In vitro transcripts of a full-length cDNA of a naturally deleted RNA2 of barley mild mosaic virus (BaMMV) replicate in BaMMV-infected plants

Abstract

The RNA2 of a German isolate of the bipartite barley mild mosaic bymovirus (BaMMV-ASL 1) is 3524 nucleotides long excluding the 3'-terminal poly(A) tail. The isolate was propagated by mechanical inoculation for several years. Electrophoretic comparison of viral nucleic acids during this period revealed that a spontaneous reduction in the length of the RNA2 occurred, resulting in the isolate BaMMV-ASLla. The deleted RNA2 of BaMMV lacked a fragment that was 630 nucleotides long. The deletion occurred in the 3' half of the single open reading flame (ORF) found in RNA2, this ORF encodes a polyprotein that has a molecular mass of 98 kDa, which is assumed to be processed autocatalytically into proteins of 25 kDa and 73 kDa. A full-length eDNA of the deleted RNA2 was synthesized and cloned under the control of the phage T7 promoter. In vitro transcripts of the BaMMV-ASLIa clone replicated in barley plants after co-inoculation with a wildtype-like isolate of BaMMV. The deletions in RNA2 of BaMMV-ASLla and those of a number of other isolates that were examined were found to affect a domain of the putative 73 kDa protein that is obviously not essential for replication but may be important for the transmission of BaMMV by its natural vector Polymyxa graminis.

Polymyxa graminis and the cereal viruses it transmits: a research challenge

“…Revers et al ., 1999) and it is likely that bymoviruses will prove to be similar but there are no experimental data on the role of the different functional proteins in, for example, virus replication, cell‐to‐cell movement or long distance transport. Following repeated mechanical inoculation, it is common to find shorter forms of BaMMV RNA2 as a result of deletions of 400–1200 nucleotides in the P2 cistron (Jacobi et al ., 1995; K. Kanyuka, unpublished; Timpe and Kühne, 1995) and similar events appear to occur in OMV (Zheng et al ., 2002) and in BaYMV (Kühne et al ., 2003). One of the deletions in BaMMV results in a loss of vector transmission (Adams et al ., 1988).…”

Bymovirus reverse genetics: requirements for RNA2‐encoded proteins in systemic infection

“…Infectious cDNA clones for RNA1 and RNA2 of Barley mild mosaic virus (BaMMV), another species in the genus Bymovirus, using the Cauliflower mosaic virus 35S promoter for the transcription of the infectious RNA in host nuclei have been described, but the systemic infectivity to barley plants was lower than 10% (Meyer and Dessens, 1997). Infectious BaMMV RNA2 transcripts from a full-length cDNA clone have also been described (Timpe and Kühne, 1995), but not for RNA1. Unfortunately, these cDNA clones have not been used successfully to study the molecular biological aspects of interactions between the virus, host plants and transmission vector (Habekuss et al, 2008;Jacobi et al, 1995;Kanyuka et al, 2004;Kashiwazaki and Hibino, 1996;Peerenboom et al, 1997).…”

The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication‐associated inclusion bodies

“…). Oat mosaic virus and Barley mild mosaic virus (both are members of the genus Furovirus ) isolates, which have deletions in the C‐terminal region of P2, are still able to replicate in their hosts (Timpe and Kuhne, ; Zheng et al ., ). In a study using infectious cDNA clones, a BaYMV mutant with the entire P2 coding region deleted replicated as efficiently as the wild‐type virus in barley protoplasts; however, when inoculated into barley plants, this mutant showed reduced efficiency in systemic infection, induced milder symptoms and accumulated to a much lower level in upper systemic leaves (You and Shirako, ).…”