Teruo Sano

Hop Stunt Viroid Strains from Dapple Fruit Disease of Plum and Peach in Japan

Viroids have been isolated from plum trees (Prunus salicina Lindley) affected with plum dapple fruit disease and from peach trees (Prunus persica Batsch) showing dapple fruit symptoms. The viroids were inoculated mechanically to cucurbitaceous plants, in which symptoms typical of hop stunt viroid (HSV) infection appeared. The complete nucleotide sequences of an isolate from plum and an isolate from peach (AF isolate) were shown to be identical, consisting of 297 nucleotides with a 93.6% sequence homology to HSV-hop. Another isolate from peach (A9 isolate) also consists of 297 nucleotides, but the sequence homology to HSV-hop is 99.7%, showing only one nucleotide replacement. These results indicate that these three viroids are strains of HSV, which we designate HSV-plum, HSV-peach (AF) and HSV-peach (A9), respectively. Comparative analysis of the nucleotide sequences of HSV strains from hop, grapevine, citrus, cucumber, plum and peach revealed variable and conserved regions in the HSV molecule. In Japan, these viroids are closely related not only to dapple fruit disease in plum cv. Taiyo, but also to dapple fruit symptoms on peach cv. Asama-Hakutou.

A revised replication cycle for viroids: the role of longer than unit length RNA in viroid replication

SummaryLonger than unit length plus and minus strand RNAs were detected in hop stunt viroid (HSV) infected cucumber leaf tissues by Northern blot hybridization analysis using strand-specific probes. To elucidate the role of these longer than unit length RNAs in the viroid replication cycle, we synthesized tandemly repeated plus and minus strand HSV RNAs in vitro from cloned HSV cDNA and assayed their infectivities. Two and four unit tandemly repeated plus strand RNAs were infectious, but one unit plus, and one, two and four unit minus strands were noninfectious. Taking these data into consideration, we propose a revised rolling circle model for viroid replication

A Viroid-like RNA Isolated from Grapevine Has High Sequence Homology with Hop Stunt Viroid

Summary Viroid-like RNA was isolated from some samples of grapevine leaves (Vitis vinifera L.). When inoculated to cucumber, this RNA induced symptoms that were identical to those induced by hop stunt viroid (HSV). It had a molecular size similar to that of HSV, and hybridized with a 32P-labelled DNA probe for the cucumber isolate of HSV (HSV-c, pCP-55). This hybridization suggests high (> 55%) sequence homology with HSV. The HSV-related sequence was detected not only in the infected cucumber plants but also directly in RNA extracted from grapevine plants. The result indicates that viroid-like RNA, closely related to HSV, is present in grapevines in Japan.

The sequence necessary for the infectivity of hop stunt viroid cDNA clones

SummaryRecombinant plasmids carrying more than two units of the hop stunt viroid (HSV) cDNA sequence linked in tandem were highly infectious. On the other hand, those carrying one unit were not. To determine the length of the HSV cDNA sequence necessary for the infectivity, we constructed series of plasmids carrying more than one but less than two units of the HSV sequence and investigated their infectivities. The results showed that cDNA clones were infectious if about 60 nucleotides of the internal part of the HSV sequence (region A) were undeleted and remained with the same arrangement as in the infectious two-unit clone. The region A can be folded into a stable secondary structure. Viroid replication is thought to involve a site-specific endonucleolytic cleavage of plus strand RNA multimers produced by a rolling circle mechanism to yield unit length molecules. Our results suggest that the region A contains the sequence or structure recognized by the putative endonuclease.

Polymerase chain-reaction-mediated cloning and expression of the coat protein gene of potato virus Y inEscherichia coli

Complementary DNAs (cDNAs) to the RNA genome of a necrotic strain of potato virus Y in Japan (Hokkaido Univ. isolate of PVY-T: PVY-TH) were synthesized and cloned into a plasmid pBR322. About 4.3 kbp of the cDNA sequence containing the 3′-poly(A) tract of PVY-TH was inserted into a recombinant plasmid pBRYT88. The coat protein coding region (CP gene) in pBRYT88 was amplified using the polymerase chain reaction (PCR) and subcloned into a plasmid pUC119. The nucleotide sequence of the CP gene was determined from both the PCR-mediated clones and pBRYT88. The CP gene of PVY-TH consisted of 801 nucleotides, corresponding to 267 amino acids of Mr 29,811. The predicted amino acid sequence of the PVY-TH CP gene was different from that of PVYN (1) in only five amino acids and displayed 98.1% sequence homology. This result indicates that PVY-TH is closely related to PVYN (1). The cDNAs of the PVY-TH CP gene containing an additional initiation codon (ATG) at the 5′ end and a stop codon at the 3′ end were constructed by PCR amplication and subcloned into anE. coli expression vector, pKK223-3. Five transformedE. coli colonies expressing the PVY-TH CP were identified by immunoscreening using both polyclonal rabbit antiserum against PVY-TH and mouse monoclonal antibody (MoAb) specific to PVY-T. The CP of PVY-TH produced in theE. coli colonies had an electrophoretic mobility identical to that of native PVY-TH CP and reacted strongly to a specific MoAb to PVY-T, but did not react to a specific MoAb to an ordinary strain of PVY (PVY-O). The maximum expression of the CP inE. coli was apapproximately 7% of the total soluble proteins. The result indicates that the CP gene cloned by PCR was functional and the PCR procedure was useful for producting biologically active cDNA clones from a single, long positive-sense RNA genome encoding a single, large polyprotein precursor, such as potyviruses.