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Advancing virus-induced gene silencing in sunflower: key factors of VIGS spreading and a novel simple protocol.

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.

SUBMITTER: Mardini M

PROVIDER: S-EPMC11318282 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Advancing virus-induced gene silencing in sunflower: key factors of VIGS spreading and a novel simple protocol.

Mardini Majd M Kazancev Mikhail M Ivoilova Elina E Utkina Victoria V Vlasova Anastasia A Demurin Yakov Y Soloviev Alexander A Kirov Ilya I

Plant methods 20240812 1

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 ...[more]

PMID: 39135113

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Project description:BackgroundVirus-induced gene silencing (VIGS) has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS) gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV).ResultsNbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A) or poly(G) homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A) tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401-500 bp in length and 99.5% lacked poly(A) tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT), with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs.ConclusionBased on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1) Insert lengths should be in the range of ~200 bp to ~1300 bp, (2) they should be positioned in the middle of the cDNA and (3) homopolymeric regions (i.e. poly(A/T) tails) should not be included. Our VIGS-cDNA library method, which incorporates these guidelines to produce sequenced, VIGS-ready cDNAs, will be useful for both fast-forward and reverse genetics experiments in TRV vectors.

Project description:BackgroundVirus-induced gene silencing (VIGS) is widely used in plant functional genomics. However, the efficiency of VIGS in young plantlets varies across plant species. Additionally, VIGS is not optimized for many plant species, especially medicinal plants that produce valuable specialized metabolites.ResultsWe evaluated the efficacy of five-day-old, etiolated seedlings of Catharanthus roseus (periwinkle) for VIGS. The seedlings were vacuum-infiltrated with Agrobacterium tumefaciens GV3101 cells carrying the tobacco rattle virus (TRV) vectors. The protoporphyrin IX magnesium chelatase subunit H (ChlH) gene, a key gene in chlorophyll biosynthesis, was used as the target for VIGS, and we observed yellow cotyledons 6 days after infiltration. As expected, the expression of CrChlH and the chlorophyll contents of the cotyledons were significantly decreased after VIGS. To validate the cotyledon based-VIGS method, we silenced the genes encoding several transcriptional regulators of the terpenoid indole alkaloid (TIA) biosynthesis in C. roseus, including two activators (CrGATA1 and CrMYC2) and two repressors (CrGBF1 and CrGBF2). Silencing CrGATA1 led to downregulation of the vindoline pathway genes (T3O, T3R, and DAT) and decreased vindoline contents in cotyledons. Silencing CrMYC2, followed by elicitation with methyl jasmonate (MeJA), resulted in the downregulation of ORCA2 and ORCA3. We also co-infiltrated C. roseus seedlings with TRV vectors that silence both CrGBF1 and CrGBF2 and overexpress CrMYC2, aiming to simultaneous silencing two repressors while overexpressing an activator. The simultaneous manipulation of repressors and activator resulted in significant upregulation of the TIA pathway genes. To demonstrate the broad application of the cotyledon-based VIGS method, we optimized the method for two other valuable medicinal plants, Glycyrrhiza inflata (licorice) and Artemisia annua (sweet wormwood). When TRV vectors carrying the fragments of the ChlH genes were infiltrated into the seedlings of these plants, we observed yellow cotyledons with decreased chlorophyll contents.ConclusionsThe widely applicable cotyledon-based VIGS method is faster, more efficient, and easily accessible to additional treatments than the traditional VIGS method. It can be combined with transient gene overexpression to achieve simultaneous up- and down-regulation of desired genes in non-model plants. This method provides a powerful tool for functional genomics of medicinal plants, facilitating the discovery and production of valuable therapeutic compounds.

Project description:Proteomic approaches were applied to identify protein spots involved in cold responses in wheat. By comparing the differentially accumulated proteins from two cultivars (UC1110 and PI 610750) and their derivatives, as well as the F10 recombinant inbred line population differing in cold-tolerance, a total of 20 common protein spots representing 16 unique proteins were successfully identified using 2-DE method. Of these, 14 spots had significantly enhanced abundance in the cold-sensitive parental cultivar UC1110 and its 20 descendant lines when compared with the cold-tolerant parental cultivar PI 610750 and its 20 descendant lines. Six protein spots with reduced abundance were also detected. The identified protein spots are involved in stress/defense, carbohydrate metabolism, protein metabolism, nitrogen metabolism, energy metabolism, and photosynthesis. The 20 differentially expressed protein spots were chosen for quantitative real-time polymerase chain reaction (qRT-PCR) to investigate expression changes at the RNA level. The results indicated that the transcriptional expression patterns of 11 genes were consistent with their protein expression models. Among the three unknown proteins, Spot 20 (PAP6-like) showed high sequence similarities with PAP6. qRT-PCR results implied that cold and salt stresses increased the expression of PAP6-like in wheat leaves. Furthermore, VIGS (virus-induced gene silencing)-treated plants generated for PAP6-like were subjected to freezing stress, these plants had more serious droop and wilt, an increased rate of relative electrolyte leakage, reduced relative water content (RWC) and decreased tocopherol levels when compared with viral control plants. However, the plants that were silenced for the other two unknown proteins had no significant differences in comparison to the BSMV0-inoculated plants under freezing conditions. These results indicate that PAP6-like possibly plays an important role in conferring cold tolerance in wheat.

Project description:BackgroundCassava mosaic disease (CMD) is a major constraint to cassava production in sub-Saharan Africa. Under field conditions, evaluation for resistance to CMD takes 12-18 months, often conducted across multiple years and locations under pressure from whitefly-mediated transmission. Under greenhouse or laboratory settings, evaluation for resistance or susceptibility to CMD involves transmission of the causal viruses from an infected source to healthy plants through grafting, or by using Agrobacterium-mediated or biolistic delivery of infectious clones. Following inoculation, visual assessment for CMD symptom development and recovery requires 12-22 weeks. Here we report a rapid screening system for determining resistance and susceptibility to CMD based on virus-induced gene silencing (VIGS) of an endogenous cassava gene.ResultsA VIGS vector was developed based on an infectious clone of the virulent strain of East African cassava mosaic virus (EACMV-K201). A sequence from the cassava (Manihot esculenta) ortholog of Arabidopsis SPINDLY (SPY) was cloned into the CP position of the DNA-A genomic component and used to inoculate cassava plants by Helios® Gene Gun microparticle bombardment. Silencing of Manihot esculenta SPY (MeSPY) using MeSPY1-VIGS resulted in shoot-tip necrosis followed by death of the whole plant in CMD susceptible cassava plants within 2-4 weeks. CMD resistant cultivars were not affected and remained healthy after challenge with MeSPY1-VIGS. Significantly higher virus titers were detected in CMD-susceptible cassava lines compared to resistant controls and were correlated with a concomitant reduction in MeSPY expression in susceptible plants.ConclusionsA rapid VIGS-based screening system was developed for assessing resistance and susceptibility to CMD. The method is space and resource efficient, reducing the time required to perform CMD screening to as little as 2-4 weeks. It can be employed as a high throughput rapid screening system to assess new cassava cultivars and for screening transgenic, gene edited and breeding lines under controlled growth conditions.

Project description:Background and aimsEschscholzia californica (California poppy) is an emerging model plant for 'evo-devo' studies from the basal eudicot clade of Papaveraceae. California poppy has a relatively small genome, a short life cycle and, most importantly, it is amenable for transformation. However, since this transformation protocol is time consuming, virus-induced gene silencing (VIGS) was evaluated as a fast method to obtain functional data for California poppy genes.MethodsCommercially available California poppy plants were infiltrated with Agrobacterium tumefaciens carrying the tobacco rattle virus plasmids pTRV1 and pTRV2. pTRV2 contained part of the eschscholzia Phytoene Desaturase (EcPDS) gene whose loss of function results in photobleaching of the green parts of the plant and in a lack of floral coloration. The degree and duration of these symptoms was evaluated for vegetative rosettes and plants in flower.Key resultsIt is shown that VIGS is able to effectively down-regulate the EcPDS gene in eschscholzia. Various degrees of silencing were observed starting <2 weeks after infiltration with Agrobacterium tumefaciens in 92 % of the plants. Tissue with silencing symptoms also showed complete or strong reduction of EcPDS transcripts. Strong silencing resulted in almost completely white petals, fruits, shoots and leaves. Plants with a strong degree of silencing will eventually die off; however, others are able to produce EcPDS gene product even after a strong initial silencing and will recover. Silencing was found to be not always systemic, but was often restricted to certain organs or parts of organs.ConclusionsVIGS is an effective, fast and transient method to down-regulate gene expression in eschscholzia. It serves well to detect prominent phenotypes which may become obvious even if some target gene transcript remains in the plant tissue. However, subtle phenotypes will be more difficult to detect, as extremely strong silencing effects occur in <10 % of all flowers from infected plants.

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Advancing virus-induced gene silencing in sunflower: key factors of VIGS spreading and a novel simple protocol.

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Advancing virus-induced gene silencing in sunflower: key factors of VIGS spreading and a novel simple protocol.

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