Composition for the development of the apple stem groove virus as a VIGS vector
The ASGV cDNA clone with a CaMV 35S promoter and nos terminator stabilizes gene silencing in diverse hosts, overcoming limitations of existing vectors by inducing efficient gene deletion with minimal symptoms, facilitating functional genomics in plants like citrus and apple.
Patent Information
- Application Number
- DE202025106993
- Authority / Receiving Office
- DE · DE
- Patent Type
- Utility models
- Current Assignee / Owner
- Filing Date
- 2025-11-14
- Publication Date
- 2025-12-31
- Estimated Expiration
- 2035-11-30
AI Technical Summary
Existing virus-induced gene silencing (VIGS) vectors like TRV and CMV have host spectrum limitations and cause symptoms that complicate phenotypic analysis, while ASGV, a capillovirus, offers potential but requires a stable silencing cassette and infectious clone for effective use.
An infectious ASGV cDNA clone is constructed with a CaMV 35S promoter and nos terminator in a binary vector, incorporating a unique cloning site and duplicate minimal CP subgenomic promoter for stable insert maintenance and efficient gene silencing, adapted for use in various host plants including citrus and apple.
The ASGV-VIGS system induces robust gene deletion with minimal symptoms, expanding VIGS applications beyond Nicotiana and enabling functional genomics in multi-year crop plants like citrus and apple.
Abstract
Description
Field of invention
[0001] The invention relates to plant molecular biology systems that utilize an infectious cDNA of the apple stem groove virus (ASGV) as a vector for virus-induced gene silencing (VIGS). This system enables functional genomics in various host plants and integrates promoter engineering and cloning techniques for stable and efficient silencing. Background of the invention
[0002] Virus-induced gene silencing (VIGS) enables the rapid inactivation of plant genes without stable transformation. However, widespread vectors such as TRV, CMV, and AMV may have host-range limitations or induce symptoms that complicate phenotyping. ASGV, a capillovirus, typically causes latent infections in apples and can infect multiple host plants. This suggests its potential as a low-symptom VIGS vector, provided an infectious clone and a stable silencing cassette are developed. Previous work established infectious ASGV cDNA clones using Gibson or agroinfectious assemblies and utilized duplicated minimal envelope (CP) promoters to control subgenomic expression in related vectors. This opened a pathway for the development of silencing payloads.Successful VIGS often utilizes duplicated or minimal subgenomic promoters for stable insert maintenance and efficient silencing of reporter genes such as phytoendesaturase (PDS), leading to albino phenotypes in susceptible host plants. ALSV provides a precedent for broadly effective VIGS systems in fruit trees. Summary of the invention:
[0003] An infectious ASGV cDNA clone was constructed by positioning the complete ASGV genome between a CaMV 35S promoter and a nos terminator in a binary vector. This allows for agro-infectious delivery and systemic infection in herbaceous host plants such as cucumber and bean without visible disease symptoms, consistent with the latent biology of ASGV. The clone was converted into a VIGS vector by introducing a unique cloning site adjacent to a duplicated minimal CP subgenomic promoter element in the 3' UTR. This stabilized foreign inserts and increased gene silencing efficiency.
[0004] Using this platform, a PDS gene fragment from bean or cucumber was inserted to generate ASGV-VIGS-PDS constructs. These induced distinct photobleached (albino) phenotypes after systemic infection, confirming robust gene silencing. Remarkably, the vector retained its infectivity in woody host plants such as citrus and apple. This expands the applicability of VIGS beyond the standard Nicotiana models and provides a tool for functional genomics in perennial crops. Detailed description
[0005] The infectious clone is assembled via Gibson- or yeast-assisted recombination with precise connections between a 35S promoter and the 5' end of the ASGV cDNA and a nos terminator in the 3' poly(A) context within a T-DNA binary scaffold suitable for Agrobacterium-mediated transmission. Infectivity is validated by agroinfiltration in cucumbers and beans, followed by RT-PCR and Northern blot confirmation of viral RNA accumulation and systemic dissemination, with minimal to no macroscopic symptoms observed. To make the clone VIGS-competent, a duplicated minimal CP promoter segment is inserted into the 3' region to generate a stable subgenomic transcription unit for inserted host gene fragments. This employs strategies described for subgenomic promoter duplication in plant VIGS systems.
[0006] A unique restriction cloning site, flanked by stabilizing sequences, allows the insertion of 100–300 bp long gene fragments in sense orientation. The integrity of the inserts is monitored before and after passage using Sanger sequencing to ensure their preservation. PDS fragments from Phaseolus and Cucumis were cloned, and plants agroinfiltrated with ASGV-VIGS-PDS showed systemic albino sectors within 10–21 days of inoculation, which correlated with the reduction in transcript amount measured by qRT-PCR. Host range testing demonstrated agroinfectivity in citrus and apple seedlings, consistent with reports of the wide host range of ASGV and its frequent asymptomatic persistence, and enabling VIGS in otherwise resistant woody plants.
[0007] To reduce recombination and insert loss, synonymous mutations were introduced into repetitive elements around the CP promoter duplication, and non-essential regions were modified to minimize sequence identity hotspots, thereby improving insert stability across serial passages. The vector scaffold supports modular promoters (e.g., doubled 35S) and selectable markers within the T-DNA without altering the viral genome. This enables high-throughput cloning and transfection, similar to established agro-infectious VIGS systems. Comparative studies with ALSV and TRV demonstrated comparable PDS silencing efficiency in cucumbers / beans and unique compatibility with citrus fruits / apples. This positions ASGV-VIGS as a complementary tool where existing vectors reach their limits.Applications include transient functional genomics in monocots and dicots where ASGV can establish infection, validation of target genes for disease resistance or metabolic traits, and silencing of genes influencing fruit quality in perennial species. The minimal symptom profile contributes to phenotypic clarity. The system is adaptable to other target genes by exchanging the insert. Guidelines for insert length and GC content are based on the general literature on VIGS vector design, which emphasizes the trade-off between stability and efficacy.
Claims
[1] Composition comprising an infectious cDNA clone of Apple Stem Grooving Virus (CaMV) positioned between a CaMV-35S promoter and a nos terminator in a binary vector enabling low-symptom systemic infection in cucumbers and beans after application in agriculture. [2] Composition according to claim 1, further comprising a duplicated minimal envelope protein subgenome promoter inserted into the 3' region of the viral genome and a unique cloning site for the inclusion of host gene fragments for virus-induced gene silencing. [3] Composition according to claim 1, wherein the insertion of a phytodesaturase gene fragment leads to systemic albino phenotypes in beans and cucumbers, indicating efficient gene silencing. [4] Composition according to claim 1, wherein the infectious construct retains host compatibility with woody species such as citrus fruits and apples, thus enabling VIGS studies in perennial crops.