High-throughput sequencing (HTS) technologies have contributed to the identification of several viroids and viroid-like RNAs, including a small (434 nt) circular RNA containing the conserved domains of hammerhead ribozymes in apple trees grown in China (Zhang et al., PLoS Pathogens 2014). Although the possible viroid nature of this RNA was anticipated, the final proof was only recently provided by bioassays confirming its autonomous replication in apple (Serra et al., Virus Res. 2018). In the same study, based on in silico modeling and co-variation analysis of viroid populations, the two polarity strands of AHVd were predicted to adopt branched conformations. Interestingly, the (+) polarity strand of AHVd, but not its (-) counterpart, was proposed to be stabilized by a kissing loop- interaction, a situation resembling that of peach latent mosaic viroid and chrysanthemum chlorotic mottle viroid (genus Pelamoviroid, family Avsunviroidae). Besides China, AHVd has been identified only in the cultivar Pacific Gale in Canada (Messmer et al., Can J. Plant Pathol. 2017). Results. In the frame of a project aimed to examine the phytosanitary status of ancient Apulian fruit tree varieties, partially purified dsRNA preparations from several fruit tree species were pooled together and analyzed by HTS (Illumina platform) for the presence of viruses and viroids. By this approach, a single contig of 303 nt sharing high sequence identity with AHVd was identified in one of the sequenced cDNA libraries. RT-PCR with AHVd- specific primers and total RNA preparations from each plant used to prepare the sequenced cDNA library allowed the identification of two apple trees of the local ancient varieties "Mela Rosa Guadagno" (MRG) and "Agostinella" (AG) as the original source plants infected by AHVd, the first report of this viroid in Italy. The natural variability of AHVd viroid populations infecting MRG and AG isolates was studied. With only five changes with respect to the master sequence, AHVd variants from MRG isolate were less variable than those from AG isolate, where polymorphic positions with respect to its master sequence were about fifty. AHVd sequence variants from the two Italian isolates were 97-93 % identical to each other, while they shared lower sequence identity with those from the China and Canada. Interestingly, most nucleotide changes in the Italian AHVd variants did not result in major modifications of the branched secondary structure recently proposed for this viroid by Serra et al. (2018). Interestingly, mutations were found in the two hairpin predicted to form a kissing- loop interaction in the AHVd (+) strand. However, nucleotide rearrangements preserved the potential kissing-loop structure, although stabilized by three instead of four interacting base pairs in the Italian isolates. Altogether, these data showing consistent co-variations preserving both the branched secondary structure and the kissing-loop interaction in AHVd variants of different origins, provide solid support for the major biological role of these structural elements. Acknowledgements. This work has been partially funded by the Apulia Regional Project "Progetto Integrato per la Biodiversita? - ReGeFruP", PSR II 2007-2013.
Molecular characterization of apple hammerhead viroid isolates from ancient Italian apple cultivars
Chiumenti M;Navarro B;Di Serio F;Minafra A
2018
Abstract
High-throughput sequencing (HTS) technologies have contributed to the identification of several viroids and viroid-like RNAs, including a small (434 nt) circular RNA containing the conserved domains of hammerhead ribozymes in apple trees grown in China (Zhang et al., PLoS Pathogens 2014). Although the possible viroid nature of this RNA was anticipated, the final proof was only recently provided by bioassays confirming its autonomous replication in apple (Serra et al., Virus Res. 2018). In the same study, based on in silico modeling and co-variation analysis of viroid populations, the two polarity strands of AHVd were predicted to adopt branched conformations. Interestingly, the (+) polarity strand of AHVd, but not its (-) counterpart, was proposed to be stabilized by a kissing loop- interaction, a situation resembling that of peach latent mosaic viroid and chrysanthemum chlorotic mottle viroid (genus Pelamoviroid, family Avsunviroidae). Besides China, AHVd has been identified only in the cultivar Pacific Gale in Canada (Messmer et al., Can J. Plant Pathol. 2017). Results. In the frame of a project aimed to examine the phytosanitary status of ancient Apulian fruit tree varieties, partially purified dsRNA preparations from several fruit tree species were pooled together and analyzed by HTS (Illumina platform) for the presence of viruses and viroids. By this approach, a single contig of 303 nt sharing high sequence identity with AHVd was identified in one of the sequenced cDNA libraries. RT-PCR with AHVd- specific primers and total RNA preparations from each plant used to prepare the sequenced cDNA library allowed the identification of two apple trees of the local ancient varieties "Mela Rosa Guadagno" (MRG) and "Agostinella" (AG) as the original source plants infected by AHVd, the first report of this viroid in Italy. The natural variability of AHVd viroid populations infecting MRG and AG isolates was studied. With only five changes with respect to the master sequence, AHVd variants from MRG isolate were less variable than those from AG isolate, where polymorphic positions with respect to its master sequence were about fifty. AHVd sequence variants from the two Italian isolates were 97-93 % identical to each other, while they shared lower sequence identity with those from the China and Canada. Interestingly, most nucleotide changes in the Italian AHVd variants did not result in major modifications of the branched secondary structure recently proposed for this viroid by Serra et al. (2018). Interestingly, mutations were found in the two hairpin predicted to form a kissing- loop interaction in the AHVd (+) strand. However, nucleotide rearrangements preserved the potential kissing-loop structure, although stabilized by three instead of four interacting base pairs in the Italian isolates. Altogether, these data showing consistent co-variations preserving both the branched secondary structure and the kissing-loop interaction in AHVd variants of different origins, provide solid support for the major biological role of these structural elements. Acknowledgements. This work has been partially funded by the Apulia Regional Project "Progetto Integrato per la Biodiversita? - ReGeFruP", PSR II 2007-2013.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
