Short 5' Untranslated Region Enables Optimal Translation of Plant Virus Tricistronic RNA via Leaky Scanning.

Regardless of the general model of translation in eukaryotic cells, a number of studies suggested that many mRNAs encode multiple proteins. Leaky scanning, which supplies ribosomes to downstream open reading frames (ORFs) by readthrough of upstream ORFs, has great potential to translate polycistronic mRNAs. However, the mRNA elements controlling leaky scanning and their biological relevance have rarely been elucidated, with exceptions such as the Kozak sequence. Here, we have analyzed the strategy of a plant RNA virus to translate three movement proteins from a single RNA molecule through leaky scanning. The in planta and in vitro results indicate thatthe significantly shorter 5' untranslated region (UTR) of the most upstream ORF promotes leaky scanning, potentially fine-tuning the translation efficiency of the three proteins in a single RNA molecule to optimize viral propagation. Our results suggest that the remarkably short length of the leader sequence, like the Kozak sequence, is a translational regulatory element with a biologically important role, as previous studies have shown biochemically. IMPORTANCEPotexvirus, a group of plant viruses, infect a variety of crops, including cultivated crops. It has been thought that the three transition proteins that are essential for the cell-to-cell transfer of potexviruses are translated from two subgenomic RNAs, sgRNA1 and sgRNA2. However, sgRNA2 has not been clearly detected. In this study, we have shown that sgRNA1, but not sgRNA2, is the major translation template for the three movement proteins. In addition, we determined the transcription start site of sgRNA1 in flexiviruses and found that the efficiency of leaky scanning caused by the short 5' UTR of sgRNA1, a widely conserved feature, regulates the translation of the three movement proteins. When we tested the infection of viruses with mutations introduced into the length of the 5' UTR, we found that the movement efficiency of the virus was affected. Our results provide important additional information on the protein translation strategy of flexiviruses, including Potexvirus, and provide a basis for research on their control as well as the need to reevaluate the short 5' UTR as a translational regulatory element with an important role in vivo.

Transfection of Protoplasts Prepared from Arabidopsis thaliana Leaves for Plant Virus Research.

Plant viruses use numerous host factors for efficient replication of the viral genome. Protoplasts, plant cells from which cell walls are removed, are the useful system to analyze the virus translation and replication in vivo. Here, we report a protocol for preparation of protoplasts from Arabidopsis thaliana leaves and transfection of plasmids to the protoplasts. Protoplasts isolated from the loss-of-function mutant of viral host factor(s) would be helpful to analyze the function of host factors in virus infection cycles.

Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses.

Since the propagation of plant viruses depends on various host susceptibility factors, deficiency in them can prevent viral infection in cultivated and model plants. Recently, we identified the susceptibility factor Essential for poteXvirus Accumulation 1 (EXA1) in Arabidopsis thaliana, and revealed that EXA1-mediated resistance was effective against three potexviruses. Although EXA1 homolog genes are found in tomato and rice, little is known about which viruses depend on EXA1 for their infection capability and whether the function of EXA1 homologs in viral infection is conserved across multiple plant species, including crops. To address these questions, we generated knockdown mutants using virus-induced gene silencing in two Solanaceae species, Nicotiana benthamiana and tomato. In N. benthamiana, silencing of an EXA1 homolog significantly compromised the accumulation of potexviruses and a lolavirus, a close relative of potexviruses, whereas transient expression of EXA1 homologs from tomato and rice complemented viral infection. EXA1 dependency for potexviral infection was also conserved in tomato. These results indicate that EXA1 is necessary for effective accumulation of potexviruses and a lolavirus, and that the function of EXA1 in viral infection is conserved among diverse plant species.

Complete Genome Sequence of a Carrot Torradovirus 1 Isolate, Obtained from Angelica keiskei in Japan.

The complete genome sequence of the first Japanese isolate of carrot torradovirus 1 (CaTV1-J), which infects Angelica keiskei, was determined. This is the first report of a CaTV1 isolate obtained from A. keiskei.

Complete Genome Sequence of Lychnis Mottle Virus Isolated in Japan.

We report here the first complete genome sequence of a Japanese isolate of lychnis mottle virus (LycMoV-J). The genome segments of LycMoV-J have a unique structure in their 3' untranslated regions, and the encoded proteins have the same structure as that of an isolate reported from South Korea.

Complete Genome Sequence of the First Isolate of Hibiscus Latent Singapore Virus Detected in Japan.

The complete genome sequence of the first Japanese isolate of hibiscus latent Singapore virus (HLSV-J) was determined. The genomes of HLSV-J and a reported isolate from Singapore had only 86.7% nucleotide identity, while the encoded proteins shared amino acid identities of more than 95%.

Complete Genome Sequence of a Lily virus X Isolate from Japan.

The complete genome sequence of Lily virus X (LVX), which infects lilies, was determined for the first time from lilies in Japan. As with previous reports, the genome of the Japanese LVX isolate lacked an AUG start codon for the triple gene block protein 3-like region.