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

Envisaging the role of plant Small RNAs: A comprehensive review on antiviral defense mechanisms

Plant RNA interference from antiviral silencing to multiplex trait engineering for climate-resilient crops

RNA interference (RNAi) in plants has evolved from an unexplained antiviral and transgene interference phenomenon into a general regulatory platform for sequence-guided gene suppression, chromatin control, systemic signaling, and phenotypic plasticity. This Review synthesizes six decades of plant RNAi, tracing its progression through conceptual bottlenecks and technological solutions. Early work established that RNA-derived homology could suppress viral infection and transgene expression. Mechanistic studies then revealed a diversified plant silencing system involving Dicer-like proteins, Argonautes, RNA-dependent RNA polymerases, systemic movement, and RNA-directed DNA methylation. In parallel, RNAi moved into crop design, enabling targeted modification of yield, fiber quality, flowering, disease resistance, allergenicity, fertility, plant architecture, lignin content, nutrient composition, and pest resistance across diverse species. Importantly, RNAi is not merely a historical precursor to genome editing. It retains distinct value because it can tune gene dosage, silence multigene families, uncover compensatory network responses, and perturb upstream regulatory nodes, such as phytochrome RNAi in cotton, where partial suppression simultaneously improves several negatively correlated traits. Most recently, host-induced silencing, spray-induced dsRNA, nanocarrier delivery, and CRISPR-associated RNA tools have repositioned RNAi as a versatile breeding platform. The future lies in convergence with genome editing, using pangenome-informed, allele-aware target design and combined RNAi-editing pipelines. The lesson learned is that useful crop engineering often requires rebalancing endogenous networks rather than permanent gene knockout. In this review, the historical developmental phases are used carefully: the formal molecular term RNA interference emerged in the late 1990s, while earlier plant work on antiviral resistance, co-suppression and post-transcriptional gene silencing anticipated the same sequence-guided logic. At the same time, practical deployment remains constrained by variable knockdown, off-target risk, construct instability, environmental degradation of sprayed RNA, delivery cost, resistance evolution in target pests or pathogens, regulatory classification, and public acceptance; these constraints are discussed as platform-specific design and risk-assessment issues rather than as generic barriers.

Virus-induced Genome Editing in Plants: Viral Vector Biology, Germline Access and the Evidence Base for Heritable, Transgene-Free Crop Improvement

Delivery, rather than nuclease chemistry, now sets the practical limit on plant genome editing. Conventional delivery depends on tissue culture and stable transformation, which remain slow, genotype-dependent and unavailable for most cultivated germplasm. Plant viruses offer an alternative because they replicate to high copy number, move systemically and can carry heterologous sequences into cells that no transformation protocol reaches. Virus-induced genome editing exploits this behaviour, and the field has moved within a decade from transient somatic mutagenesis in a model tobacco to heritable, transgene-free mutations in hexaploid bread wheat. This critical review evaluates the strength and the limits of that evidence. Vector chassis are compared on the properties that actually determine outcome, namely cargo capacity, insert stability, host range, movement behaviour and access to reproductive tissue, rather than on reported somatic editing percentages, which are shown to be poor predictors of germline transmission. The central unresolved problem is identified as the disjunction between efficient editing in infected somatic tissue and rare, stochastic entry of editing reagents into cells that give rise to gametes. Three engineering responses to that problem, namely fusion of guide RNAs to mobile RNA motifs, control of nuclease expression through meristem-competent promoters, and exploitation of axillary or adventitious growth points, are assessed against the evidence for each. Reagent miniaturisation, guide array design and virus-delivered precision editing are examined as partial solutions to the cargo constraint. Recurrent weaknesses in the literature are documented, including reliance on visible reporter loci, small progeny samples, inconsistent definitions of editing efficiency, near-absence of independent replication and the complete absence of field evaluation. The claim that virus-derived products are transgene-free is examined against the regulatory frameworks that will govern them, and the biosafety implications of releasing engineered, potentially insect-transmissible vectors are considered. Prioritised research directions are proposed, emphasising standardised reporting of progeny-level outcomes, biocontained vector design, and genotype-spanning validation in elite crop backgrounds.

RNA interference for hemipteran pest control: mechanisms, resistance factors, and advances in delivery systems

RNA interference (RNAi) represents a species-specific and environmentally sustainable strategy for the control of hemipteran pests, including aphids, whiteflies, psyllids, and stinkbugs. This review integrates current knowledge on the molecular mechanisms of RNAi in insects and its translational applications in crop protection, with particular focus on host-induced gene silencing (HIGS, transgenic expression of dsRNA in planta) and spray-induced gene silencing (SIGS). The biological effects of gene knockdown – such as reduced survival, impaired development, and disrupted feeding – are discussed alongside physiological, genetic, and epigenetic factors limiting RNAi efficacy in Hemiptera. Key challenges include dsRNA degradation in the digestive tract, inefficient cellular uptake, and limited systemic spread. Recent innovations – including nanocarrier-based delivery, biodegradable formulations, and transplastomic expression – are critically evaluated. Similarly, advances in target gene identification using transcriptomic and proteomic approaches, as well as risk assessment tools for minimizing non-target effects, support the rational design of RNAi-based biopesticides. As RNAi technologies advance, their incorporation into integrated pest management strategies may facilitate species-specific, residue-free control of hemipteran pests while supporting the conservation of agroecosystem biodiversity.

RNA interference mediated disease resistance in vegetable crops: fundamental discovery to current trends

Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement