Causal Validation of Mobile RNA and Extracellular Vesicle Signals in Plants: Experimental Frameworks for Sequential...

Long-distance molecular transport contributes to coordination between locally stressed tissues and distal organs, but assigning causal systemic-signaling functions to mobile RNAs and extracellular vesicle (EV)-associated molecules remains experimentally challenging. This review critically evaluates the evidence required to distinguish molecular association, source assignment, physical movement, recipient activity, persistence, and causal function in sequential-stress systems. We assess source-resolved RNA tracking, graft-based mobility assays, sequence-assignment and contamination errors, molecular integrity, route validation, EV source-fluid quality, particle identity and heterogeneity, cargo topology, vesicular versus non-vesicular extracellular RNA, recipient uptake, and functional delivery. Recent studies on mobile-mRNA assignment, stress-responsive graft-transmissible transcripts, plant EV proteomes and markers, EV isolation and characterization, and extracellular small-RNA carrier states are integrated to identify which inferences are experimentally supported and which remain provisional. Sequential-stress designs are considered separately because persistence must be distinguished from residual injury, prolonged acute responses, generalized sensitization, and stress-order effects. The objective of this review is to establish reproducible experimental and analytical criteria for assigning systemic-signaling functions to RNA and EV-associated carriers. We therefore propose an evidence hierarchy progressing from association and source assignment to movement, recipient activity, persistence, necessity, sufficiency, and rescue. This framework defines the minimum evidence needed to advance from molecular detection to causal assignment in studies of systemic plant acclimation.