A Novel Approach to Engineering Tomato Spotted Wilt Virus Infectious Clones by Disarming Key Nodes in Anti-Viral Defenses.

A geminivirus betasatellite subverts the viral coat protein-mediated resistance to promote systemic infection

Viral coat protein (CP)-mediated resistance represents an effective strategy that confers robust antiviral defense in plants. However, the functional relevance of this resistance mechanism during mixed infections by distinct viral species remains largely elusive. Mixed viral infections in agricultural crops frequently lead to exacerbated disease symptoms and substantial yield losses, posing a severe threat to global agricultural production. In this study, we demonstrate that expression of tomato leaf curl New Delhi virus (ToLCNDV) CP confers effective resistance against tomato yellow leaf curl China virus (TYLCCNV), another prominent geminivirus. Strikingly, the βC1 protein encoded by TYLCCNV betasatellite can fully counteract this CP-mediated resistance through direct interaction with ToLCNDV CP and promotion of its proteasome-dependent degradation. We further identified the residue Tyr110 of βC1 as essential for the interaction and subsequent CP degradation. Consequently, βC1-mediated suppression of CP resistance promotes efficient systemic infection of ToLCNDV DNA A. Collectively, our results uncover a novel mechanism by which a viral effector antagonizes viral CP-mediated host resistance during mixed viral infections, offering important insights into viral pathogenesis and the development of sustainable disease control strategies.

Reverse genetics platform for tomato mottle mosaic virus: infectious clone construction and resistance screening in tomato

Comparative evaluation of pKSE401 and pHSE401 gRNA constructs for CRISPR/Cas9-Mediated resistance to cotton leaf curl virus

Cotton leaf curl disease (CLCuD), caused by begomoviruses and their betasatellites, is a major threat to cotton production, especially in South Asia, where periodic viral outbreaks continue to affect cotton yields, quality, and livelihoods. The advent of CRISPR/Cas9 gene-editing technology has transformed plant biotechnology, offering efficient, accurate, and programmable methods for combating viral pathogens at the genetic level. Here, the antiviral efficacy of two most widely used CRISPR/Cas9 binary plant expression vectors, pKSE401 and pHSE401, was tested in Nicotiana benthamiana against Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan betasatellite (CLCuMuB). The guide-RNAs (gRNAs) were designed to target viral genes that play significant roles in pathogenicity and replication, with pHSE401 encoding a single gRNA and pKSE401 a multiplex of two gRNAs. Agrobacterium-mediated transient transformation and viral inoculation experiments revealed that both CRISPR/Cas9 vectors effectively delayed symptom onset and reduced virus titers relative to infected controls. Remarkably, the multiplex pKSE401 system was more effective at suppressing viral infection, achieving about a 90% reduction in viral accumulation compared with a 75% reduction by the single gRNA pHSE401 construct. pKSE401-treated plants showed delayed symptom development, reduced severity, and partial recovery, demonstrating the improved efficiency of multiplex genome editing. The results demonstrate the cutting-edge potential of CRISPR/Cas9 multiplex approaches as next-generation methods for designing sustainable resistance to multifaceted plant virus diseases. This research not only contributes to our understanding of CRISPR-based antiviral response mechanisms but also provides a promising avenue for designing broad-spectrum, sustainable resistance against viral epidemics in cotton and other commercially valuable crops.

Reduced risk of a next-generation recombinant viral vector engineered from a plant rhabdovirus genome

The risk of environmental escape of a bioengineered, recombinant maize mosaic virus (MMV-GFP) that encodes green fluorescent protein as a test case was examined and MMV-GFP was less fit than MMV-WT with regards to transmission biology and plant infection.

Genomic Component Interactions Shape Host-Specific Systemic Infection in Tomato Leaf Curl New Delhi Virus.

These findings identify DNA-B as a major determinant of tomato infectivity and reveal an essential role for CP in systemic movement of the Mediterranean ToLCNDV isolate.

Virus-induced gene silencing as a tool for functional genomics in weeds: Challenges and future directions.

Virus-induced gene silencing (VIGS) has evolved from a conceptual demonstration of antiviral defense into a pivotal reverse-genetics platform for plant functional genomics. By exploiting engineered DNA- or RNA-based viral vectors, VIGS enables rapid, sequence-specific transcript knockdown through RNA-mediated degradation of target transcripts. Recent refinements in vector design, inoculation strategies, and viral species selection, such as TRV, BSMV, and FoMV, have expanded its application to previously recalcitrant plants, including major crops and emerging weed models. In weeds, functional genomics remains particularly challenging due to high genetic variability, limited genomic resources, and incompatibility with conventional viral vectors and transformation systems. In this context, VIGS provides a tractable approach to investigate genes associated with herbicide resistance, metabolic adaptation, and stress tolerance. Beyond weed biology, its application to studies of immune signaling, hormonal crosstalk, and secondary metabolism highlights VIGS as a versatile biotechnology for elucidating gene function and supporting next-generation strategies in plant improvement and integrated pest management.