Complete genome sequence of a novel ophiovirus associated with chlorotic disease of pepper (Capsicum annuum L.) in Japan.

In July 2018, pepper plants (Capsicum annuum L.) with chlorotic leaves and fruits were observed in Kochi prefecture, Japan. High-throughput sequencing (HTS) identified the possible presence of an ophiovirus-like virus possessing three RNA segments in a chlorotic leaf. Using Sanger sequencing with primers designed based on the HTS results and a different source of RNA from the one used for HTS, the complete nucleotide sequences of three RNA segments encoding four proteins on their complementary strand were determined. The amino acid sequences of these four proteins showed similarity to those of the RNA-dependent RNA polymerase, RNA-silencing suppressor protein, movement protein, and coat protein, respectively, of ophioviruses, which are negative-sense single-stranded RNA viruses. However, the coat protein amino acid sequence of the virus found on pepper plants was no more than 61.9% identical to those of any known ophioviruses, which is lower than the species demarcation threshold of 85 %. Hence, we suggest that this virus, which we have named "pepper chlorosis associated virus" (PepCaV) should be considered a member of a new species in the genus Ophiovirus, for which we propose the name "Ophiovirus capsici". The results of phylogenetic analysis using coat protein amino acid sequences of PepCaV and other ophioviruses also supported this conclusion. PepCaV RNA was found to have conserved nucleotide sequences at both the 5' and 3' termini of the different RNA segments, and the conserved terminal nucleotide sequences were predicted to form a self-complementary double-stranded region, resulting in a panhandle structure in each of the genomic RNAs.

Perilla Mosaic Virus Is a Highly Divergent Emaravirus Transmitted by Shevtchenkella sp. (Acari: Eriophyidae).

Shiso (Perilla frutescens var. crispa) is widely grown as an important vegetable or herb crop in Japan. Beginning around the year 2000, occurrences of severe mosaic symptoms on shiso were documented and gradually spread across Kochi Prefecture, one of four major shiso production areas in Japan. Next generation sequencing and cloning indicated the presence of a previously unknown virus related to the members of the genus Emaravirus, for which we proposed the name Perilla mosaic virus (PerMV). The genome of PerMV consists of 10 RNA segments, each encoding a single protein in the negative-sense orientation. Of these proteins, P1, P2, P3a, P3b, P4, and P5 show amino acid sequence similarities with those of known emaraviruses, whereas no similarities were found in P6a, P6b, P6c, and P7. Characteristics of the RNA segments as well as phylogenetic analysis of P1 to P4 indicate that PerMV is a distinct and highly divergent emaravirus. Electron microscopy observations and protein analyses corresponded to presence of an emaravirus. Transmission experiments demonstrated that an eriophyid mite, Shevtchenkella sp. (family Eriophyidae), transmits PerMV with a minimum 30-min acquisition access period. Only plants belonging to the genus Perilla tested positive for PerMV, and the plant-virus-vector interactions were evaluated. The nucleotide sequences reported here are available in the DDBJ/ENA/GenBank databases under accession numbers LC496090 to LC496099.

Development and Application of Attenuated Plant Viruses as Biological Control Agents in Japan.

In 1929, it was reported that yellowing symptoms caused by a tobacco mosaic virus (TMV) yellow mosaic isolate were suppressed in tobacco plants that were systemically infected with a TMV light green isolate. Similar to vaccination, the phenomenon of cross-protection involves a whole plant being infected with an attenuated virus and involves the same or a closely related virus species. Therefore, attenuated viruses function as biological control agents. In Japan, many studies have been performed on cross-protection. For example, the tomato mosaic virus (ToMV)-L11A strain is an attenuated isolate developed by researchers and shows high control efficiency against wild-type ToMV in commercial tomato crops. Recently, an attenuated isolate of zucchini yellow mosaic virus (ZYMV)-2002 was developed and registered as a biological pesticide to control cucumber mosaic disease. In addition, attenuated isolates of pepper mild mottle virus (PMMoV), cucumber mosaic virus (CMV), tobacco mild green mosaic virus (TMGMV), melon yellow spot virus (MYSV), and watermelon mosaic virus (WMV) have been developed in Japan. These attenuated viruses, sometimes called plant vaccines, can be used not only as single vaccines but also as multiple vaccines. In this review, we provide an overview of studies on attenuated plant viruses developed in Japan. We also discuss the application of the attenuated strains, including the production of vaccinated seedlings.

Amino acids in Tobamovirus coat protein controlling pepper L(1a) gene-mediated resistance.

In pepper plants (genus Capsicum), the resistance against Tobamovirus spp. is conferred by L gene alleles. The recently identified L variant L(1a) can recognize coat proteins (CPs) of Tobacco mild green mosaic virus Japanese strain (TMGMV-J) and Paprika mild mottle virus Japanese strain (PaMMV-J), but not of Pepper mild mottle virus (PMMoV), as the elicitor to induce resistance at 24 °C. Interestingly, L(1a) gene-mediated resistance against TMGMV-J, but not PaMMV-J, is retained at 30 °C. This observation led us to speculate that L(1a) can discriminate between CPs of TMGMV-J and PaMMV-J. In this study, we aimed to determine the region(s) in CP by which L(1a) distinguishes TMGMV-J from PaMMV-J. By using chimeric CPs consisting of TMGMV-J and PaMMV-J, we found that the chimeric TMGMV-J CP, whose residues in the ß-sheet domain were replaced by those of PaMMV-J, lost its ability to induce L(1a) gene-mediated resistance at 30 °C. In contrast, the chimeric PaMMV-J CP with the ß-sheet domain replaced by TMGMV-J CP was able to induce L(1a) gene-mediated resistance at 30 °C. Furthermore, viral particles were not detected in the leaves inoculated with either chimeric virus. These observations indicated that the amino acids within the ß-sheet domain were involved in both the induction of L(1a) gene-mediated resistance and virion formation. Further analyses using chimeric CPs of TMGMV-J and PMMoV indicated that amino acids within the ß-sheet domain alone were not sufficient for the induction of L(1a) gene-mediated resistance by TMGMV-J CP. These results suggest that multiple regions in Tobamovirus CP are implicated in the induction of L(1a) gene-mediated resistance.