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Potato spindle tuber viroid (PSTVd) is a circular non-coding RNA of 359 nucleotides that replicates and spreads systemically in host plants, thus all functions required to establish an infection are mediated by sequence and structural elements in the genome. The PSTVd secondary structure contains 26 Watson-Crick base-paired stems and 27 loops. Most of the loops are believed to form three-dimensional (3D) structural motifs through non-Watson-Crick base pairing, base stacking, and other local interactions. Homology-based prediction using the JAR3D online program revealed that loop 27 (nucleotides 177–182) most likely forms a 3D structure similar to the loop of a conserved hairpin located in the 3' untranslated region of histone mRNAs in animal cells. This stem-loop, which is involved in 3'-end maturation, is not found in polyadenylated plant histone mRNAs. Mutagenesis showed that PSTVd genomes containing base substitutions in loop 27 predicted by JAR3D to disrupt the 3D structure were unable to replicate in Nicotiana benthamiana leaves following mechanical rub inoculation, with one exception: a U178G/U179G double mutant was replication-competent and able to spread within the upper epidermis of inoculated leaves, but was confined to this cell layer. Remarkably, direct delivery of the U178G/U179G mutant into the vascular system by needle puncture inoculation allowed it to spread systemically and enter mesophyll cells and epidermal cells of upper leaves. These findings highlight the importance of RNA 3D structure for PSTVd replication and intercellular trafficking and indicate that loop 27 is required for epidermal exit, but not epidermal entry or transit between other cell types. Thus, requirements for RNA trafficking between epidermal and underlying palisade mesophyll cells are unique and directional. Our findings further suggest that 3D structure and RNA-protein interactions constrain RNA sequence evolution, and validate JAR3D as a tool to predict RNA 3D structure., Author summary Potato spindle tuber viroid (PSTVd) is a small, circular non-coding RNA that systemically infects host plants. All functions required for replication as well as cell-to-cell and systemic spread are mediated by the RNA genome, whose 2D structure consists of multiple base paired stems and loops. However, a wealth of evidence indicates that most loops in RNA molecules are structured by non-canonical base pairs and other interactions. Here, we show that PSTVd loop 27 forms a 3D structure that is essential for replication. Analysis of an exceptional mutant revealed that loop 27 is also required for transit from infected epidermal cells to underlying palisade mesophyll cells, but is not needed for transport in the reverse direction or into other cell types. Thus, loop 27 is necessary for epidermal exit but not entry, and requirements for transit between epidermal cells and palisade mesophyll cells are unique and directional. PSTVd moves between cells through specialized structures known as plasmodesmata. Combined with previous findings, these studies suggest that plasmodesmal gates interconnecting most and perhaps all plant cell types are unique, allowing precise regulation of RNA transport and the establishment of distinct cellular boundaries.