NAC transcription factor SlNOR-like1 plays a dual regulatory role in tomato fruit cuticle formation.

The plant cuticle is an important protective barrier on the plant surface, constructed mainly by polymerized cutin matrix and a complex wax mixture. Although the pathway of plant cuticle biosynthesis has been clarified, knowledge of the transcriptional regulation network underlying fruit cuticle formation remains limited. In the present work, we discovered that tomato fruits of the NAC transcription factor SlNOR-like1 knockout mutants (nor-like1) produced by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] displayed reduced cutin deposition and cuticle thickness, with a microcracking phenotype, while wax accumulation was promoted. Further research revealed that SlNOR-like1 promotes cutin deposition by binding to the promoters of glycerol-3-phosphate acyltransferase6 (SlGPAT6; a key gene for cutin monomer formation) and CUTIN DEFICIENT2 (SlCD2; a positive regulator of cutin production) to activate their expression. Meanwhile, SlNOR-like1 inhibits wax accumulation, acting as a transcriptional repressor by targeting wax biosynthesis, and transport-related genes 3-ketoacyl-CoA synthase1 (SlKCS1), ECERIFERUM 1-2 (SlCER1-2), SlWAX2, and glycosylphosphatidylinositol-anchored lipid transfer protein 1-like (SlLTPG1-like). In conclusion, SlNOR-like1 executes a dual regulatory effect on tomato fruit cuticle development. Our results provide a new model for the transcriptional regulation of fruit cuticle formation.

Transcription factor SlBEL2 interferes with GOLDEN2-LIKE and influences green shoulder formation in tomato fruits.

Chloroplasts play a crucial role in plant growth and fruit quality. However, the molecular mechanisms of chloroplast development are still poorly understood in fruits. In this study, we investigated the role of the transcription factor SlBEL2 (BEL1-LIKE HOMEODOMAIN 2) in fruit of Solanum lycopersicum (tomato). Phenotypic analysis of SlBEL2 overexpression (OE-SlBEL2) and SlBEL2 knockout (KO-SlBEL2) plants revealed that SlBEL2 has the function of inhibiting green shoulder formation in tomato fruits by affecting the development of fruit chloroplasts. Transcriptome profiling revealed that the expression of chloroplast-related genes such as SlGLK2 and SlLHCB1 changed significantly in the fruit of OE-SlBEL2 and KO-SlBEL2 plants. Further analysis showed that SlBEL2 could not only bind to the promoter of SlGLK2 to inhibit its transcription, but also interacted with the SlGLK2 protein to inhibit the transcriptional activity of SlGLK2 and its downstream target genes. SlGLK2 knockout (KO-SlGLK2) plants exhibited a complete absence of the green shoulder, which was consistent with the fruit phenotype of OE-SlBEL2 plants. SlBEL2 showed an expression gradient in fruits, in contrast with that reported for SlGLK2. In conclusion, our study reveals that SlBEL2 affects the formation of green shoulder in tomato fruits by negatively regulating the gradient expression of SlGLK2, thus providing new insights into the molecular mechanism of fruit green shoulder formation.

A tomato NAC transcription factor, SlNAM1, positively regulates ethylene biosynthesis and the onset of tomato fruit ripening.

Fruit ripening in tomato (Solanum lycopersicum) is the result of selective expression of ripening-related genes, which are regulated by transcription factors (TFs). The NAC (NAM, ATAF1/2, and CUC2) TF family is one of the largest families of plant-specific TFs and members are involved in a variety of plant physiological activities, including fruit ripening. Fruit ripening-associated NAC TFs studied in tomato to date include NAC-NOR (non-ripening), SlNOR-like1 (non-ripening like1), SlNAC1, and SlNAC4. Considering the large number of NAC genes in the tomato genome, there is little information about the possible roles of other NAC members in fruit ripening, and research on their target genes is lacking. In this study, we characterize SlNAM1, a NAC TF, which positively regulates the initiation of tomato fruit ripening via its regulation of ethylene biosynthesis. The onset of fruit ripening in slnam1-deficient mutants created by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology was delayed, whereas fruit ripening in OE-SlNAM1 lines was accelerated compared with the wild type. The results of RNA-sequencing (RNA-seq) and promoter analysis suggested that SlNAM1 directly binds to the promoters of two key ethylene biosynthesis genes (1-aminocyclopropane-1-carboxylate synthase: SlACS2 and SlACS4) and activates their expression. This hypothesis was confirmed by electrophoretic mobility shift assays and dual-luciferase reporter assay. Our findings provide insights into the mechanisms of ethylene production and enrich understanding of the tomato fruit ripening regulatory network.

The role and interaction between transcription factor NAC-NOR and DNA demethylase SlDML2 in the biosynthesis of tomato fruit flavor volatiles.

Flavor-imparting volatile chemicals accumulate as fruits ripen, making major contributions to taste. The NAC transcription factor nonripening (NAC-NOR) and DNA demethylase 2 (SlDML2) are essential for tomato fruit ripening, but details of the potential roles and the relationship between these two regulators in the synthesis of volatiles are lacking. Here, we show substantial reductions in fatty acid and carotenoid-derived volatiles in tomato slnor and sldml2 mutants. An unexpected finding is the redundancy and divergence in volatile profiles, biosynthetic gene expression, and DNA methylation in slnor and sldml2 mutants relative to wild-type tomato fruit. Reduced transcript levels are accompanied by hypermethylation of promoters, including the NAC-NOR target gene lipoxygenase (SlLOXC) that is involved in fatty acid-derived volatile synthesis. Interestingly, NAC-NOR activates SlDML2 expression by directly binding to its promoter both in vitro and in vivo. Meanwhile, reduced NAC-NOR expression in the sldml2 mutant is accompanied by hypermethylation of its promoter. These results reveal a relationship between SlDML2-mediated DNA demethylation and NAC-NOR during tomato fruit ripening. In addition to providing new insights into the metabolic modulation of flavor volatiles, the outcome of our study contributes to understanding the genetics and control of fruit ripening and quality attributes in tomato.

Re-evaluation of the nor mutation and the role of the NAC-NOR transcription factor in tomato fruit ripening.

The tomato non-ripening (nor) mutant generates a truncated 186-amino-acid protein (NOR186) and has been demonstrated previously to be a gain-of-function mutant. Here, we provide more evidence to support this view and answer the open question of whether the NAC-NOR gene is important in fruit ripening. Overexpression of NAC-NOR in the nor mutant did not restore the full ripening phenotype. Further analysis showed that the truncated NOR186 protein is located in the nucleus and binds to but does not activate the promoters of 1-aminocyclopropane-1-carboxylic acid synthase2 (SlACS2), geranylgeranyl diphosphate synthase2 (SlGgpps2), and pectate lyase (SlPL), which are involved in ethylene biosynthesis, carotenoid accumulation, and fruit softening, respectively. The activation of the promoters by the wild-type NOR protein can be inhibited by the mutant NOR186 protein. On the other hand, ethylene synthesis, carotenoid accumulation, and fruit softening were significantly inhibited in CR-NOR (CRISPR/Cas9-edited NAC-NOR) fruit compared with the wild-type, but much less severely affected than in the nor mutant, while they were accelerated in OE-NOR (overexpressed NAC-NOR) fruit. These data further indicated that nor is a gain-of-function mutation and NAC-NOR plays a significant role in ripening of wild-type fruit.

BEL1-LIKE HOMEODOMAIN 11 regulates chloroplast development and chlorophyll synthesis in tomato fruit.

Chloroplast development and chlorophyll(Chl)metabolism in unripe tomato contribute to the growth and quality of the fruit, however these mechanisms are poorly understood. In this study, we initially investigated seven homeobox-containing transcription factors (TFs) with specific ripening-associated expression patterns using virus-induced gene silencing (VIGS) technology and found that inhibiting the expression of one of these TFs, BEL1-LIKE HOMEODOMAIN11 (SlBEL11), significantly increased Chl levels in unripe tomato fruit. This enhanced Chl accumulation was further validated by generating stable RNA interference (RNAi) transgenic lines. RNA sequencing (RNA-seq) of RNAi-SlBEL11 fruit at the mature green (MG) stage showed that 48 genes involved in Chl biosynthesis, photosynthesis and chloroplast development were significantly upregulated compared with the wild type (WT) fruit. Genomic global scanning for Homeobox TF binding sites combined with RNA-seq differential gene expression analysis showed that 22 of these 48 genes were potential target genes of SlBEL11 protein. These genes included Chl biosynthesis-related genes encoding for protochlorophyllide reductase (POR), magnesium chelatase H subunit (CHLH) and chlorophyllide a oxygenase (CAO), and chloroplast development-related genes encoding for chlorophyll a/b binding protein (CAB), homeobox protein knotted 2 (TKN2) and ARABIDOPSIS PSEUDO RESPONSE REGULATOR 2-LIKE (APRR2-like). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation quantitative polymerase chain reaction (PCR) (ChIP-qPCR) assays were employed to verify that SlBEL11 protein could bind to the promoters for TKN2, CAB and POR. Taken together, our findings demonstrated that SlBEL11 plays an important role in chloroplast development and Chl synthesis in tomato fruit.

Cooperative functioning between phenylalanine ammonia lyase and isochorismate synthase activities contributes to salicylic acid biosynthesis in soybean.

Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL) or the isochorismate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We investigated the relative contributions of PAL and ICS to defense-related SA accumulation in soybean (Glycine max). Soybean plants silenced for five PAL isoforms or two ICS isoforms were analyzed for SA concentrations and SA-derived defense responses to the hemibiotrophic pathogens Pseudomonas syringae and Phytophthora sojae. We show that, unlike in Arabidopsis, PAL and ICS pathways are equally important for pathogen-induced SA biosynthesis in soybean. Knock-down of either pathway shuts down SA biosynthesis and abrogates pathogen resistance. Moreover, unlike in Arabidopsis, pathogen infection is associated with the suppression of ICS gene expression. Pathogen-induced biosynthesis of SA via the PAL pathway correlates inversely with phenylalanine concentrations. Although infections with either virulent or avirulent strains of the pathogens increase SA concentrations, resistance protein-mediated response to avirulent P. sojae strains may function in an SA-independent manner. These results show that PAL- and ICS-catalyzed reactions function cooperatively in soybean defense and highlight the importance of PAL in pathogen-induced SA biosynthesis.

Composition, metabolism and postharvest function and regulation of fruit cuticle: A review.

The cuticle of plants, a hydrophobic membrane that covers their aerial organs, is crucial to their ability to withstand biotic and abiotic stressors. Fruit is the reproductive organ of plants, and an important dietary source that can offer a variety of nutrients for the human body, and fruit cuticle performs a crucial protective role in fruit development and postharvest quality. This review discusses the universality and diversity of the fruit cuticle composition, and systematically summarizes the metabolic process of fruit cuticle, including the biosynthesis, transport and regulatory factors (including transcription factors, phytohormones and environmental elements) of fruit cuticle. Additionally, we emphasize the postharvest functions and postharvest regulatory technologies of fruit cuticle, and propose future research directions for fruit cuticle.

Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species.

UNLABELLED: Virus-induced gene silencing (VIGS) is a robust technique for identifying the functions of plant genes. Tobacco rattle virus (TRV)-mediated VIGS has been commonly used in many plants. In order to overcome the limitations of existing agroinoculation methods, we report an easy and effective method of agroinoculation for virus-induced gene silencing-sprout vacuum-infiltration (SVI). Using sprout vacuum-infiltration, we have successfully silenced the expression of phytoene desaturase and Mg-protoporphyrin chelatase genes in four important solanaceous crops, including tomato, eggplant, pepper, and Nicotiana benthamiana. The gene-silenced phenotypes are conspicuous in 1-week-old plants. The method is simple, low cost and rapid compared to other techniques such as leaf infiltration or agrodrench. It may be more practical for studying gene function in the early stages of plant growth. An important aspect of SVI is that it will be used for high-throughput VIGS screens in the future. SVI will be an effective tool to overcome the limitations of current inoculation methods and to facilitate large-scale VIGS analysis of cDNA libraries. KEY MESSAGE: SVI is a simple, low cost agroinoculation method for VIGS. It is practical for studying the function of genes expressed in early stages of plant growth and high-throughput VIGS screens.

NAC Transcription Factor Family Regulation of Fruit Ripening and Quality: A Review.

The NAC transcription factor (TF) family is one of the largest plant-specific TF families and its members are involved in the regulation of many vital biological processes during plant growth and development. Recent studies have found that NAC TFs play important roles during the ripening of fleshy fruits and the development of quality attributes. This review focuses on the advances in our understanding of the function of NAC TFs in different fruits and their involvement in the biosynthesis and signal transduction of plant hormones, fruit textural changes, color transformation, accumulation of flavor compounds, seed development and fruit senescence. We discuss the theoretical basis and potential regulatory models for NAC TFs action and provide a comprehensive view of their multiple roles in modulating different aspects of fruit ripening and quality.

Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean.

Omega-3 fatty acid desaturase (FAD3)-catalyzed conversion of linoleic acid to linolenic acid (18:3) is an important step for the biosynthesis of fatty acids as well as the phytohormone jasmonic acid (JA) in plants. We report that silencing three microsomal isoforms of GmFAD3 enhanced the accumulation of Bean pod mottle virus (BPMV) in soybean. The GmFAD3-silenced plants also accumulated higher levels of JA, even though they contained slightly reduced levels of 18:3. Consequently, the GmFAD3-silenced plants expressed JA-responsive pathogenesis-related genes constitutively and exhibited enhanced susceptibility to virulent Pseudomonas syringae. Increased accumulation of BPMV in GmFAD3-silenced plants was likely associated with their JA levels, because exogenous JA application also increased BPMV accumulation. The JA-derived increase in BPMV levels was likely not due to repression of salicylic acid (SA)-derived signaling because the GmFAD3-silenced plants were enhanced in SA-dependent defenses. Furthermore, neither exogenous SA application nor silencing the SA-synthesizing phenylalanine ammonia lyase gene altered BPMV levels in soybean. In addition to the altered defense responses, the GmFAD3-silenced plants also produced significantly larger and heavier seed. Our results indicate that loss of GmFAD3 enhances JA accumulation and, thereby, susceptibility to BPMV in soybean.

GmRAR1 and GmSGT1 are required for basal, R gene-mediated and systemic acquired resistance in soybean.

RAR1, SGT1, and HSP90 are important components of effector-triggered immunity (ETI) in diverse plants, where RAR1 and SGT1 are thought to serve as HSP90 co-chaperones. We show that ETI in soybean requires RAR1 and SGT1 but not HSP90. Rsv1-mediated extreme resistance to Soybean mosaic virus (SMV) and Rpg-1b-mediated resistance to Pseudomonas syringae were compromised in plants silenced for GmRAR1 and GmSGT1-2 but not GmHSP90. This suggests that RAR1- or SGT1-dependant signaling is not always associated with a dependence on HSP90. Unlike in Arabidopsis, SGT1 in soybean also mediates ETI against the bacterial pathogen P. syringae. Similar to Arabidopsis, soybean RAR1 and SGT1 proteins interact with each other and two related HSP90 proteins. Plants silenced for GmHSP90 genes or GmRAR1 exhibited altered morphology, suggesting that these proteins also contribute to developmental processes. Silencing GmRAR1 and GmSGT1-2 impaired resistance to virulent bacteria and systemic acquired resistance (SAR) in soybean as well. Because the Arabidopsis rar1 mutant also showed a defect in SAR, we conclude that RAR1 and SGT1 serve as a point of convergence for basal resistance, ETI, and SAR. We demonstrate that, although soybean defense signaling pathways recruit structurally conserved components, they have distinct requirements for specific proteins.

Metabolomic and Transcriptomic Analyses Reveal That a MADS-Box Transcription Factor TDR4 Regulates Tomato Fruit Quality.

Tomato fruit ripening is a complex process, which determines the formation of fruit quality. Many factors affect fruit ripening, including environmental conditions and genetic factors. Transcription factors (TFs) play key roles in regulating fruit ripening and quality formation. Current studies have found that the TDR4 gene is an important TF for tomato fruit ripening, but its effects on fruit metabolism and quality are less well studied. In this study, suppression of TDR4 gene expression obtained through virus-induced gene silencing (VIGS) technology resulted in an orange pericarp phenotype. Transcriptomic analysis of TDR4-silenced fruit showed changes in the expression of genes involved in various metabolic pathways, including amino acid and flavonoid biosynthesis pathways. Metabolomic analysis showed that levels of several amino acids including phenylalanine and tyrosine, and organic acids were reduced in TDR4-silenced fruit, while α-tomatine accumulated in TDR4-silenced fruit. Taken together, our RNA-seq and metabolomics analyses of TDR4-silenced fruit showed that TDR4 is involved in ripening and nutrient synthesis in tomato fruit, and is therefore an important regulator of fruit quality.

Diversity and redundancy of the ripening regulatory networks revealed by the fruitENCODE and the new CRISPR/Cas9 CNR and NOR mutants.

Tomato is considered as the genetic model for climacteric fruits, in which three major players control the fruit ripening process: ethylene, ripening transcription factors, and DNA methylation. The fruitENCODE project has now shown that there are multiple transcriptional circuits regulating fruit ripening in different species, and H3K27me3, instead of DNA methylation, plays a conserved role in restricting these ripening pathways. In addition, the function of the core tomato ripening transcription factors is now being questioned. We have employed CRISPR/Cas9 genome editing to mutate the SBP-CNR and NAC-NOR transcription factors, both of which are considered as master regulators in the current tomato ripening model. These plants only displayed delayed or partial non-ripening phenotypes, distinct from the original mutant plants, which categorically failed to ripen, suggesting that they might be gain-of-function mutants. Besides increased DNA methylation genome-wide, the original mutants also have hyper-H3K27me3 in ripening gene loci such as ACS2, RIN, and TDR4. It is most likely that multiple genetic and epigenetic factors have contributed to their strong non-ripening phenotypes. Hence, we propose that the field should move beyond these linear and two-dimensional models and embrace the fact that important biological processes such as ripening are often regulated by highly redundant network with inputs from multiple levels.

An oleic acid-mediated pathway induces constitutive defense signaling and enhanced resistance to multiple pathogens in soybean.

Stearoyl-acyl carrier protein-desaturase (SACPD)-catalyzed synthesis of oleic acid (18:1) is an essential step in fatty acid biosynthesis. Arabidopsis mutants (ssi2) with reduced SACPD activity accumulate salicylic acid (SA) and exhibit enhanced resistance to multiple pathogens. We show that reduced levels of 18:1 induce similar defense-related phenotypes in soybean. A Bean pod mottle virus (BPMV)-based vector was employed to effectively silence soybean SACPDs. The silenced plants contained reduced 18:1 and increased stearic acid, developed spontaneous cell death lesions, increased SA accumulation, and constitutively expressed pathogenesis-related genes. These plants also expressed elevated levels of resistance-like genes and showed resistance to bacterial and oomycete pathogens. Exogenous application of glycerol induced similar phenotypes, mimicking the effect of silencing SACPDs in healthy soybean plants. Overexpression of a soybean SACPD increased 18:1 levels in ssi2 but not in wild-type Arabidopsis plants, suggesting that the soybean enzyme was under feedback regulation similar to that of the Arabidopsis isozymes. These results suggest that soybean and Arabidopsis respond similarly to 18:1-derived cues by inducing a novel broad-spectrum resistance-conferring pathway, even though they differ significantly in their lipid biosynthetic pathways. We also demonstrate the efficacy of BPMV-induced gene silencing as a tool for functional studies in soybean.

Manipulation of Light Signal Transduction Factors as a Means of Modifying Steroidal Glycoalkaloids Accumulation in Tomato Leaves.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived specialized metabolites produced by Solanaceous plant species. They contribute to pathogen defense but are considered as anti-nutritional compounds and toxic to humans. Although the genes involved in the SGA biosynthetic pathway have been successfully cloned and identified, transcription factors regulating this pathway are still poorly understood. We report that silencing tomato light signal transduction transcription factors ELONGATED HYPOCOTYL 5 (SlHY5) and PHYTOCHROME INTERACTING FACTOR3 (SlPIF3), by virus-induced gene silencing (VIGS), altered glycoalkaloids levels in tomato leaves compared to control plant. Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) analysis confirmed that SlHY5 and SlPIF3 bind to the promoter of target genes of GLYCOALKALOID METABOLISM (GAME1, GAME4, GAME17), affecting the steady-state concentrations of transcripts coding for SGA pathway enzymes. The results indicate that light-signaling transcription factors HY5 and PIF3 regulate the abundance of SGAs by modulating the transcript levels of these GAME genes. This insight into the regulation of SGA biosynthesis can be used for manipulating the level of these metabolites in crops.

A NAC transcription factor, NOR-like1, is a new positive regulator of tomato fruit ripening.

Ripening of the model fruit tomato (Solanum lycopersicum) is controlled by a transcription factor network including NAC (NAM, ATAF1/2, and CUC2) domain proteins such as No-ripening (NOR), SlNAC1, and SlNAC4, but very little is known about the NAC targets or how they regulate ripening. Here, we conducted a systematic search of fruit-expressed NAC genes and showed that silencing NOR-like1 (Solyc07g063420) using virus-induced gene silencing (VIGS) inhibited specific aspects of ripening. Ripening initiation was delayed by 14 days when NOR-like1 function was inactivated by CRISPR/Cas9 and fruits showed obviously reduced ethylene production, retarded softening and chlorophyll loss, and reduced lycopene accumulation. RNA-sequencing profiling and gene promoter analysis suggested that genes involved in ethylene biosynthesis (SlACS2, SlACS4), color formation (SlGgpps2, SlSGR1), and cell wall metabolism (SlPG2a, SlPL, SlCEL2, and SlEXP1) are direct targets of NOR-like1. Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), and dual-luciferase reporter assay (DLR) confirmed that NOR-like1 bound to the promoters of these genes both in vitro and in vivo, and activated their expression. Our findings demonstrate that NOR-like1 is a new positive regulator of tomato fruit ripening, with an important role in the transcriptional regulatory network.

The role of phytochromes in regulating biosynthesis of sterol glycoalkaloid in eggplant leaves.

Glycoalkaloids are toxic compounds that are synthesized by many Solanum species. Glycoalkaloid biosynthesis is influenced by plant genetic and environmental conditions. Although many studies have shown that light is an important factor affecting glycoalkaloid biosynthesis, the specific mechanism is currently unknown. Chlorophyll and carotenoid biosynthesis depend on light signal transduction and share some intermediate metabolites with the glycoalkaloid biosynthetic pathway. Here, we used virus-induced gene silencing to silence genes encoding phytoene desaturase (PDS) and magnesium chelatase (CHLI and CHLH) to reduce chlorophyll and carotenoid levels in eggplant leaves. Quantification of carotenoid and chlorophyll levels is analyzed by LC/PDA/APCI/MS and semipolar metabolite profiling by LC/HESI/MS. Notably, the resulting lines showed decreases in glycoalkaloid production. We further found that the expression of some genes involved in the production of glycoalkaloids and other metabolites were suppressed in these silenced lines. Our results indicate that photosynthetic pigment accumulation affects steroidal glycoalkaloid biosynthesis in eggplant leaves. This finding lays the foundation for reducing the levels of endogenous antinutritional compounds in crops.

Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity.

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of a phytoene desaturase (PDS) gene is maintained throughout TRV-PDS-inoculated tomato plants as well as in their flowers and fruit and is enhanced by low temperature (15 degrees C) and low humidity (30%). RT-PCR analysis of the PDS gene revealed a dramatic reduction in the level of PDS mRNA in leaves, flowers and fruits. Silencing of PDS results in the accumulation of phytoene, the desaturase substrate. In addition, the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants was reduced by more than 90%. We also silenced the LeEIN2 gene by infecting seedlings, and this suppressed fruit ripenning. We conclude that this VIGS approach should facilitate large-scale functional analysis of genes involved in the development and ripening of tomato.