Precisely Monomeric Linear RNAs of Viroids Belonging to <i>Pospiviroid</i> and <i>Hostuviroid</i> Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a pre...

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Autores principales:	Tatsuji Hataya, Takashi Naoi
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	viroids cDNA clone transcripts monomeric RNA infectious RNA 5′-terminus Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/cea66b687b824cf49403ad682b3a4046
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spelling	oai:doaj.org-article:cea66b687b824cf49403ad682b3a40462021-11-25T17:09:55ZPrecisely Monomeric Linear RNAs of Viroids Belonging to <i>Pospiviroid</i> and <i>Hostuviroid</i> Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure10.3390/cells101129712073-4409https://doaj.org/article/cea66b687b824cf49403ad682b3a40462021-11-01T00:00:00Zhttps://www.mdpi.com/2073-4409/10/11/2971https://doaj.org/toc/2073-4409Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.Tatsuji HatayaTakashi NaoiMDPI AGarticleviroidscDNA clonetranscriptsmonomeric RNAinfectious RNA5′-terminusBiology (General)QH301-705.5ENCells, Vol 10, Iss 2971, p 2971 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	viroids cDNA clone transcripts monomeric RNA infectious RNA 5′-terminus Biology (General) QH301-705.5
spellingShingle	viroids cDNA clone transcripts monomeric RNA infectious RNA 5′-terminus Biology (General) QH301-705.5 Tatsuji Hataya Takashi Naoi Precisely Monomeric Linear RNAs of Viroids Belonging to <i>Pospiviroid</i> and <i>Hostuviroid</i> Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure
description	Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.
format	article
author	Tatsuji Hataya Takashi Naoi
author_facet	Tatsuji Hataya Takashi Naoi
author_sort	Tatsuji Hataya
title	Precisely Monomeric Linear RNAs of Viroids Belonging to <i>Pospiviroid</i> and <i>Hostuviroid</i> Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure
title_short	Precisely Monomeric Linear RNAs of Viroids Belonging to <i>Pospiviroid</i> and <i>Hostuviroid</i> Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure
title_full	Precisely Monomeric Linear RNAs of Viroids Belonging to <i>Pospiviroid</i> and <i>Hostuviroid</i> Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure
title_fullStr	Precisely Monomeric Linear RNAs of Viroids Belonging to <i>Pospiviroid</i> and <i>Hostuviroid</i> Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure
title_full_unstemmed	Precisely Monomeric Linear RNAs of Viroids Belonging to <i>Pospiviroid</i> and <i>Hostuviroid</i> Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure
title_sort	precisely monomeric linear rnas of viroids belonging to <i>pospiviroid</i> and <i>hostuviroid</i> genera are infectious regardless of transcription initiation site and 5′-terminal structure
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/cea66b687b824cf49403ad682b3a4046
work_keys_str_mv	AT tatsujihataya preciselymonomericlinearrnasofviroidsbelongingtoipospiviroidiandihostuviroidigeneraareinfectiousregardlessoftranscriptioninitiationsiteand5terminalstructure AT takashinaoi preciselymonomericlinearrnasofviroidsbelongingtoipospiviroidiandihostuviroidigeneraareinfectiousregardlessoftranscriptioninitiationsiteand5terminalstructure
_version_	1718412679657291776

Precisely Monomeric Linear RNAs of Viroids Belonging to <i>Pospiviroid</i> and <i>Hostuviroid</i> Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

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