Abstract
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.
Highlights
The ability of multicellular organisms to grow, develop, and respond to the environment requires efficient, targeted, and regulated cell-to-cell and systemic communication involving the exchange of both small molecules and macromolecules
We show that Potato spindle tuber viroid (PSTVd) loop 27 forms a 3D structure that is essential for replication
Analysis of an exceptional mutant revealed that loop 27 is 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
Summary
The ability of multicellular organisms to grow, develop, and respond to the environment requires efficient, targeted, and regulated cell-to-cell and systemic communication involving the exchange of both small molecules and macromolecules. Cell boundaries play critical roles in balancing information transfer and cell autonomy, in part by acting as barriers that either facilitate or prevent the exchange of specific nucleic acids and proteins. Developmental, physiological, and defensive processes involve the spread of specific mRNAs and proteins from the cells in which they are produced into neighboring and distant cells [3]. Trafficking of siRNAs mediates local and systemic gene silencing through pathways such as RNA interference and RNA-directed DNA methylation [15,16,17], which are essential for antiviral defense [18,19,20]. Natural inter-organism exchanges of RNA molecules have been observed between host and parasitic plants [21, 22], as well as fungal pathogens and their hosts [23, 24]
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