A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana

Autoři: Jian Wu aff001;  Neocles B. Leontis aff002;  Craig L. Zirbel aff003;  David M. Bisaro aff001;  Biao Ding aff001
Působiště autorů: Department of Molecular Genetics, Center for Applied Plant Sciences, Center for RNA Biology, Infectious Diseases Institute, and Graduate Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, Ohio, United States of aff001;  Department of Chemistry and Center for Biomolecular Sciences, Bowling Green State University, Bowling Green, Ohio, United States of America aff002;  Department of Mathematics and Statistics, Bowling Green State University, Bowling Green, Ohio, United States of America aff003
Vyšlo v časopise: A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana. PLoS Pathog 15(10): e32767. doi:10.1371/journal.ppat.1008147
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008147


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.

Klíčová slova:

Histones – Leaves – RNA extraction – RNA structure – Sequence motif analysis – RNA stem-loop structure – Viroids – Palisade mesophyll


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