Abstract
Viroid discovery as well as the economic significance of viroids and biological properties are presented. Next-generation sequencing (NGS) technologies combined with informatics have been applied to viroid research and diagnostics for almost a decade. NGS provides highly efficient, rapid, low-cost high-throughput sequencing of viroid genomes and of the 21–24 nt vd-sRNAs generated by the RNA silencing defense of the host. NGS has been utilized in various viroid studies which are presented. The discovery during the last few years that prokaryotes have heritable adaptive immunity mediated through clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated Cas proteins, have led to transformative advances in molecular biology, notably genome engineering and most recently molecular diagnostics. The potential application of the CRISPR-Cas13a system for engineering viroid interference in plants is suggested by targeting specific motifs of three economically important viroids. The CRISPR-Cas13 system has been utilized recently for the accurate detection of human RNA viruses by visual read out in 90 min or less and by paper-based assay. Multitarget RNA tests by this technology have a good potential for application as a rapid and accurate diagnostic assay for known viroids. The CRISPR/Cas system will work only for known viroids in contrast to NGS, but it should be much faster.
Highlights
The cone yields of hop plants infected with hop stunt viroid (HSVd) in Japan were reduced by 50% or more [12] and infected hop vines could be reduced in height by 35% at the end of the seventh growing season [13]
Some economically important viroids such as apple scar skin viroid (ASSVd), coconut cadang-cadang viroid (CCCVd), chrysanthemum stunt viroid (CSVd), HSVd, and potato spindle tuber viroid (PSTVd) are transmitted by seed and pollen
next-generation sequencing (NGS) platforms became available in the market for the first time in 2000, and their use since 2004 has changed the approach to both basic and applied research in many biological disciplines, including plant virology, which deals with viruses and viroids
Summary
Some economically important viroids such as ASSVd, CCCVd, CSVd, HSVd, and PSTVd are transmitted by seed and pollen The frequency of their occurrence in infected hosts and the harm they cause have created additional pathways for the introduction of these and other viroids in new areas as well as for the emergence of novel viroid variants. It has been shown that the utilization of next-generation sequencing (NGS) in viroid research and diagnosis is sensitive, accurate, and fast using different viroid host plant species, including woody perennial crops such as pome and stone fruits and citrus which have low titers of these pathogens. In this article we discuss NGS and CRISPR-Cas systems as related to viroid research and molecular diagnostics
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