silencing efficiency of an inoculation method on different tissues was different, and the highest silencing efficiency on the leaves and roots were injection method and injection plus agroinoculation, respectively. [ABSTRACT FROM AUTHOR]

The nucleolus and Cajal bodies (CBs) are sub-nuclear domains with well-known roles in RNA metabolism and RNA-protein assembly. However, they also participate in other important aspects of cell functioning. This study uncovers a previously unrecognised mechanism by which these bodies and their components regulate host defences against pathogen attack. We show that the CB protein coilin interacts with poly(ADP-ribose) polymerase 1 (PARP1), redistributes it to the nucleolus and modifies its function, and that these events are accompanied by substantial increases in endogenous concentrations of salicylic acid (SA), activation of SA-responsive gene expression and callose deposition leading to the restriction of tobacco rattle virus (TRV) systemic infection. Consistent with this, we also find that treatment with SA subverts the negative effect of the pharmacological PARP inhibitor 3-aminobenzamide (3AB) on plant recovery from TRV infection. Our results suggest that PARP1 could act as a key molecular actuator in the regulatory network which integrates coilin activities as a stress sensor for virus infection and SA-mediated antivirus defence. [ABSTRACT FROM AUTHOR]

Virus-induced gene silencing (VIGS) is a fast and efficient method for assaying gene function in plants. At present, the VIGS system mediated by Tobacco rattle virus (TRV) has been successfully practiced in some species such as cotton and tomato. However, little research of VIGS systems has been reported in woody plants, nor in Chinese jujube. In this study, the TRV-VIGS system of jujube was firstly investigated. The jujube seedlings were grown in a greenhouse with a 16 h light/8 h dark cycle at 23 °C. After the cotyledon was fully unfolded, Agrobacterium mixture containing pTRV1 and pTRV2-ZjCLA with OD600 = 1.5 was injected into cotyledon. After 15 days, the new leaves of jujube seedlings showed obvious photo-bleaching symptoms and significantly decreased expression of ZjCLA, indicating that the TRV-VIGS system had successfully functioned on jujube. Moreover, it found that two injections on jujube cotyledon could induce higher silencing efficiency than once injection. A similar silencing effect was then also verified in another gene, ZjPDS. These results indicate that the TRV-VIGS system in Chinese jujube has been successfully established and can be applied to evaluate gene function, providing a breakthrough in gene function verification methods. [ABSTRACT FROM AUTHOR]

Ilex dabieshanensis is not only an important ornamental plant, but can also be used to produce Kuding tea, owing to its lipid-lowering and anti-inflammatory medicinal properties. The genetic transformation of I. dabieshanensis is currently difficult, which restricts functional gene studies and molecular breeding research on this species. Virus-induced gene silencing (VIGS) is a powerful tool for determining gene functions in plants. The present study reports the first application of VIGS mediated by a tobacco rattle virus (TRV) vector in I. dabieshanensis. We tested the efficiency of the VIGS system to silence Mg-chelatase H subunit (ChlH) gene through agroinfiltration. The agroinfiltrated leaves of I. dabieshanensis exhibited a typical yellow-leaf phenotype of ChlH gene silencing at 21 days post infiltration. Endogenous ChlH expression levels in the leaves of yellow-leaf phenotype plants were all significantly lower than that in the leaves of mock-infected and control plants. Overall, our results indicated that the TRV-based VIGS system can efficiently silence genes in I. dabieshanensis, and this system will contribute to efficient functional genomics research in I. dabieshanensis. [ABSTRACT FROM AUTHOR]

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.

Root parasitic weed Phelipanche aegyptiaca is an obligate plant parasite that causes severe damage to host crops. Agriculture crops mainly belong to the Brassicaceae, Leguminosae, Cruciferae, and Solanaceae plant families affected by this parasitic weed, leading to the devastating loss of crop yield and economic growth. This root-specific parasitic plant is not able to complete its life cycle without a suitable host and is dependent on the host plant for nutrient uptake and germination. Therefore, selected parasitic genes of P. aegyptiaca which were known to be upregulated upon interaction with the host were chosen. These genes are essential for parasitism, and reduced activity of these genes could affect host-parasitic interaction and provide resistance to the host against these parasitic weeds. To check and examine the role of these parasitic genes which can affect the development of host resistance, we silenced selected genes in the P. aegyptiaca using the tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS) method. Our results demonstrated that the total number of P. aegyptiaca parasite tubercles attached to the root of the host plant Nicotiana benthamiana was substantially decreased in all the silenced plants. However, silencing of the P. aegyptiaca MNT1 gene which encodes the mannitol transporter showed a significantly reduced number of germinated shoots and tubercles. Thus, our study indicates that the mannitol transport gene of P. aegyptiaca plays a crucial role in parasitic germination, and silencing of the PaMNT1 gene abolishes the germination of parasites on the host roots.

Cotton (Gossypium hirsutum) is the world's most important fiber crop, critical to global textile industries and agricultural economies. However, cotton yield and harvest quality are undermined by the challenges introduced from invading pathogens and pests. Plant-synthesized oxylipins, specifically 9-hydroxy fatty acids resulting from 9-lipoxygenase activity (9-LOX), enhance the growth and development of many microbes and pests. We hypothesized that targeted disruption of 9-LOX-encoding genes in cotton could bolster crop resilience against prominent agronomic threats. Fusarium oxysporum f. sp. vasinfectum (FOV), Aphis gossypii (cotton aphid), and Tobacco rattle virus induced the expression of 9-oxylipin biosynthesis genes, suggesting that the 9-LOX gene products were susceptibility factors to these stressors. Transiently disrupting the expression of the 9-LOX-encoding genes by virus-induced gene silencing significantly reduced target transcript accumulation, and this correlated with impaired progression of FOV infections and a significant decrease in the fecundity of cotton aphids. These findings emphasize that the cotton 9-LOX-derived oxylipins are leveraged by multiple pathogens and pests to enhance their virulence in cotton, and reducing the expression of 9-LOX-encoding genes can benefit cotton crop vitality., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Virus-induced gene silencing (VIGS) is a common reverse genetics strategy for characterizing the function of genes in plants. The detailed mechanism governing RNA silencing efficiency triggered by viruses is largely unclear. Here, we reveal that a petunia (Petunia hybrida) ocs element binding factor, PhOBF1, one of the basic leucine zipper (bZIP) transcription factors, was up-regulated by Tobacco rattle virus (TRV) infection. Simultaneous silencing of PhOBF1 and a reporter gene, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV-based VIGS led to a failure of the development of leaf photobleaching or the white-corollas phenotype. PhOBF1 silencing caused down-regulation of RNA silencing-related genes, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs). After inoculation with the TRV-PhPDS, PhOBF1-RNAi lines exhibited a substantially impaired PDS silencing efficiency, whereas overexpression of PhOBF1 resulted in a recovery of the silencing phenotype (photobleaching) in systemic leaves. A compromised resistance to TRV and Tobacco mosaic virus was found in PhOBF1-RNAi lines, while PhOBF1-overexpressing lines displayed an enhanced resistance to their infections. Compared with wild-type plants, PhOBF1-silenced plants accumulated lower levels of free salicylic acid (SA), salicylic acid glucoside, and phenylalanine, contrarily to higher levels of those in plants overexpressing PhOBF1. Furthermore, transcripts of a number of genes associated with the shikimate and phenylpropanoid pathways were decreased or increased in PhOBF1-RNAi or PhOBF1-overexpressing lines, respectively. Taken together, the data suggest that PhOBF1 regulates TRV-induced RNA silencing efficiency through modulation of RDRs, DCLs, and AGOs mediated by the SA biosynthesis pathway.

Viral vectors are being engineered to deliver CRISPR/Cas9 components systemically in plants to induce somatic or heritable site-specific mutations. It is hypothesized that RNA mobility signals facilitate entry of viruses or single guide RNAs (sgRNAs) into the shoot apical meristem where germline mutations can occur. Our objective was to understand the impact of RNA mobility signals on virus-induced somatic and germline gene editing in Nicotiana benthamiana and Zea mays. Previously, we showed that foxtail mosaic virus (FoMV) expressing sgRNA induced somatic mutations in N. benthamiana and Z. mays expressing Cas9. Here, we fused RNA mobility signals to sgRNAs targeting the genes encoding either N. benthamiana phytoene desaturase (PDS) or Z. mays high affinity potassium transporter 1 (HKT1). Addition of Arabidopsis thaliana Flowering Locus T (AtFT) and A. thaliana tRNA-Isoleucine (AttRNAIle) did not improve FoMV-induced somatic editing, and neither were sufficient to facilitate germline mutations in N. benthamiana. Maize FT homologs, Centroradialus 16 (ZCN16) and ZCN19, as well as AttRNAIle were found to aid somatic editing in maize but did not enable sgRNAs delivered by FoMV to induce germline mutations. Additional viral guide RNA delivery systems were assessed for somatic and germline mutations in N. benthamiana with the intention of gaining a better understanding of the specificity of mobile signal-facilitated germline editing. Potato virus X (PVX), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV) were included in this comparative study, and all three of these viruses delivering sgRNA were able to induce somatic and germline mutations. Unexpectedly, PVX, a potexvirus closely related to FoMV, expressing sgRNA alone induced biallelic edited progeny, indicating that mobility signals are dispensable in virus-induced germline editing. These results show that PVX, BSMV, and TRV expressing sgRNA all have an innate ability to induce mutations in the germline. Our results indicate that mobility signals alone may not be sufficient to enable virus-based delivery of sgRNAs using the viruses, FoMV, PVX, BSMV, and TRV into cell types that result in germline mutations.

Virus-induced gene silencing (VIGS) is a genetic tool used to assess gene function. Tobacco rattle virus (TRV) is a VIGS vector commonly used to induce endogenous gene silencing in plants. However, there is no VIGS system established for Centaurea spp. We evaluated the effectiveness of a TRV-based VIGS system using phytoene desaturase (PDS) as a reporter gene in Centaurea cyanus. Three methods including pressure-, vacuum- and apical meristem-infiltration were tested to infect C. cyanus seedlings. Photobleached leaves were only obtained using apical meristem-infiltration after a 14 d treatment. The CcPDS transcripts in photobleached leaves were significantly reduced compared with that in green leaves treated with empty TRV. Four C. cyanus cultivars were tested to detect their VIGS responses, and ‘Dwarf Tom Pouce Blue’ was the most sensitive. The agro-infiltration condition was optimized by screening for the optimal seedling stage as well as the optimum Agrobacterium density for efficient silencing. Seedlings with four true leaves and infiltration with an Agrobacterium density of OD600 0.5 were optimal conditions to obtain more photobleached leaves and more intense photobleached phenotype. The results demonstrated the feasibility of TRV-based VIGS for functional analysis of genes in C. cyanus.

The nucleolus and Cajal bodies (CBs) are sub-nuclear domains with well-known roles in RNA metabolism and RNA-protein assembly. However, they also participate in other important aspects of cell functioning. This study uncovers a previously unrecognised mechanism by which these bodies and their components regulate host defences against pathogen attack. We show that the CB protein coilin interacts with poly(ADP-ribose) polymerase 1 (PARP1), redistributes it to the nucleolus and modifies its function, and that these events are accompanied by substantial increases in endogenous concentrations of salicylic acid (SA), activation of SA-responsive gene expression and callose deposition leading to the restriction of tobacco rattle virus (TRV) systemic infection. Consistent with this, we also find that treatment with SA subverts the negative effect of the pharmacological PARP inhibitor 3-aminobenzamide (3AB) on plant recovery from TRV infection. Our results suggest that PARP1 could act as a key molecular actuator in the regulatory network which integrates coilin activities as a stress sensor for virus infection and SA-mediated antivirus defence.

Virus-induced gene silencing (VIGS) is a fast and efficient method for assaying gene function in plants. At present, the VIGS system mediated by Tobacco rattle virus (TRV) has been successfully practiced in some species such as cotton and tomato. However, little research of VIGS systems has been reported in woody plants, nor in Chinese jujube. In this study, the TRV-VIGS system of jujube was firstly investigated. The jujube seedlings were grown in a greenhouse with a 16 h light/8 h dark cycle at 23 °C. After the cotyledon was fully unfolded, Agrobacterium mixture containing pTRV1 and pTRV2-ZjCLA with OD600 = 1.5 was injected into cotyledon. After 15 days, the new leaves of jujube seedlings showed obvious photo-bleaching symptoms and significantly decreased expression of ZjCLA, indicating that the TRV-VIGS system had successfully functioned on jujube. Moreover, it found that two injections on jujube cotyledon could induce higher silencing efficiency than once injection. A similar silencing effect was then also verified in another gene, ZjPDS. These results indicate that the TRV-VIGS system in Chinese jujube has been successfully established and can be applied to evaluate gene function, providing a breakthrough in gene function verification methods.

Systemic necrosis, induced by plant virus-derived elicitors, is considered as one of the most severe symptoms. It has never been reported that the elicitors encoded by wheat yellow mosaic virus (WYMV) can induce systemic necrosis in plant. In this study, we discovered that the WYMV-encoded 14K protein localized to the endoplasmic reticulum (ER) membrane and triggered a necrotic symptom in Nicotiana benthamiana at 5 days post-infiltration (dpi). Meanwhile, overexpression of WYMV 14K suppressed the replication of tobacco rattle virus (TRV) and potato virus X (PVX). Additionally, deletion of the transmembrane domain and substitution of two non-conserved regions in 14K resulted in the loss of the ability of this protein to induce systemic necrosis. Moreover, the 14K homologous proteins of other bymoviruses failed to induce systemic necrosis. Our results, for the first time, reveal that WYMV 14K induces systemic necrosis and suppresses the replication of other viruses. [ABSTRACT FROM AUTHOR]

RNA silencing is a common antiviral mechanism in eukaryotic organisms. However, the transcriptional regulatory mechanism that controls the RNA silencing process remains elusive. Here, we performed high-depth transcriptome analysis on petunia (Petunia hybrida) leaves infected with tobacco rattle virus (TRV) strain PPK20. A total of 7,402 differentially expressed genes (DEGs) were identified. Of them, some RNA silencingrelated transcripts, such as RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs), were induced by viral attack. Furthermore, we performed TRV-based virus-induced gene silencing (VIGS) assay on 39 DEGs encoding putative transcription factors (TFs), using green fluorescent protein (GFP) and phytoene desaturase (PhPDS) as reporters. Results showed that the down-regulation of PhbHLH41, PhbHLH93, PhZPT4-3, PhCOL4, PhHSF-B3A, PhNAC90, and PhWRKY75 led to enhanced TRV accumulation and inhibited PhPDS-silenced photobleaching phenotype. In contrast, silencing of PhERF22 repressed virus accumulation and promoted photobleaching development. Thus, these TFs were identified as potential positive and negative regulators of antiviral RNA silencing, respectively. One positive regulator PhCOL4, belonging to the B-box zinc finger family, was selected for further functional characterization. Silencing and transient overexpression of PhCOL4 resulted in decreased and increased expression of several RNA silencing-related genes. DNA affinity purification sequencing analysis revealed that PhCOL4 targeted PhRDR6 and PhAGO4. Dual luciferase and yeast one-hybrid assays determined the binding of PhCOL4 to the PhRDR6 and PhAGO4 promoters. Our findings suggest that TRV-GFPPhPDS- based VIGS could be helpful to identify transcriptional regulators of antiviral RNA silencing. [ABSTRACT FROM AUTHOR]

Tobacco rattle virus (TRV) is an important soil-borne virus of potato that is transmitted by stubby-root nematodes. TRV causes corky ringspot, a tuber disease of economic importance to potato production. Utilizing protein-coding regions of the whole genome and a range of computational tools, the genetic diversity, and population structure of TRV isolates from several potato-growing regions (Colorado, Idaho, Indiana, Minnesota, Nebraska, North Dakota, and Washington State) in the USA were determined. Phylogenetic analyses based on RNA2 nucleotide sequences, the coat protein (CP) and nematode transmission (2b) genes, showed geographical clustering of USA isolates with previously known American isolates, while European isolates grouped in a distinct cluster. This was corroborated by the observed genetic differentiation and infrequent gene flow between American and European isolates. Low genetic diversity was revealed among American isolates compared to European isolates. Phylogenetic clustering based on RNA1 genes (RdRp, RdRp-RT, and 1a) were all largely incongruent to that of 1b gene (virus suppressor of RNA silencing). This genetic incongruence suggested the influence of recombination. Furthermore, the RdRp, RdRp-RT, and 1a genes were predicted to be more conserved and under negative selection, while the 1b gene was less constrained. Different evolutionary lineages between TRV RNA1 and RNA2 genomic segments were revealed. [ABSTRACT FROM AUTHOR]

Abstract Background Antirrhinum (snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Transient gene expression from viral vectors and virus-induced gene silencing (VIGS) offer transformation-free alternatives. Here we investigate the utility of Tobacco rattle virus (TRV) for homologous gene expression in Antirrhinum and VIGS in Antirrhinum and its relative Misopates. Results A. majus proved highly susceptible to systemic TRV infection. TRV carrying part of the Phytoene Desaturase (PDS) gene triggered efficient PDS silencing, visible as tissue bleaching, providing a reporter for the extent and location of VIGS. VIGS was initiated most frequently in young seedlings, persisted into inflorescences and flowers and was not significantly affected by the orientation of the homologous sequence within the TRV genome. Its utility was further demonstrated by reducing expression of two developmental regulators that act either in the protoderm of young leaf primordia or in developing flowers. The effects of co-silencing PDS and the trichome-suppressing Hairy (H) gene from the same TRV genome showed that tissue bleaching provides a useful marker for VIGS of a second target gene acting in a different cell layer. The ability of TRV-encoded H protein to complement the h mutant phenotype was also tested. TRV carrying the native H coding sequence with PDS to report infection failed to complement h mutations and triggered VIGS of H in wild-type plants. However, a sequence with 43% synonymous substitutions encoding H protein, was able to complement the h mutant phenotype when expressed without a PDS VIGS reporter. Conclusions We demonstrate an effective method for VIGS in the model genus Antirrhinum and its relative Misopates that works in vegetative and reproductive tissues. We also show that TRV can be used for complementation of a loss-of-function mutation in Antirrhinum. These methods make rapid tests of gene function possible in these species, which are difficult to transform genetically, and opens up the possibility of using additional cell biological and biochemical techniques that depend on transgene expression.

Ilex dabieshanensis is not only an important ornamental plant, but can also be used to produce Kuding tea, owing to its lipid-lowering and anti-inflammatory medicinal properties. The genetic transformation of I. dabieshanensis is currently difficult, which restricts functional gene studies and molecular breeding research on this species. Virus-induced gene silencing (VIGS) is a powerful tool for determining gene functions in plants. The present study reports the first application of VIGS mediated by a tobacco rattle virus (TRV) vector in I. dabieshanensis. We tested the efficiency of the VIGS system to silence Mg-chelatase H subunit (ChlH) gene through agroinfiltration. The agroinfiltrated leaves of I. dabieshanensis exhibited a typical yellow-leaf phenotype of ChlH gene silencing at 21 days post infiltration. Endogenous ChlH expression levels in the leaves of yellow-leaf phenotype plants were all significantly lower than that in the leaves of mock-infected and control plants. Overall, our results indicated that the TRV-based VIGS system can efficiently silence genes in I. dabieshanensis, and this system will contribute to efficient functional genomics research in I. dabieshanensis.

Ralstonia pseudosolanacearum , a notorious phytopathogen, is responsible for causing bacterial wilt, leading to significant economic losses globally in many crops within the Solanaceae family. Despite various cultural and chemical control strategies, managing bacterial wilt remains a substantial challenge. This study demonstrates, for the first time, the effective use of plant-induced bacterial gene silencing against R. pseudosolanacearum , facilitated by Tobacco rattle virus -mediated gene silencing, to control bacterial wilt symptoms in Nicotiana benthamiana . The methodology described in this study could be utilized to identify novel phytobacterial virulence factors through both forward and reverse genetic approaches. To validate plant-induced gene silencing, small RNA fractions extracted from plant exudates were employed to silence bacterial gene expression, as indicated by the reduction in the expression of GFP and virulence genes in R. pseudosolanacearum . Furthermore, treatment of human and plant pathogenic Gram-negative and Gram-positive bacteria with plant-generated small RNAs resulted in the silencing of target genes within 48 hours. Taken together, the results suggest that this technology could be applied under field conditions, offering precise, gene-based control of target bacterial pathogens while preserving the indigenous microbiota., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Jang, Kim, Lee and Ryu.)

Specialized metabolism is an evolutionary answer that fortifies plants against a wide spectrum of (a) biotic challenges. A plethora of diversified compounds can be found in the plant kingdom and often constitute the basis of human pharmacopeia. Olive trees (Olea europaea) produce an unusual type of secoiridoids known as oleosides with promising pharmaceutical activities. Here, we transiently silenced oleuropein β-glucosidase (OeGLU), an enzyme engaged in the biosynthetic pathway of secoiridoids in the olive trees. Reduction of OeGLU transcripts resulted in the absence of both upstream and downstream secoiridoids in planta , revealing a regulatory loop mechanism that bypasses the flux of precursor compounds toward the branch of secoiridoid biosynthesis. Our findings highlight that OeGLU could serve as a molecular target to regulate the bioactive secoiridoids in olive oils. [ABSTRACT FROM AUTHOR]

Kalmegh [Andrographis paniculata (Burm.f.) Wall. ex Nees] is one of the most studied medicinal plants for pharmaceutical properties and phytochemistry. However, functional genomics studies in kalmegh are so far limited due to the unavailability of a robust tool for gene silencing. Here, we tested the application of virus-induced gene silencing (VIGS) in kalmegh using the well-known Tobacco rattle virus (TRV)-based vectors and achieved targeted silencing of phytoene desaturase (ApPDS) which is essential in plants for carotenoid biosynthesis that protects chlorophyll from photooxidation. ApPDS silencing in kalmegh leaves developed a typical photobleaching phenotype. The silencing of ApPDS was confirmed by analysing ApPDS transcript level and determining chlorophyll content in the leaves of VIGS seedlings. The analysis revealed ~30% reduction in chlorophyll content, and 40 to 60% reduction in ApPDS transcript level in the leaves of VIGS seedlings. These findings clearly demonstrated the applicability of VIGS in kalmegh using TRV-based vectors. The VIGS protocol presented in this study might be useful for studying gene function related to medicinal and agricultural traits in kalmegh. [ABSTRACT FROM AUTHOR]

Being an important garden plant, Dianthus chinensis flower has a great variety of colors and color patterns. Chalcone synthase (CHS) is the key enzyme in the anthocyanin biosynthetic pathway. Although CHS genes have been isolated and characterized in ornamental plants, the CHS gene is still unknown in D. chinensis. In our study, three CHS genes, DchCHS1 (KX893854), DchCHS2 (MK404175) and DchCHS3 (MK416198) were isolated in D. chinensis. Their deduced amino acid sequences show high homology with the known CHS sequences in Caryophyllaceae. The phylogenetic tree suggests that the DchCHS1 and the DchCHS3 have a close relation with the known CHS sequences in Caryophyllaceae and the DchCHS2 is different from them. The DchCHSs were characterized by the Tobacco Rattle Virus (TRV)-based virus-induced gene silencing (VIGS) system. We obtained white or pale purple flowers in the DchCHS1-silenced flowers and reducing purple flowers in the DchCHS2-silenced and the DchCHS3-silenced flowers. The anthocyanin content and the transcript level of the silenced DchCHS were significantly reduced in accordance with the silencing phenotypes. The DchCHSs showed different expression patterns during floral bud developments, among flower colors and in organs. Their expression levels in the purple flower were significantly higher than those in the white flower. Compared with DchCHS2 and DchCHS3, DchCHS1 was abundantly expressed at each floral bud stage, in each flower color and in the flower organ. In conclusion, the three DchCHSs are all involved in the anthocyanin synthesis and the flower coloration, and DchCHS1 probably plays a major role in D. chinensis flowers. Key message: At least three DchCHSs are involved in the anthocyanin synthesis and the flower coloration in D. chinensis. It may be the reason for its richness in flower colors and color patterns. [ABSTRACT FROM AUTHOR]

The Persian walnut (Juglans regia L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV-JrPDS recombinant clone. A co-culture inoculation method utilizing Agrobacterium was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for J. regia fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the JrPDS transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related J. regia polyphenol oxidase (PPO) genes JrPPO1 and JrPPO2 decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating J. regia fruit with efficient silence efficiency via co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in J. regia fruits.

The Persian walnut (Juglans regia L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV- JrPDS recombinant clone. A co-culture inoculation method utilizing Agrobacterium was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for J. regia fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the JrPDS transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related J. regia polyphenol oxidase (PPO) genes JrPPO1 and JrPPO2 decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating J. regia fruit with efficient silence efficiency via co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in J. regia fruits. [ABSTRACT FROM AUTHOR]

Cape gooseberry (Physalis peruviana, L.) is a herbaceous plant belonging to the Solanaceae family that produces an edible berry appreciated for its nutraceutical and pharmaceutical properties. Its production is often limited by diseases and reproducible fruit quality. Recent studies have reported genes associated with fruit quality and resistance response to the root-infecting fungus Fusarium oxysporum f. sp. physali (Foph,) which causes vascular wilt. In order to standardize a method to validate the biological function of candidate genes in the non-model species P. peruviana, we tested the robust approach in reverse genetics, virus-induced gene silencing (VIGS). In this study, we validated and optimized VIGS using an insert of the phytoene desaturase (PDS) gene in a silencing viral vector generated from tobacco rattle virus (TRV). Leaves infiltrated with Agrobacterium (GV3101 strain) showed photo-bleached segments, which were distinctive for PDS suppression at 7 days post-infection (dpi). More than half of the treated plants showed photo-bleaching, indicating an efficiency rate of 50 % of the VIGS protocol. The results of this study showed that VIGS can be used for future functional gene characterization implicated in the immune response, disease resistance and fruit quality in capegooseberry.

Virus-induced gene silencing (VIGS) is a powerful gene-silencing tool that has been intensively applied in plants. To data, the application of VIGS in rubber tree has not yet been reported. In this study, we described the efficient gene silencing in rubber tree by VIGS. The gene encoding Hevea brasiliensis phytoene desaturase (HbPDS) was identified in rubber tree genome. Small interfering RNAs from HbPDS and the silencing gene fragment were predicted and a length of 399 bp was selected to be tested. We showed that the tobacco rattle virus (TRV)-VIGS could induce effective HbPDS silencing in rubber tree. This study was the first to report VIGS in rubber tree. The present TRV-VIGS method could be used to perform reverse genetic approaches to identify unknown gene functions and might be further applied to produce gene silenced rubber tree plants, to advance functional gene of rubber tree. [ABSTRACT FROM AUTHOR]

In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used Agrobacterium -mediated VIGS inoculation methods include stem scratching, leaf infiltration, use of agrodrench, and air-brush spraying. In this study, we developed a root wounding-immersion method in which 1/3 of the plant root (length) was cut and immersed in a tobacco rattle virus (TRV)1:TRV2 mixed solution for 30 min. We optimized the procedure in Nicotiana benthamiana and successfully silenced N. benthamiana , tomato ( Solanum lycopersicum ), pepper ( Capsicum annuum L.), eggplant ( Solanum melongena ), and Arabidopsis thaliana phytoene desaturase ( PDS ), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of PDS in N. benthamiana and tomato was 95-100%. In addition, we successfully silenced two disease-resistance genes, SITL5 and SITL6 , to decrease disease resistance in tomatoes (CLN2037E). The root wounding-immersion method can be used to inoculate large batches of plants in a short time and with high efficiency, and fresh bacterial infusions can be reused several times. The most important aspect of the root wounding-immersion method is its application to plant species susceptible to root inoculation, as well as its ability to inoculate seedlings from early growth stages. This method offers a means to conduct large-scale functional genome screening in plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Tao, Xu, Xu, Yang, Jiang, Zhou, Deng, Lv and Zhao.)

Background: Antirrhinum (snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Transient gene expression from viral vectors and virus-induced gene silencing (VIGS) offer transformation-free alternatives. Here we investigate the utility of Tobacco rattle virus (TRV) for homologous gene expression in Antirrhinum and VIGS in Antirrhinum and its relative Misopates. Results: A. majus proved highly susceptible to systemic TRV infection. TRV carrying part of the Phytoene Desaturase (PDS) gene triggered efficient PDS silencing, visible as tissue bleaching, providing a reporter for the extent and location of VIGS. VIGS was initiated most frequently in young seedlings, persisted into inflorescences and flowers and was not significantly affected by the orientation of the homologous sequence within the TRV genome. Its utility was further demonstrated by reducing expression of two developmental regulators that act either in the protoderm of young leaf primordia or in developing flowers. The effects of co-silencing PDS and the trichome-suppressing Hairy (H) gene from the same TRV genome showed that tissue bleaching provides a useful marker for VIGS of a second target gene acting in a different cell layer. The ability of TRV-encoded H protein to complement the h mutant phenotype was also tested. TRV carrying the native H coding sequence with PDS to report infection failed to complement h mutations and triggered VIGS of H in wild-type plants. However, a sequence with 43% synonymous substitutions encoding H protein, was able to complement the h mutant phenotype when expressed without a PDS VIGS reporter. Conclusions: We demonstrate an effective method for VIGS in the model genus Antirrhinum and its relative Misopates that works in vegetative and reproductive tissues. We also show that TRV can be used for complementation of a loss-of-function mutation in Antirrhinum. These methods make rapid tests of gene function possible in these species, which are difficult to transform genetically, and opens up the possibility of using additional cell biological and biochemical techniques that depend on transgene expression. [ABSTRACT FROM AUTHOR]

Many members of the family Gesneriaceae are cultivated as ornamental plants, including Cape primrose (Streptocarpus) species. The range of plant architecture found in this genus has also made it a model to study leaf and meristem development and their evolution. However, the lack of tools to study gene functions through reverse genetics in Streptocarpus has limited the exploitation of its genetic potential. To aid functional genomic studies in Streptocarpus rexii, we sought to investigate virus-induced gene silencing (VIGS). Using the broad host range Tobacco Rattle Virus (TRV) to target the PHYTOENE DESATURASE (PDS) gene of S. rexii, we show that infection with sap from Nicotiana benthamiana triggered VIGS efficiently. VIGS was most effective in the seedling leaves 8 weeks after sowing, but was limited in duration and systemic spread. This study reports the first successful use of VIGS in Streptocarpus and in the family Gesneriaceae. The inoculation of viral sap derived from N. benthamiana was able to overcome the difficulties of standard Agrobacterium-mediated transformation in this genus. Irrespective of its transient effect, this VIGS system will be useful to assess gene function at the cellular level and represent an important tool for further understanding molecular mechanisms in Streptocarpus. [ABSTRACT FROM AUTHOR]

Corky ringspot (CRS) is a widespread potato tuber necrotic disease caused by Tobacco rattle virus (TRV) infection. In the Pacific Northwest, this virus is transmitted by the stubby root nematode (SRN) within the genus Paratrichodorus. Remediating CRS affected fields is a major challenge that can be mitigated by growing plant varieties that are resistant to TRV infection. Growing alfalfa has been shown to reduce TRV levels in CRS infested fields over time but the development of a potato cultivar with these same capabilities would be of great economic benefit to potato growers. Castle Russet is a new potato clone that does not develop symptoms of CRS disease. To assess its ability to reduce soil virus load, Castle Russet, tobacco var. “Samsun NN”, alfalfa var. “Vernema”, and Russet Burbank potato were grown for a period of 1 to 3 months in soils containing viruliferous SRN populations at two different inoculation pressures (60 nematodes/pot and 1060 nematodes/pot) in greenhouse pot experiments. SRN population size and the presence of TRV were assessed over several months post inoculation. Results indicate that plant host and length of exposure significantly influence SRN population dynamics, whereas the TRV infection status of bait plants was significantly affected by both of these factors as well as inoculation pressure. These results suggest that both alfalfa var. “Vernema” and Castle Russet are resistant to TRV infection and may potentially be used to eliminate the virus from fields affected by CRS. [ABSTRACT FROM AUTHOR]

Abstract Background Virus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a high-throughput manner. However, the specificity and efficiency for knocking down members of a highly homologous gene family have remained to date a significant challenge in VIGS due to silencing of off-targets. Results Here we present an improved method for the selection and evaluation of gene fragments used for VIGS to specifically and efficiently knock down members of a highly homologous gene family. Using this method, we knocked down twelve and four members, respectively of group III of the gene family encoding ubiquitin-conjugating enzymes (E2) in Nicotiana benthamiana. Assays using these VIGS-treated plants revealed that the group III E2s are essential for plant development, plant immunity-associated reactive oxygen species (ROS) production, expression of the gene NbRbohB that is required for ROS production, and suppression of immunity-associated programmed cell death (PCD) by AvrPtoB, an effector protein of the bacterial pathogen Pseudomons syringae. Moreover, functional redundancy for plant development and ROS production was found to exist among members of group III E2s. Conclusions We have found that employment of a gene fragment as short as approximately 70 base pairs (bp) that contains at least three mismatched nucleotides to other genes within any 21-bp sequences prevents silencing of off-target(s) in VIGS. This improved approach in the selection and evaluation of gene fragments allows for specific and efficient knocking down of highly homologous members of a gene family. Using this approach, we implicated N. benthamiana group III E2s in plant development, immunity-associated ROS production, and suppression of multiple immunity-associated PCD by AvrPtoB. We also unraveled functional redundancy among group III members in their requirement for plant development and plant immunity-associated ROS production.

The whitefly B. tabaci is a global pest and transmits extremely important plant viruses especially begomoviruses, that cause substantial crop losses. B. tabaci is one of the top invasive species worldwide and have developed resistance to all major pesticide classes. One of the promising alternative ways for controlling this pest is studying its genetic makeup for identifying specific target proteins which are critical for its development and ability to transmit viruses. Tomato yellow leaf curl virus (TYLCV) is the most economically important and well-studied begomovirus transmitted by B. tabaci, in a persistent-circulative manner. Recently, we reported that B. tabaci Cyclophilin B (CypB) and heat shock protein 70 proteins (hsp70) interact and co-localize with TYLCV in the whitefly midgut, on the virus transmission pathway, and that both proteins have a significant role in virus transmission. Here, we extended the previous work and used the Tobacco rattle virus (TRV) plant-mediated RNA silencing system for knocking down both genes and testing the effect of their silencing on whitefly viability and virus transmission. Portions of these two genes were cloned into TRV constructs and tomato plants were infected and used for whitefly feeding and transmission experiments. Following whitefly feeding on TRV-plants, the expression levels of cypB and hsp70 in adult B. tabaci significantly decreased over 72 h feeding period. The knockdown in the expression of both genes was further shown in the first generation of silenced whiteflies, where phenotypic abnormalities in the adult, wing, nymph and bacteriosomes development and structure were observed. Additionally, high mortality rates that reached more than 80% among nymphs and adults were obtained. Finally, silenced whitefly adults with both genes showed decreased ability to transmit TYLCV under lab conditions. Our results suggest that plant-mediated silencing of both cypB and hsp70 have profound effects on whitefly development and its ability to transmit TYLCV.

Tree peony is a perennial deciduous shrub with great ornamental and medicinal value. A limitation of its current functional genomic research is the lack of effective molecular genetic tools. Here, the first application of a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) in the tree peony species Paeonia ostii is presented. Two different approaches, leaf syringe-infiltration and seedling vacuum-infiltration, were utilized for Agrobacterium-mediated inoculation. The vacuum-infiltration was shown to result in a more complete Agrobacterium penetration than syringe-infiltration, and thereby determined as an appropriate inoculation method. The silencing of reporter gene PoPDS encoding phytoene desaturase was achieved in TRV-PoPDS-infected triennial tree peony plantlets, with a typical photobleaching phenotype shown in uppermost newly-sprouted leaves. The endogenous PoPDS transcripts were remarkably down-regulated in VIGS photobleached leaves. Moreover, the green fluorescent protein (GFP) fluorescence was detected in leaves and roots of plants inoculated with TRV-GFP, suggesting the capability of TRV to silence genes in various tissues. Taken together, the data demonstrated that the TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in tree peony.

Summary: In addition to well‐known roles in RNA metabolism, the nucleolus and Cajal bodies (CBs), both located within the nucleus, are involved in plant responses to biotic and abiotic stress. Previously we showed that plants in which expression of the CB protein coilin is downregulated are more susceptible to certain viruses including tobacco rattle virus (TRV), suggesting a role of coilin in antiviral defence.Experiments with coilin‐deficient plants and the deletion mutant of the TRV 16K protein showed that both 16K and coilin are required for restriction of systemic TRV infection. The potential mechanisms of coilin‐mediated antiviral defence were elucidated via experiments involving co‐immunoprecipitation, use of NahG transgenic plants deficient in salicylic acid (SA) accumulation, measurement of endogenous SA concentrations and assessment of SA‐responsive gene expression.Here we show that TRV 16K interacts with and relocalizes coilin to the nucleolus. In wild‐type plants these events are accompanied by activation of SA‐responsive gene expression and restriction of TRV systemic infection. By contrast, viral systemic spread was enhanced in NahG plants, implicating SA in these processes.Our findings suggest that coilin is involved in plant defence, responding to TRV infection by recognition of the TRV‐encoded 16K protein and activating SA‐dependent defence pathways. [ABSTRACT FROM AUTHOR]

Summary: DNA methylation is an important epigenetic mechanism for controlling innate immunity against microbial pathogens in plants. Little is known, however, about the manner in which viral infections interact with DNA methylation pathways. Here we investigate the crosstalk between epigenetic silencing and viral infections in Arabidopsis inflorescences. We found that tobacco rattle virus (TRV) causes changes in the expression of key transcriptional gene silencing factors with RNA‐directed DNA methylation activities that coincide with changes in methylation at the whole genome level. Viral susceptibility/resistance was altered in DNA (de)methylation‐deficient mutants, suggesting that DNA methylation is an important regulatory system controlling TRV proliferation. We further show that several transposable elements (TEs) underwent transcriptional activation during TRV infection, and that TE regulation likely involved both DNA methylation‐dependent and ‐independent mechanisms. We identified a cluster of disease resistance genes regulated by DNA methylation in infected plants that were enriched for TEs in their promoters. Interestingly, TEs and nearby resistance genes were co‐regulated in TRV‐infected DNA (de)methylation mutants. Our study shows that DNA methylation contributes to modulate the outcome of viral infections in Arabidopsis, and opens up new possibilities for exploring the role of TE regulation in antiviral defence. [ABSTRACT FROM AUTHOR]