An all-in-one plant virus-based vector toolkit for streamlined gene silencing, overexpression, and genome editing

Dear Editor,

Plant expression vectors are instrumental tools used to assess plant functional genomics and facilitate breeding improvements. Plant viruses may contain exogenous genes for replication and systemic movement in host plants, allowing them to be engineered into plant expression vectors for systemic and enhanced gene expression manipulation (Abrahamian et al., 2020). These unique advantages have been leveraged across a range of applications developed using plant virus expression vectors, including virus-induced gene silencing (VIGS), virus-mediated overexpression (VOX), virus-assisted transient expression (VATE), and virus-induced genome editing (VIGE) (Rössner et al., 2022). However, the cloning approaches and multiple cloning sites (MCSs) used vary among viral vectors developed by different laboratories, complicating their integration into a unified toolkit. Although multiple conventional plant expression vector toolkits have recently been developed (Han et al., 2022; Yan et al., 2023), there are no published reports on the integration of multiple viruses into a streamlined toolkit with diverse functions.

Bipartite and multipartite viruses are frequently utilized in plant biology research (Dommes et al., 2019). The traditional strategy used for generating infectious clones for agroinfiltration involves cloning each viral genome into separate binary T-DNA vectors. However, this mixed-bacteria strategy necessitates additional steps to culture and prepare uniformly mixed genomes, increasing the overall workload, especially in high-throughput experiments (Figure 1A). An alternative strategy involves the transformation of two compatible plasmids into a single disarmed Agrobacterium strain, enabling genome delivery via two T-DNAs (Pasin et al., 2017; Aragonés et al., 2022). A simpler strategy for transforming all genomes via a single T-DNA has not yet been reported.

Figure 1.

All-in-one plant viral vector toolkit.

(A) Workflow comparison between a traditional strategy and the all-in-one viral vector strategy, exemplified by bipartite Cotton leaf crumple virus (CLCrV). Compared to conventional methods, the all-in-one strategy simplifies the procedures at the Agrobacterium stage by eliminating the need for separate transformation or resuscitation culture of helper bacteria, adjustment of bacterial suspension concentration, and mixing steps.

(B) Architecture of the original and modified all-in-one vectors for CLCrV. Mutated restriction enzyme sites are indicated in gray font. A 158-bp deletion within the common region (CR; originally 901 bp) on one side was used to verify viral genome cyclization via Sanger sequencing.

(C) Photobleaching phenotype of cotton inoculated with VS-GhPDS at 90 days post-inoculation (dpi).

(D) Gel electrophoresis results of circular viral genomes from plants inoculated with VS or VA plus VB. “M” indicates the uninoculated mock control. The red and blue arrows indicate primers specific for amplification of the circular VA and VB genomes, respectively.

(E) Architecture of the original and modified all-in-one vectors for TRV. Mutated restriction enzyme sites are indicated in gray font.

(F) Photobleaching phenotype of VS2-GhCLA-inoculated cotton plants at 21 dpi.

(G) Diagram of the recombination-based molecular cloning method for all-in-one viral vectors. The 2×BsaI-containing MCS and its flanking adapter sequences are highlighted for each application.

(H) Schematic diagrams and applications of the all-in-one viral vector toolkit. pVS/pVO, pVS2, and pVS3/pVO3 for virus-induced gene silencing (VIGS)/virus-mediated overexpression (VOX) (top left). pVT, pVTCLs, and pVTHV for virus-assisted transient expression (VATE) (top middle). NU, nucleus; PM, plasma membrane; ER, endoplasmic reticulum; GB, Golgi body. pVE was used for virus-induced genome editing (VIGE) (top right). Co-transformation of pVS and pVSr was used to evaluate VIGS in protoplasts (bottom left). Two strategies for constructing pVM for virus-induced gene manipulation combination (VIGM-Combo) (bottom right). GOI, gene of interest.

To address these issues, we focused on two widely used bipartite viral vector systems: the DNA virus Cotton leaf crumple virus (CLCrV) and the RNA virus Tobacco rattle virus (TRV) (Liu et al., 2002; Tuttle et al., 2012), which were modified to generate two all-in-one VIGS vector systems, designated VS and VS2, respectively. In the CLCrV-based VS system, the VA and VB genomes were arranged in tandem and flanked by their respective common regions, ensuring reconstruction of their circular genomes (Figure 1B). After several rounds of modification, the final plasmid size of VS was comparable to that of the VA-containing plasmid (see the supplemental methods for details). To assess the feasibility of the VS system, we delivered VS-GhPDS, which contained a fragment of the cotton Phytoene desaturase (GhPDS) gene, into cotton cotyledons via agroinfiltration. As anticipated, the photobleaching phenotype resulting from GhPDS silencing was observed and persisted through late developmental stages (Figure 1C). Additionally, circularized viral genome DNA was detected by PCR and confirmed by Sanger sequencing in non-inoculated young leaves, indicating that tandem-arranged viral genomes could be recovered from a single linear T-DNA without mis-splicing (Figure 1D; Supplemental Figure 1A). In the TRV-based VS2 system, the 35S expression cassettes of the RNA1 and RNA2 genomes were arranged bidirectionally, and all five BsaI sites within the viral coding sequences were modified through synonymous mutations (see supplemental methods for details) (Figure 1E). As with the VS system, cotton plants infiltrated with VS2-GhCLA exhibited a phenotype consistent with the Cloroplastos alterados (GhCLA) gene-silencing phenotype (Figure 1F). To further evaluate the effectiveness of the VS and VS2 vectors, we constructed empty vectors pVSe and pVS2e as negative controls and tested the silencing of multiple additional marker genes. Our results demonstrated that both VS and VS2 vectors showed VIGS efficiencies comparable to, or greater than, those of the original bipartite vectors, possibly due to more effective genome co-delivery into single cells via a single T-DNA (Supplemental Figures 1B–1L). Although the cloning efficiency of the all-in-one plasmids decreased (Supplemental Figure 1M), particularly for pVS2 that contained the toxic RNA1 genome, it remained sufficient for single-gene research (VS2 system) and high-throughput gene research (VS system). Notably, both VS and VS2 plasmids shared a unified 2×BsaI-containing MCS and homologous arms, facilitating simultaneous recombination-based molecular cloning for CLCrV and TRV (Figure 1G). Collectively, these results demonstrate that the all-in-one viral vector strategy is applicable to bipartite DNA and RNA viruses without compromising viral replication or VIGS efficiency. To further expand the utility of the all-in-one silencing vector, we modified pVS to generate pVT, pVO, and pVE for intended use in VATE, VOX, and VIGE, respectively. As before, unified MCS adapters were employed for homologous-recombination-based vector assembly for each application (Figure 1G). For VATE, the VB-encoded genes responsible for systemic movement were replaced with a 35S–MCS–NOS expression cassette to generate pVT. This expression cassette, flanked by the common region of VB, could be spliced to form an artificial replicon, potentially increasing its copy number and expression level (Supplemental Figures 2A and 2B). The EGFP, β-Glucuronidase (GUS), Luciferase (LUC), and Ruby reporters were successfully expressed in tobacco and cotton leaves (Supplemental Figures 2C–2E). Additionally, we modified pVT into pVTCLs and pVTHV3 as all-in-one vectors for protein subcellular localization and Bimolecular Fluorescence Complementation (BiFC) assays, respectively (Figure 1H). To this end, the previously characterized protein AtbZIP63 was used as a test case, and its nuclear localization and self-interaction were confirmed (Supplemental Figures 3A–3F). For VOX, pVS could be directly utilized as pVO when the protein-coding gene was arranged in-frame after the start codon of the residual viral AV gene (Supplemental Figures 4A and 4B). For VIGE, the AtU6-26 promoter was used to drive Single-guide RNA (sgRNA) expression, resulting in systemic somatic editing in Cas9-expressing cotton plants (Supplemental Figures 4C and 4G). Taking these results together, we illustrated that the all-in-one viral vector can be further developed into a multifunctional toolkit featuring a unified and streamlined cloning method.

Next, we explored the all-in-one vector strategy for multiple gene manipulation. To simultaneously achieve VIGS and VOX, we incorporated an additional VA component into the MCS of pVS/pVO and successfully manipulated the expression of cotton SINGLE FLOWER TRUSS (GhSFT) and SELF-PRUNING (GhSP) through a single T-DNA delivery (Supplemental Figures 5A–5C). We also successfully demonstrated that two VIGS fragments could be arranged in tandem within the MCS of pVS2, enabling simultaneous gene silencing (Supplemental Figures 5D–5G). PCR and nanopore sequencing results confirmed that the all-in-one vectors were stable in both Escherichia coli and Agrobacterium (Supplemental Figures 5H–5K). We termed these strategies “virus-induced gene manipulation combination.” This system not only enables the study of functionally redundant genes and related genetic pathways, but also serves as a pre-screening program for designed breeding, particularly for the majority of plants that currently lack efficient heritable multiplex genome editing techniques (Figure 1H).

In planta screening of efficient VIGS fragments via agroinfiltration typically requires 2–3 weeks. Inspired by our serendipitous discovery that circular viral genomes could be detected as early as 12 h post-protoplast transformation with pVS, we inferred that VIGS could be rapidly evaluated in protoplasts within 2 days, an assay herein referred to as proto-VIGS (Supplemental Figure 6A). We designed a dual-luciferase-based VIGS reporter plasmid, named pVSr, to monitor proto-VIGS (Supplemental Figure 6B). In this reporter, a homologous sequence containing the PDS fragment of pVS-GhPDS was fused in-frame with LUC, resulting in a significant decrease in the LUC/Renilla luciferase (REN) ratio upon co-transformation with pVS-GhPDS compared to pVSe, indicative of proto-VIGS occurrence (Supplemental Figures 6B–6G). Demonstrating the utility of this reporter, we identified two highly efficient cotton flavonoid O-methyltransferase (GhOMT1) fragments and further validated their efficiency using agroinfiltration-mediated VIGS (Supplemental Figures 6H–6J). In future studies, pVSr can serve not only to screen highly efficient VIGS fragments but also to identify VIGS enhancers and repressors in a high-throughput and time-saving manner (Supplemental Figure 6K).

To further expand our toolkit, Cotton chlorotic spot virus was engineered de novo as the VS3 system using the all-in-one strategy, and its effectiveness as a VIGS vector was confirmed (Supplemental Figures 7A–7F) (de Almeida et al., 2013). VS3-inoculated cotton plants showed preferential silencing at growing points, and VS3 was not transmitted to the next generation, suggesting its potential as an efficient VIGE tool (Supplemental Figures 7G–7J). Finally, we demonstrated that the CLA fragment from cotton successfully induced TRV-based VIGS in other Malvoideae species, demonstrating the effectiveness of our all-in-one viral vector toolkit in related species (Supplemental Figures 8A–8J). Considering the extensive applicability of the TRV-based VIGS tool across diverse plant species and in a multitude of functions, enhancing the cloning efficiency of the VS2 system is expected to further augment its utility for assessing plant functional genomics.

In summary, we have developed a multifunctional all-in-one viral vector toolkit that simplifies the use of bipartite viral vectors, serving as a valuable tool due to its ease of use, versatility, compatibility, and extensibility. In addition to facilitating basic research, our all-in-one strategy may facilitate large-scale agronomic trait reprogramming in the field, thus advancing plant virus commercialization (Pasin et al., 2024).

Data and code availability

All plasmids generated in this study can be requested directly via email (zdy041@163.com) or through Addgene (https://www.addgene.org/depositing/85611/).

Funding

This work was supported by the Biological Breeding of Early Maturing and Disease Resistant Cotton Varieties (grant no. 2023ZD04041), the China Agricultural Research System (grant no. CARS-15-06), the Natural Science Foundation of Henan Province (grant no. 232300421041), and the Nanfan Special Project (grant nos. YBXM2316 and YBXM2441).

Acknowledgments

We thank Prof. Mingbao Luan at the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, for providing kenaf seedlings. No conflict of interest declared.

Author contributions

G.S. and Y.H. designed the experiments and wrote the manuscript. Y.H., Z.Y., Y.L., H.C., Q.W., D.Z., Y.Z., L.L., and J.L. conducted the experiments and revised the manuscript.

Published: May 19, 2025