Soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, contains a bipartite positive-stranded RNA genome (RNA1 and RNA2) encapsidated in separate rod-shaped particles. The vector that transmits this virus in soil is a plasmodiophoraceous protozoan, Polymyxa graminis (Shirako and Wilson, 1993). The RNA1 of genomic RNA, which is 7099 nucleotides (nt) in the U.S. strain-Nebraska isolate (SBWMV-NE) and 7226 nt in the Japanese strain- JT isolate (SBWMV-JT), codes two N-terminally overlapping putative replicase proteins in the 5'-terminal region (150 and 209 kDa for SBWMV-NE, and 152 and 211 kDa for SBWMV-JT) and a 37-kDa cell-to-cell movement protein (MP) in the 3'-terminal region. The C-terminal 59-kDa regions of the 209- or 211-kDa proteins are expressed by translational readthrough. In the 3'-terminal region of RNA1, the 37-kDa MP is probably expressed from subgenomic RNA. RNA2 (3593 nt in SBWMV-NE and 3574 nt in SBWMV-JT) codes the 19-kDa capsid protein (CP) in the 5'-terminal region; an 83-kDa protein that is a readthrough product of the 19-kDa protein, which is hypothesized to be required for transmission of the virus by the vector P. graminis (Shirako and Brakke, 1984); and a 19-kDa cysteine-rich protein in the 3'-terminal region. In addition to these proteins, RNA2 codes a 24-kDa protein that has 40 amino acids extension towards the N-terminus of the CP. The initiation codon of the 24-kDa protein was identified as a CUG (Shirako, 1998).

The optimum temperature for the propagation of this virus is 17℃, and systemically infected plants can recover from the virus if the temperature increases (Rao and Brakke, 1970). The inoculation of infectious in vitro transcripts of RNA1 and RNA2 into barley mesophyll protoplasts showed that the CP most abundantly accumulated at 17℃, but was undetectable at 25℃ (Ohsato et al., 2003), suggesting that replication of the virus is temperature sensitive. However, little is known about the involvement of the movement protein in the temperature sensitivity of SBWMV. Considering that cell-to-cell movement of Tobacco mosaic virus (TMV) RNA is temperature-dependent (Boyko et al. 2000) and mutations in the MP gene of TMV have been reported to be involved in temperature sensitivity (Boyko et al., 2000; 2007), thus, it could be possible that both replication and cell-to-cell movement of the virus are implicated in SBWMV temperature sensitivity.

Under laboratory conditions, when the infected plants are subjected to a shift in temperature (17℃ to 22℃ to 25℃), both plants without symptoms (i.e., plants recovered from the viral infection) and plants retaining the symptoms appear (Shirako, 2005). The latter plants are thought to harbor SBWMV variants with altered temperature sensitivity. Mutations were examined that could occur in the MP gene of variants that can propagate under higher temperatures in order to determine the possible involvement of mutations in the MP gene of SBWMV in temperature sensitivity. Two isolates, SBWMV-NE and SBWMV-JT, which are genetically related and belonging to a single species (Miyanishi et al. 2002) have been used in this study.

Plant materials and temperature shifting

Wheat cv. Fukuho (65 plants) and barley cv. Ryoufu (80 plants) were seeded into pots, kept at 17℃ for 10 days until the two-leaf stage, and then used for mechanical inoculation of SBWMV-NE. Temperature was shifted from 17℃ to 25℃ as described by Shirako (2005) with minor modification by keeping the plants at each temperature for one month. Inoculated plants were kept at 17℃ for 1 month, transferred to and kept at 22℃ for 1 month, and finally transferred to and grown at 25℃ for 1 month. The plants that still had disease symptoms at 25℃ were used for virion purification. In the case of SBWMV-JT, 4 barley plants (cultivar Mokusekko provided by Barley and Wild Plant Resource Center, Research Institute for Bioresources, Okayama University, Kurashiki, Japan) were seeded into pots followed by mechanical inoculation and temperature shifting as described for SBWMV-NE.

Western blotting

Ground plant tissue or purified virus in sample buffer was treated at 95℃ for 3 min and used for SDS-PAGE and Western blots. A 10-μl aliquot of sample per lane was loaded onto 12.5% SDS-polyacrylamide gel. After electrophoresis, the proteins were electroblotted onto a nitrocellulose membrane. The CP of SBWMV was detected using anti-SBWMV CP as the primary antibody (raised against SBWMV-NE or SBWMV-JT) and goat anti-rabbit IgG-alkaline phosphatase-conjugated antibody as the secondary antibody and visualized by BCIP (bromo-chloro-indryl phosphate) and NBT (nitro blue tetrazolium).

Virus purification

Leaf tissue (100-300 mg) were ground with a mortar and pestle in 5 ml of 0.5 M sodium borate buffer (pH = 9.0) containing 1 mM EDTA, followed by centrifugation at 4000 rpm for 5 min. One-tenth volume of 20% Triton X-100 was added to the supernatant and centrifuged at 38000 rpm for 2 h. The pellet was resuspended in 250 μl of water to prepare the purified virion solution.

RNA extraction and RT-PCR

Purified virion was used for RNA extraction followed by reverse transcription-polymerase chain reaction (RT-PCR) of the 3'-terminal 1.8-kb region of RNA1. To amplify the sequences, primers TN16 and TN202 were used for SBWMV-NE, and primers TP3 and TP25 were used for SBWMV-JT (Fig. 1), followed by PCR and cloning the PCR products into pGEM-T (Promega) to examine the sequence of the MP gene of the independent variant or were sequenced directly.

Preparation of independent sequence clones

RT-PCR products were cloned into the pGEM-T vector according to the manufacturer's instructions. The ligation products were introduced to strain MC1061 of Escherichia coli to obtain clones of the sequence of the MP gene from the independent mutants.

Plasmid DNA extraction and sequence analysis

Plasmids were extracted and used for direct sequencing using the BigDye Terminator v3.1 Reaction mix (ABI). The primers used for sequencing are shown in Fig. 1. Sequence data were analyzed by the ABI PRISM sequence analysis program and assembled using the ABI Auto Assembler (Perkin Elmer).

RESULTS AND DISCUSSION

Symptom development of SBWMV by temperature shifting

Sixteen out of 65 wheat cv. Fukuho plants and 3 out of 80 barley cv. Ryoufu were systemically infected with SBWMV-NE at 17℃ among which, 3 wheat plants (F22, F30, and F63) and 1 barley plant (R80) kept the symptoms at 25℃ and were selected for viral purification followed by SDS-PAGE and Western blotting. Symptom severity was increased at higher temperatures (such as yellow mosaic of the leaves and severe stunting of the plants at 25℃ comparing to the green mosaic and minimal stunting at 17℃) (data not shown). In the case of the JT isolate, all of the 4 inoculated barely plants that showed disease symptoms at 25℃ were further analyzed.

Determination of the sequence of the 3'-terminal region of RNA 1 containing the MP gene of SBWMV-NE

In the case of SBWMV-NE, purified RT-PCR products from variants, F22, F30, F63 and R80 were sequenced directly. Amino acid substitutions were observed in the variants propagated in wheat F30 and F63 and in barley R80, whereas only a silent mutation was observed in the variants in F22 (Fig. 2). In the variants in F30, nucleotide 5831, which was originally adenine (A), was changed to a mixture of A and guanine (G). These changes contained a mixed population of the variants that have glutamine or arginine as the 60th amino acid (Gln-60 or Arg-60) in the MP (Fig. 2). Also, a mutation of G to A at nt 6483 was observed, which is a silent mutation. In the variants in F63, C at nt 6173 (C6173) was mutated to T, causing amino acid substitution of threonine at position 174 (Thr-174) to methionine (Met) (Fig. 2). In R80, the variants had a mixture of A and G at nt 5759, which leads Gln-36 to become a mixture of Gln and Arg. Also, a mixture of A and T at nt 6172 was observed, indicating the presence of a mixture of variants harboring Ser or Thr at position 174. The amino acid substitutions observed in these variants may play a role in changing the temperature sensitivity of SBWMV-NE, especially the substitution at amino acid position 174, which occurred in variants of two independent plants (F63 and R80). However, considering that the variants in F22 only had a silent mutation in the MP gene, mutations of another gene(s) of SBWMV-NE may also be involved in changing the temperature sensitivity of this virus.

Determination of sequence of the 3'-terminal region of RNA 1 containing the MP gene of SBWMV-JT

For mutations in SBWMV-JT, the sequences of independent clones (10-11 clones for each virus sample) of the RT-PCR products of the MP region of each virus sample (#6-9) were analyzed. Among the various mutations observed in the independent clones, most clones showed a mutation from A to G at nt 6205, which caused a Thr-172 to Ala substitution (Fig. 2). Seven out of 11 clones of #6, 8 out of 10 clones of #7, 9 out of 10 clones of #8, and 7 out of 10 clones of #9 had this mutation. These results strongly suggest the possible role of the amino acid substitution of Thr-172 to Ala-172 of the MP in the change in temperature sensitivity of SBWMV-JT.

In conclusion, it was shown that mutation of Thr174 (in SBWMV-NE) or Thr172 (in SBWMV-JT) occurred probably due to adaptation of virus to the new environment (higher temperature) for cell-to-cell and long-distance movement as well as complete systemic infection, and possible involvement of the movement protein in the temperature sensitivity of SBWMV was revealed.

Fig. 1. RNA 1. Schematic diagram of the position of the MP gene and the primers used for sequencing. The leaky UGA codon of readthrough is indicated by arrowhead.

Fig. 2. Comparison of amino acid mutations among variants of two SBWMV isolates. Closed gray circles: Mutations causing changes in amino acids; Open circles: Silent mutations; Closed black circles: Amino acid 172 that 63-90% of the cloned sequences had the mutation.

ムギ類萎縮ウイルス(Soil-borne wheat mosaic virus: SBWMV)は、Furovirus属の植物ウイルスで、原生動物界に属する土壌菌類であるPolymyxa graminis によって媒介される。本ウイルスは2分節の一本鎖RNA(+)ゲノム(RNA1 and RNA2)を持つ。RNA1は2種のRNA複製酵素とウイルスの細胞間移行に関与するタンパク質(MP)をコードしており、RNA2は19-kDa の外被タンパク質(capsid protein:CP)とその終止コドンのリードスールー産物である83-kDaタンパク質、さらに19-kDaの cysteine-richなタンパク質をコードする。本ウイルスは低温を好み、圃場では温度が20℃以下の春先に全身感染し、モザイク症状や萎縮症状などの病徴を発現するが、その後温度が高くなると、地上部のウイルスは消え、病徴も消失する現象が知られている。しかしながら、実験室条件において、17℃から22℃、次いで25℃へと温度条件を徐々に上昇させた場合に、高温下でも病徴示す個体が出現することが報告されている。これは本ウイルスの変異による高温適応と考えられ、今後予想される気候温暖化を考慮すると農業生産上大きな問題を引き起こす可能性がある。本研究では、地理的に由来の異なるアメリカ-ネブラスカ(NE)株と日本(JT)株の2種類のSBWMVを材料に、ウイルスの移行に関わるMP遺伝子に着目し、高温適応に伴う遺伝子レベルの変異について解析を行った。

1.アメリカ株ムギ類萎縮ウイルス(SBWMV-NE)の解析

コムギ(品種Fukuho)65個体と、オオムギ(品種Ryoufu)80個体にSBWMV-NEを接種し、17 - 22 - 25℃と温度を徐々に変化させて病徴を観察した。その結果、3個体のコムギ(F30,F63)、1個体のオオムギ(R80)において、25℃の高温条件下でも発病し、むしろ激しい病徴を呈するものが認められた。これらの個体に感染しているウイルスのゲノムRNAについて、RT-PCRならびにダイレクトシークエンス法により、MP遺伝子の塩基配列解析を行った。F30より分離されたウイルスには60番目のアミノ酸(AA60)のグルタミンからアルギニンへの変異が、F63ではAA174 のスレオニンからメチオニンへの変異が、R80ではAA36のグルタミンのアルギニンへの変異とAA174 のスレオニンからセリンへの変異が認められた。これらの変異は、ウイルスの移行における温度感受性に関与する可能性を示すものであり、言い換えれば、病徴発現の温度感受性に関係している可能性を示すものである。

2.日本系ムギ類萎縮ウイルス(SBWMV-JT)の解析

オオムギ(品種Mokusekko)を用い、SBWMV-JTを接種した植物個体に対して、SBWMV-NEと同様な温度処理を行った。 その結果、4個体が25℃の条件下で典型的な病徴を示したため、これらの個体より分離したウイルスについてもMP遺伝子部分の塩基配列解析を行った。その結果、大半のウイルスにおいてAA172がスレオニンからアラニンに変異していた。このことは、SBWMV-JTにおいては、AA172のスレオニンからアラニンへの変異が高温条件下におけるウイルスの全身感染に関与している可能性を示すものである。

以上を要するに、地理的な由来の異なるSBWMV-NEと-JTの2つの系統を材料に、実験室条件下において、高温条件下で全身感染する変異ウイルスの出現が認められ、それらのウイルスの多くは移行因子のタンパク質の特定のアミノ酸に変異を生じていた。これらの研究結果により、ムギ類萎縮ウイルスの高温条件下における全身感染に関わる分子レベルの知見を得ることが出来た。今後は、MPのアミノ酸変異に伴う構造変異を明らかにすることにより、SBWMVの高温適応のメカニズム解明が期待されるとともに、高温抵抗性の変異ウイルスの遺伝子診断による検出が可能となる。

本研究は、ムギ類萎縮ウイルスの高温条件下において全身感染し、激しい病徴発現に関与すると予想される移行因子における遺伝子レベルの変異を明らかにした。SBWMVにおいては、その複製過程が温度感受性に関与することはすでに示されているが、移行に関わるタンパク質遺伝子の変異が本ウイルスの全身移行と病徴発現の温度適応性に関与していることを示したのは本研究がはじめてである。今後は、逆遺伝学的検証が求められるが、本ウイルスは下等菌類に媒介され土壌伝染性する実験の非常に困難なウイルスであり、気象温暖化が農業に与える影響が懸念される今日、この成果は、今後の土壌伝染性ウイルスの研究に一石を投じるものである。よって審査委員一同は本論文が博士(農学)に値するものと認めた。