Analysis of Nucleotide Sequences and Multimeric Forms of a Novel Satellite RNA Associated with Beet Black Scorch Virus

Abstract

The full-length sequence of a satellite RNA (sat-RNA) of Beet black scorch virus isolate X (BBSV-X) was determined. This agent is 615 nucleotides long and lacks extensive sequence homology with its helper virus or with other reported viruses. Purified virus particles contained abundant single-stranded plus-sense monomers and smaller amounts of dimers. Single-stranded RNAs from total plant RNA extracts also included primarily monomers and smaller amounts of dimers that could be revealed by hybridization, and preparations of purified double-stranded RNAs also contained monomers and dimers. Coinoculation of in vitro transcripts of sat-RNA to Chenopodium amaranticolor with BBSV RNAs was used to assess the replication and accumulation of various forms of sat-RNA, including monomers, dimers, and tetramers. Dimeric sat-RNAs with 5-or 10-base deletions or 15-base insertions within the junction regions accumulated preferentially. In contrast, the replication of monomeric sat-RNA was severely inhibited by five-nucleotide deletions in either the 5 or the 3 termini. Therefore, sequences at both the 5 and the 3 ends of the monomers or the presence of intact juxtaposed multimers is essential for the replication of sat-RNA and for the predomination of monomeric progeny. Comparisons of the time courses of replication initiated by in vitro-synthesized monomeric or multimeric sat-RNAs raised the possibility that the dimeric form has an intermediate role in replication. We propose that replication primarily involves multimers, possibly as dimeric forms. These forms may revert to monomers by a termination of replication at 5 end sequences and/or by internal initiation at the 3 ends of multimeric junctions.Beet black scorch virus (BBSV) was first reported in the late 1980s (13,14). The virus is responsible for serious damage to the sugar beet crop in the Xinjiang, Ningxia, Inner Mongolia, Gansu, and Heilongjiang provinces of China. BBSV infects sugar beet plants systemically through the roots after transmission with Olpidium brassicae zoospores (25) and causes black scorch symptoms on the leaves (6). Mechanical inoculation in a greenhouse resulted in local infections of 15 plant species, including several Chenopodium spp., producing inapparent to extremely mild symptoms or necrotic local lesions (7). Sugar beets on different plantations contaminated by BBSV isolates in China developed similar severe black scorch symptoms on leaves, and most plants died within a week after the appearance of symptoms. Normally, only a single RNA was associated with infections, but a small single-stranded RNA (ssRNA) that is similar to the satellite RNAs (sat-RNA) of other necroviruses (39) has been found in isolates from the Xinjiang province (6). This small RNA multiplied in test plants only when associated with BBSV genomic RNA, either from the same viral isolate or from other sources of BBSV (5). The presence of sat-RNA resulted in more local lesions on the leaves of Chenopodium amaranticolor plants than did inoculation with BBSV RNA alone when the...

Genetic diversity and population structure of beet necrotic yellow vein virus in China

“…Then total RNA was extracted as described previously (Guo et al, 2005). The primers used for cDNA synthesis and amplification of the CP gene, full-length RNA3, RNA4 and RNA5 are listed in Supplementary Table S2.…”

Phylogenetic analysis of Beet necrotic yellow vein virus isolates from China

“…Viruses were purified from 2 g root tissues and viral RNAs were extracted [21,22]. BNYVV infection surveyed in all the six regions was confirmed by RT-PCR with specific primers designed to the CP gene (CP-F, ATG TCG AGT GAA GGT AGA TA and CP-R, CTA TTG TCC GGG TGG ACT GG, designed according to D84411).…”

Identification and phylogenetic analysis of orf virus from goats in Taiwan

“…Total RNA were extracted from 500 mg liquid nitrogen powdered leaf tissue, following the method of Guo et al [8], using 500 ll of extraction buffer (20 mM Tris-HCl [pH 8.0], 1% sodium dodecyl sulfate, 200 mM NaC1, 5 mM EDTA) and phenol-chloroform (1:1). The resulting RNA was dissolved in diethyl pyrocarbonate-treated distilled water.…”