Applications of Virus Induced Gene Silencing (VIGS) in Plant Functional Genomics Studies

Bekele, Dagnachew; Tesfaye, Kassahun; Fikre, Asnake

doi:10.4172/2329-9029.1000229

Cited by 25 publications

(13 citation statements)

References 54 publications

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“…In the past two decades, over 50 plant VIGS vectors have been generated (Dommes et al, 2019). However, most VIGS vectors functioning in eudicots are not effective in monocots (Bekele et al, 2019;Dommes et al, 2019;Kant and Dasgupta, 2019). Among those developed for monocotyledons, three have been applied to silence genes in wheat.…”

Section: Introductionmentioning

confidence: 99%

An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.)

et al. 2021

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Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

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Section: Introductionmentioning

confidence: 99%

An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.)

et al. 2021

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“…VIGS is sensitive to the genotypes of specific plant species (Bekele et al, 2019). Thus, three types of watermelon cultigens were evaluated in the current work.…”

Section: Discussionmentioning

confidence: 99%

Induction of virus-induced gene silencing andin plantavalidation in cucurbits using the CFMMV-Cm vector

Lee

Rhee

Jang

et al. 2021

Preprint

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Virus-induced gene silencing (VIGS) has been employed for the high-throughput analysis of endogenous gene function. We developed a CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) for the efficient generation of viral transcripts in plants. Using the novel pCF93 vector, we identified genes related to male sterility in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. We previously reported reference-based and de novo transcriptomic profiling for the detection of differentially expressed genes between a male fertile line (DAH3615) and its near isogenic male sterile line (DAH3615-MS). Based on the RNA-seq results, we identified 38 de novo-exclusive differentially expressed genes (DEDEGs) that are potentially responsible for male sterility. Partial genes of 200∼300bp were cloned into pCF93 which was then inoculated into DAH, a small type of watermelon that enables high-throughput screening with a small cultivation area. In this manner, we simultaneously characterized phenotypes associated with the 38 candidate genes in a common-sized greenhouse. Eight out of the 38 gene-silenced plants produced male sterile flowers with abnormal stamens and no pollens. Gene expression levels in flowers were validated via RT-qPCR. Stamen histological sections from male sterile floral buds and mature flowers showed developmental disruption and shrunken pollen sacs. Based on the current findings, we believe that the novel pCF93 vector and our VIGS system facilitate high-throughput analysis for the study of gene function in watermelons.One sentence summaryThe CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) facilitates large-scale validation of male sterility-related gene functions in watermelon.

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“…In the context of insect pest control, plant viruses or insect viruses can potentially be engineered for VIGS against insect pests. Many VIGS systems have been widely developed to specifically suppress target genes in plants (Bekele et al 2019;Abrahamian et al 2020). Interestingly, these systems can also be modified for pest control by inserting insect target genes into the plant viral vectors (Kolliopoulou et al 2020), with the aim of ultimately silencing essential genes in the targeted insect pests.…”

Section: Virus-induced Gene Silencing (Vigs) Systemsmentioning

confidence: 99%

Engineering strategies for insect viruses and microbes for dsRNA production and delivery to insects for targeted gene silencing

Xue¹,

Santos²,

Smagghe³

et al. 2023

entomologia

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RNA interference (RNAi)-based pest control is viewed as a safe and effective method for insect pest control. However, the stability and efficient cellular delivery of the double stranded RNA (dsRNA) active molecule remains crucial to its success in pest control. Among the different approaches that have been proposed to improve dsRNA delivery in insects, the use of nano-carriers, engineered viruses and engineered microbes have been successfully employed in proofof-concept studies. This review focuses on engineering approaches that have been successfully employed to modify insect viruses and different microbes for dsRNA production and delivery in insects. First, the different viral vector systems based on insect RNA and DNA viruses for dsRNA delivery are discussed. Second, a comprehensive overview of different microbe-based (bacteria, fungi, microalgae) dsRNA production and delivery systems that have been developed till date are presented. Finally, future perspectives on the development of other delivery systems with potential in the context of insect pest control are provided. Overall, this review is expected to stimulate awareness, interest, insight and research into promising sources of biotechnologically interesting viruses and microbes for protecting agriculture and human health against insect pests.

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Applications of Virus Induced Gene Silencing (VIGS) in Plant Functional Genomics Studies

Cited by 25 publications

References 54 publications

An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.)

An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.)

Induction of virus-induced gene silencing andin plantavalidation in cucurbits using the CFMMV-Cm vector

Engineering strategies for insect viruses and microbes for dsRNA production and delivery to insects for targeted gene silencing

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