Plant viruses and viroids in Japan

An increasing number of plant viruses and viroids have been reported from all over the world due largely to metavirogenomics approaches with technological innovation. Herein, the official changes of virus taxonomy, including the establishment of megataxonomy and amendments of the codes of virus classification and nomenclature, recently made by the International Committee on Taxonomy of Viruses were summarized. The continued efforts of the plant virology community of Japan to index all plant viruses and viroids occurring in Japan, which represent 407 viruses, including 303 virus species and 104 unclassified viruses, and 25 viroids, including 20 species and 5 unclassified viroids, as of October 2021, were also introduced. These viruses and viroids are collectively classified into 81 genera within 26 families of 3 kingdoms (Shotokuvirae, Orthornavirae, Pararnavirae) across 2 realms (Monodnaviria and Riboviria). This review also overviewed how Japan’s plant virus/viroid studies have contributed to advance virus/viroid taxonomy

An Improved Method for the Extraction of Nucleic Acids from Plant Tissue without Grinding to Detect Plant Viruses and Viroids

Gene amplification techniques such as polymerase chain reaction (PCR) are widely used for the diagnosis of plant diseases caused by viruses and viroids. It is preferable that sample preparation methods for PCR or reverse transcription (RT) PCR are rapid, straightforward, and inexpensive. We previously reported a method for the extraction of nucleic acids without mechanical tissue grinding using a buffer containing potassium ethyl xanthogenate (PEX) to detect viroid RNAs. In the present report, the previous PEX method was improved and simplified. In the simplified PEX (SPEX) method, the process of PEX buffer treatment for plant cell wall disruption is improved to one step of incubation at 80 °C for 10 min, instead of three steps that took more than 26 min at 65 °C in the previous method. Total nucleic acids could be extracted from fresh, frozen, or dried leaves of a cultivar or wild species of tobacco, tomato, citron, hop plants, and pericarps of persimmon fruits by the SPEX method. Several RNA viruses and viroids were successfully detected from the extracted nucleic acids together with an internal mRNA by RT-PCR. The SPEX method may be useful for detecting not only viruses and viroids, but also other plant pathogens

An Improved Method for the Extraction of Nucleic Acids from Plant Tissue without Grinding to Detect Plant Viruses and Viroids

Gene amplification techniques such as polymerase chain reaction (PCR) are widely used for the diagnosis of plant diseases caused by viruses and viroids. It is preferable that sample preparation methods for PCR or reverse transcription (RT) PCR are rapid, straightforward, and inexpensive. We previously reported a method for the extraction of nucleic acids without mechanical tissue grinding using a buffer containing potassium ethyl xanthogenate (PEX) to detect viroid RNAs. In the present report, the previous PEX method was improved and simplified. In the simplified PEX (SPEX) method, the process of PEX buffer treatment for plant cell wall disruption is improved to one step of incubation at 80 degrees C for 10 min, instead of three steps that took more than 26 min at 65 degrees C in the previous method. Total nucleic acids could be extracted from fresh, frozen, or dried leaves of a cultivar or wild species of tobacco, tomato, citron, hop plants, and pericarps of persimmon fruits by the SPEX method. Several RNA viruses and viroids were successfully detected from the extracted nucleic acids together with an internal mRNA by RT-PCR. The SPEX method may be useful for detecting not only viruses and viroids, but also other plant pathogens

Tolerance Even to Lethal Strain of Potato Spindle Tuber Viroid Found in Wild Tomato Species Can Be Introduced by Crossing

To date, natural resistance or tolerance, which can be introduced into crops by crossing, to potato spindle tuber viroid (PSTVd) has not been reported. Additionally, responses to PSTVd infection in many wild tomato species, including some species that can be crossed with PSTVd-susceptible cultivated tomatoes (Solanum lycopersicum var. lycoperaicum), have not been ascertained. The aim of this study was to evaluate responses to PSTVd infection including resistance and tolerance. Accordingly, we inoculated several cultivated and wild tomato species with intermediate and lethal strains of PSTVd. None of the host plants exhibited sufficient resistance to PSTVd to render systemic infection impossible; however, these plants displayed other responses, including tolerance. Further analysis of PSTVd accumulation revealed low accumulation of PSTVd in two wild species, exhibiting high tolerance, even to the lethal strain. Additionally, F1 hybrids generated by crossing a PSTVd-sensitive wild tomato (Solanum lycopersicum var. cerasiforme) with these wild relatives also exhibited tolerance to the lethal PSTVd strain, which is accompanied by low PSTVd accumulation during early infection. These results indicate that the tolerance toward PSTVd in wild species is a dominant trait and can be utilized for tomato breeding by crossing

Construction and characterization of an infectious cDNA clone of potato virus S developed from selected populations that survived genetic bottlenecks

Abstract Background Infectious cDNA clones are a powerful tool for studies on RNA viruses using reverse genetics. Potato virus S (PVS) is a carlavirus with a worldwide distribution. Although the complete genome sequences of many PVS isolates have been reported, the construction of an infectious cDNA clone of PVS is yet to be reported. The aim of this study is the development and molecular characterization of an infectious cDNA clone of PVS. Methods A full-length cDNA clone pPVS-H-FL-AB was constructed by connecting eight cDNA clones of PVS isolate H95. Capped RNA transcripts from pPVS-H-FL-AB and a modified clone pPVS-H-FL-H, containing the consensus genome sequence of PVS-H95, proved to be non-infectious. Therefore, a full-length cDNA clone pPVS-H-FL-β was reconstructed from PVS-H00, isolated from PVS-H95 populations by repeating a single local lesion isolation in Chenopodium quinoa three times; PVS-H00 appeared to be a selected variant that survived genetic bottlenecks. The sequence of cDNA clone pPVS-H-FL-β was determined as the genome sequence of PVS-H00 and compared with the consensus sequence of PVS-H95 genome. Results All Nicotiana occidentalis plants inoculated with ≥0.2 μg capped RNA transcripts from pPVS-H-FL-β developed symptoms on upper leaves, as observed with PVS-H00 inoculation. Similar levels of viral genomic and subgenomic RNAs and coat protein were detected in systemically infected leaves. Sequence comparison of PVS-H95 and PVS-H00 revealed 370 nucleotide polymorphisms (4.4% of the entire genome sequence), causing 91 amino acid substitutions in six open reading frames (ORFs). The infectivity of chimeric RNAs derived from recombinants between the two cDNA clones revealed that the lack of infectivity of pPVS-H-FL-H transcripts was due to ORF1, which encodes replicase and harbors 80 amino acid substitutions compared with pPVS-H-FL-β. Approximately 71.3% amino acid substitutions in replicase were located within the variable region of unknown function between the putative methyltransferase and ovarian tumor-like protease domains. Conclusions This is the first report of the development of an infectious cDNA clone of PVS. Our analyses suggest that PVS population within a plant exists as quasispecies and the replicase sequence diversity of PVS obstruct the construction of a full-length infectious cDNA clone

Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid 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 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 & PRIME;-triphosphate and 3 & PRIME;-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 & PRIME;-monophosphate, 5 & PRIME;-hydroxyl, or 5 & PRIME;-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5 & PRIME;-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5 & PRIME;-monophosphate RNA inoculation, followed by the plants with 5 & PRIME;-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