SummaryThe single‐stranded, positive‐sense RNA genome of viruses in the genus Potyvirus encodes a large polyprotein that is cleaved to yield 10 mature proteins. The first three cleavage products are P1, HCpro and P3. An additional short open reading frame (ORF), called pipo, overlaps the P3 region of the polyprotein ORF. Four related potyviruses infecting sweet potato (Ipomoea batatas) are predicted to contain a third ORF, called pispo, which overlaps the 3′ third of the P1 region. Recently, pipo has been shown to be expressed via polymerase slippage at a conserved GA6 sequence. Here, we show that pispo is also expressed via polymerase slippage at a GA6 sequence, with higher slippage efficiency (∼5%) than at the pipo site (∼1%). Transient expression of recombinant P1 or the ‘transframe’ product, P1N‐PISPO, in Nicotiana benthamiana suppressed local RNA silencing (RNAi), but only P1N‐PISPO inhibited short‐distance movement of the silencing signal. These results reveal that polymerase slippage in potyviruses is not limited to pipo expression, but can be co‐opted for the evolution and expression of further novel gene products.

Sweetpotato (Ipomoea batatas) plants become infected with over 30 RNA or DNA viruses in different parts of the world but little is known about viruses infecting sweetpotato crops in Central America, the center of sweetpotato domestication. Small-RNA deep-sequencing (SRDS) analysis was used to detect viruses in sweetpotato in Honduras and Guatemala, which detected Sweet potato feathery mottle virus strain RC and Sweet potato virus C (Potyvirus spp.), Sweet potato chlorotic stunt virus strain WA (SPCSV-WA; Crinivirus sp.), Sweet potato leaf curl Georgia virus (Begomovirus sp.), and Sweet potato pakakuy virus strain B (synonym: Sweet potato badnavirus B). Results were confirmed by polymerase chain reaction and sequencing of the amplicons. Four viruses were detected in a sweetpotato sample from the Galapagos Islands. Serological assays available to two of the five viruses gave results consistent with those obtained by SRDS, and were negative for six additional sweetpotato viruses tested. Plants coinfected with SPCSV-WA and one to two other viruses displayed severe foliar symptoms of epinasty and leaf malformation, purpling, vein banding, or chlorosis. The results suggest that SRDS is suitable for use as a universal, robust, and reliable method for detection of plant viruses, and especially useful for determining virus infections in crops infected with a wide range of unrelated viruses.

Raspberry (Rubus idaeus L.) is a valuable and widely grown softfruit that is a host for 40 viruses and virus-like agents, of which many are not characterized at the molecular level. Recently, Raspberry leaf blotch virus (RLBV, putative emaravirus species) was described from raspberries (cv. Glen Ample) in the United Kingdom and Serbia. Plants displayed conspicuous yellow blotches on leaves and abnormal development of leaf hairs in the corresponding areas of the abaxial side (3). Similar symptoms were observed in ‘Glen Ample’ grown in protective plastic tunnels and open fields in the main berry growing area in eastern Finland in June 2011. In three farms, leaves were sampled from symptomatic and symptomless plants of ‘Glen Ample’ and also cv. Polka displaying no symptoms. Total RNA was extracted using CTAB reagent. Equal amounts of RNA were pooled from 13 samples and subjected to small-RNA (sRNA) deep sequencing (Fasteris SA, Plan-les-Ouates, Switzerland) to detect viruses without advance information (2). Contigs were built on 21- to 24-nt sRNA reads using Velvet. Contigs larger than 50 nt were used to search homologous sequences in GenBank by BLAST and significant similarity (up to 99%) was observed with RLBV RNA3 and RNA4. Mapping sRNA reads to the genome of RLBV (1) by MAQ resulted in significant coverage of RNA1 (16%), RNA2 (37%), RNA3 (46%), RNA4 (65%), and RNA5 (27%). cDNA was synthesized on RNA of one symptom-expressing plant using random hexamer primers and the cDNA tested by PCR with a forward primer (RLBV-F 5′-TCAAATCCACTTGCATAGAACC-3′, nt 723 to 744) and reverse primer (RLBV-R1 5′-CCTCAAACCTTGCAAACACA-3′, nt 1,318 to 1,337) designed according to the nucleocapsid (NP) gene of the Scottish RLBV isolate (3). The sequence of the amplified partial NP gene (576 nt; GenBank Accession No. JQ684678) was 92.8% and 94.8% identical to the Scottish isolate at nt and amino acid levels, respectively. The forward primer RLBV-F and a new reverse primer (RLBV-R2 5′-GCCGAAAGTCAAACCTGGTG-3′, nt 943 to 962) were used to test additional plants for RBLV and to make a probe (198 nt) to detect RLBV using digoxigen-labeled sense and antisense RNA probes, as described for European mountain ash ringspot associated virus (1). RLBV was detected in all tested plants of ‘Glen Ample’ with yellow leaf blotch symptoms in the three farms, but not in any symptomless plants of ‘Glen Ample’ and ‘Polka.’ The sense probes gave strong signals, in contrast to the antisense probes, which gave only weak or no detectable signals in the virus-positive plants, consistent with the negative RNA strand of RLBV being encapsidated in virus particles. The results show RLBV is associated with severe, distinct, and characteristic symptoms in raspberries of cv. Glen Ample grown in plastic tunnels and open fields in Finland and has an apparent negative impact on plant growth and yield. Our observations in 2011 also suggest that the incidence of diseased plants is much higher in plastic tunnels than in open fields, perhaps because the conditions for the vector of RLBV (raspberry leaf and bud mite, Phyllocoptes gracilis Nalepa) (1) are more favorable in plastic tunnels. These results clarify the etiology of raspberry leaf blotch disease in Finland and emphasize the need to inspect raspberry planting materials for RLBV for better control of the disease. References: (1) A. K. Kallinen et al. Phytopathology 99:344, 2009. (2) J. F. Kreuze et al. Virology 388:1, 2009. (3) W. J. McGavin et al. J. Gen. Virol. 93:430, 2012.

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