On the road with RNA interference: Missed stops and future directions in leveraging spray-induced gene silencing for sustainable fungal management in agriculture.

Spray-induced gene silencing (SIGS) has moved from proof-of-concept to one of the most compelling RNA-based routes for crop protection, yet its field deployment still rests on unresolved biological interfaces. In this review, we revisit SIGS against fungal pathogens by asking where exogenous double-stranded RNAs go, which organisms perceive them, and how delivery technologies may reshape their ecological footprint. We first examine double-stranded RNA (dsRNA) uptake as a limiting and still unevenly understood step: clathrin-mediated endocytosis is emerging as a recurrent entry route in fungi, whereas plant perception, transport across surface barriers, and systemic movement remain mechanistically obscure. We then discuss evidence that dsRNAs are not inert silencing triggers. Beyond sequence-specific RNA interference, they can activate pathogen-associated molecular pattern (PAMP)-like and stress-response pathways, including fungal high osmolarity glycerol (HOG) signaling, implying that dose, formulation, and exposure context may influence both efficacy and nontarget effects. This perspective is extended to the plant holobiont, where direct off-target silencing and indirect microbiome remodeling represent distinct but often conflated risk layers, particularly for endophytic and beneficial fungi exposed through systemic RNAi. Finally, we evaluate how nanocarriers, BioClay, chitosan particles, and artificial vesicles are being developed to protect dsRNAs from degradation, improve uptake and enable more precise delivery. We argue that the next phase of SIGS research must integrate molecular uptake biology, concentration-aware risk assessment, and scalable formulation design. Such integration will determine whether SIGS becomes merely another promising laboratory technology or a robust, ecologically informed platform for sustainable fungal disease management in agriculture.