Comprehensive Secondary Structure Elucidation of Four Genera of the Family Pospiviroidae

Abstract

Viroids are small, circular, single stranded RNA molecules that infect plants. Since they are non-coding, their structures play a critical role in their life cycles. To date, little effort has been spend on elucidating viroid structures in solution due to both the experimental difficulties and the time-consuming nature of the methodologies implicated. Recently, the technique of high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) was adapted for the probing of the members of family Avsunviroidae, all of whom replicate in the chloroplast and demonstrate ribozyme activity. In the present work, twelve viroid species belonging to four different genera of the family Pospiviroidae, whose members are characterized by the presence of a central conserved region (CCR) and who replicate in nucleus of the host, were probed. Given that the structures of five distinct viroid species from the family Pospiviroidae have been previously reported, an overview of the different structural characteristics for all genera and the beginning of a manual classification of the different viroids based on their structural features are presented here.

Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants

“…The dimeric construct of PSTVd-Dahlia (GenBank accession number AB623143: the term PSTVd-M was used as this variant induces mild symptoms in tomato plants), which was previously used to study structure in solution, was used to synthesize infectious dimeric transcripts (Giguère et al, 2014). In the case of the PSTVd-I variant, the head-to-tail dimers were constructed using monomeric PSTVd-I (GenBank accession number AY937179) obtained from gBlock Gene Fragments (Integrated DNA Technologies) in the pBluescript KS+ vector (Stratagene) as described previously (Giguère et al, 2014).…”

“…In the case of the PSTVd-I variant, the head-to-tail dimers were constructed using monomeric PSTVd-I (GenBank accession number AY937179) obtained from gBlock Gene Fragments (Integrated DNA Technologies) in the pBluescript KS+ vector (Stratagene) as described previously (Giguère et al, 2014). Monomeric mutants of PSTVd were generated by PCR amplification using PSTVd-81F primers combined with appropriate reverse primer as listed in Supplemental Table 8.…”

“…Secondary structure predictions revealed that these two changes affect the sizes of loops 7 and 8, which are required for the initiation of systemic trafficking and cell-to-cell movement through PD of PSTVd (Supplemental Figure 1; Zhong et al, 2008). Recently, new methods, such as high-throughput SHAPE, have been used on viroids in solution, in combination with prediction algorithms, to elucidate viroid structure (Giguère et al, 2014). We used SHAPE to verify and compare the structure of two PSTVd variants in solution.…”

“…We used SHAPE to verify and compare the structure of two PSTVd variants in solution. As the secondary structure of PSTVd-M in solution had been previously described (Giguère et al, 2014), transcripts of PSTVd-I were prepared for benzoyl cyanide treatment followed by primer extension, as previously described (Giguère et al, 2014). All resulting mapping data were inputted into the RNAstructure software to determine the secondary structures ( Figure 1A; Reuter and Mathews, 2010).…”

“…Recently, the structure of PSTVd in solution was resolved by highthroughput selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). PSTVd-infected tomato plants have also been used to both identify small RNAs (sRNAs) derived from PSTVd upon infection, as well as to monitor tomato gene regulation by microarray analysis (Owens et al, 2012;Giguère et al, 2014). Furthermore, different PSTVd variants induce an array of symptoms in S. lycopersicum cv Rutgers, ranging from mild to severe (Tsushima et al, 2015).…”