Barley yellow mosaic virus (BaYMV), the type species of the genus Bymovirus in the family Potyviridae, is one of the major viral pathogens of barley in Europe and Easy Asia. It is transmitted by Polymyxa graminis in soil and causes a yellow mosaic disease of winter barley with significant yield losses. The virus has a bipartite positive-sense RNA genome. RNA1 (7.6 kb) encodes a large polyprotein which is processed into eight products (P3/6K1/CI/6K2/VPg/NIa-Pro/NIb/CP). RNA2 (3.6 kb) also encodes a polyprotein which is cleaved into two products (Pl/P2). Breeding resistant cultivars using rym resistant genes from barley landlaces is the only practical way to avoid crop losses due to BaYMV infection in fields. However, resistance-breaking BaYMV variants are continuing to emerge. To study molecular biological aspects of interactions among the virus, host plants and the transmission vector, establishment of reverse genetics of BaYMV is a prerequisite.

In this study, full-length cDNA clones of RNA1 and RNA2 of a Japanese K05 isolate of BaYMV (BaYMV-JK05) were constructed. In vitro transcripts were systemically infectious. Using the in vitro transcripts system, functions of P1, P2 and VPg in systemic infection and resistance and susceptibility of barley cultivars were studied.

1. Establishment of reverse genetics of BaYMV

Based on the genome information previously studied during my Master' s course, full-length cDNA inserts for RNA1 and RNA2 were individually cloned into a pBR322-derived plasmid and propagated in Escherichia coli MC1061. Multiple cDNA clones for RNA1 and RNA2 were examined by infectivity assay in barley mesophyll protoplasts (cvs. Minori and Ryofu) . Using the highest expression level of CP in transfected protoplasts as a criterion, one each clone for RNA1 and RNA2 were selected and designated as pBY1 and pBY2, respectively.

RNA1, which encodes eight proteins including a viral proteinase NIa-Pro, the RNA-dependent RNA polymerase NIb, a genome-linked protein VPg and the capsid protein CP, replicated autonomously in protoplasts optimally at 15 ° C, a temperature similar to the optimum for causing disease in barley fields. P1 was detected from protoplasts transfected with RNA1 and RNA2 transcripts, but not from those transfected with RNA2 transcripts alone, indicating that RNA2 replication depends on RNA1. The accumulation level of CP increased several folds in the presence of RNA2, indicating that RNA2 or RNA2-encoded proteins enhance RNA1 replication and gene expression in host cells.

Barley plants (cv. Ryofu) were mechanically inoculated with RNA1 and RNA2 transcripts and grown at 15 ° C. Three weeks after inoculation, upper uninoculated leaves of 53. 5% inoculated plants started showing yellow mosaic symptoms. From these leaves, CP, VPg, VPg-containing polyproteins and P1 were detected by Western blot analysis. The progeny viral RNA1 and RNA2 had identical nucleotide sequences as in cDNA inserts of pBY1 and pBY2, respectively. Thus in vitro transcripts of RNA1 and RNA2 were proven to be systemically infectious in barley. None of the plants inoculated with RNA1 transcripts alone were infected, indicating that RNA2 is essential for systemic infection.

2. Analysis of RNA2-encoded proteins in systemic infection

Four pBY2-derivative mutants were constructed, in which the P1-coding region was deleted (pBY2. AP1), the P2-coding region was deleted (pBY2. AP2), a termination codon was inserted after the P1-coding region (pBY2. P1Tp,,) and the -1 amino acid at the Pl/P2 boundary (Gly/Ser) was mutated to abolish cleavage (pBY2.P12V). The expected protein products from these mutant constructs were confirmed by cell-free translation of in vitro transcripts.

Protoplasts transfected with RNA1 and WT RNA2 or AP2 RNA2 transcripts gave similar strong bands of CP and P1 on Western blots. However, from protoplasts transfected with RNA1 together with ΔP1 RNA2, P1T, RNA2, or P12V RNA2 transcripts, CP was only weakly detectable, similar to the situation when protoplasts were transfected with RNA1 transcripts alone. P12 protein was readily detected in the case of P12V RNA2. P1 was not detected in the case of P1TAA RNA2, probably due to the additional P2-coding sequence in the 3' UTR having a deleterious effect in replication. These results indicated that expression of P1 from RNA2 leads to efficient RNA1 replication and the gene expression in host cells.

Barley plants (cv. Ryofu) were inoculated with RNA1 and AP2 RNA2 transcripts. Five out of 29 plants showed small chlorotic spots on upper uninoculated leaves at 5 weeks after inoculation, and CP and P1 were weakly detectable from these leaves, indicating that the plants were systemically infected. Sequencing analysis confirmed that the original AP2 mutation was retained in progeny virus genome. On the other hand, plants inoculated with RNA1 transcripts alone or together with AP1 RNA2 or P12V RNA2 transcripts were not infected. One out of 28 plants inoculated with RNA1 and P1TAA RNA2 transcripts was systemically infected, in which the inserted UAA termination codon was mutated to a UCA serine codon, regenerating a Gly/Ser cleavage site. Therefore, both P1 and P2 are required for efficient systemic infection: P1 is essential whereas P2 may facilitate virus movement and WT symptom development.

3. Analysis of VPg132 RNA1 mutants in virus replication and systemic infection

In the case of European isolates of BaYMV, a substitution of Lys at the position 132 of VPg with Asn or His was suspected to be responsible for breaking rym4―resistance. The Japanese BaYMV JK05 isolate has a His at the same position of VPg. To investigate the importance of the VPg132 amino acid in BaYMV replication and systemic infection, four VPg132 mutant RNA1 constructs were prepared with His codon replaced with Ala, Lys, Asn or Tyr codons. Protoplasts prepared from susceptible cvs. Hadakal and New Golden and a rym4―posessing Franka were transfected with WT or VPg132 mutant RNA1 together with WT RNA2 transcripts. From Hadakal and New Golden protoplasts, CP and P1 were detected strongly with WT His RNA1, lesser with Tyr RNA1, much lesser with Asn RNA1, and almost undetectable with Lys or Ala RNA1. From Franka (rym4) protoplasts, CP and P1 could not be detected in all cases. Thus, using the Japanese BaYMV JK05 isolate, none of His, Asn, Lys, Ala or Tyr at the position 132 of VPg could break the rym4 resistance.

When Hadaka1 and New Golden plants were inoculated with the Tyr RNA1 transcripts with WT RNA2 transcripts, one Hadaka1 plant and two New Golden plants developed the wild-type symptoms in five weeks. Sequencing analysis of the progeny viral genome showed that the mutated Tyr codon was retained or reverted to the WT His codon partially or completely in the progeny virus population. Hadaka1 and New Golden plants inoculated with Asn RNA1 and RNA2 transcripts were not infected. These results indicated that VPg is one of the essential viral proteins in replication and systemic infection and that the amino acid at the position 132 is important for the function of VPg.

4. Analysis of resistance and susceptibility of barley cultivars using transcripts

The infectious in vitro transcripts system enables analysis of resistance and susceptibility of barley cultivars against BaYMV infection at the molecular level. Barley cultivars with ryml, 2, 3, 4 ,5 or 6 genes as well as those without known rym genes were subjected to infectivity assay at the cellular level and at the whole plant level using the in vitro transcripts. The result showed that cultivars with ryml, 2, 4, 5 or 6 were immune to BaYMV JK05 at the cellular level. Two cultivars with rym3, Haganemugi and Ishukushirazu, were susceptible at the cellular level but not at the whole plant level, indicating that these cultivars are resistant at either cell-to-cell or long-distance movement levels. Haruna Nijo without known resistance genes was shown to be immune at the cellular level, implying that Haruna Nijo has an unidentified resistance gene. Kashimamugi without known resistance genes was susceptible at the cellular level but did not develop recognizable symptoms. However, CP was detected from symptomless leaves as well as from roots, indicating that Kashimamugi is systemically infected with JK05 BaYMV. Thus, the in vitro transcripts system was proven to enable precise analysis of the virus-host compatibility and finding resistant cultivars at the cellular level.

This is the first report of establishment of a reverse genetics system for a virus in the genus Bymovirus and the first studies on biology of bymoviruses and host resistance and susceptibility at the molecular level. The obtained infectious cDNA clones of BaYMV open new avenues for the studies on bymovirus-plant host-transmission vector interactions, leading to development of efficient control measures for bymovirus diseases.

日本、韓国および中国などの東アジア地域と欧州各国で栽培されるオオムギには、土壌伝染性オオムギ縞萎縮ウイルス(Barley yellow mosaic virus)による縞萎縮病が多発し、大きな被害を及ぼしている。被害地域は年々拡大する傾向にあり、栽培品種の抵抗性を打破する変異株の発生も既に各地で報告されている。オオムギ縞萎縮ウイルスは、圃場での感染発病が13 °C~16 °Cと低温で、実験的な接種試験が困難であり、これまでウイルス病原側からの分子生物学的な研究は滞っていた。本ウイルスによる病害を防除するために、ウイルスの病原性、伝搬性および宿主抵抗性に関する詳細な研究が必要である。そこで本論文では、オオムギ縞萎縮ウイルスが属ずるバイモウイルス属で初めて、2分節ゲノムに対する感染性全長cDNAクローンpBY1およびpBY2を構築し、RNA1とRNA2にコードされた合計10種類のタンパク質のうち、全身感染性に関与する3種のタンパク質についてその機能を解析し、さらにオオムギ品種の罹病性と抵抗性を細胞レベルと個体レベルで解析した。

第一章では、オオムギ縞萎縮ウイルスに関する既知のゲノム情報、生物学的性状、品種抵抗性、ウイルス逆遺伝学の重要性と研究の経緯および本研究の意義が述べられている。

第二章では、2005年春、岡山県倉敷市内で採集したオオムギ縞萎縮ウイルスJK05分離株の遺伝子構造解析を行い、JK05株の遺伝型が日本、韓国および中国で発生しているアジア型のオオムギ縞萎縮ウイルスと同じクラスターに含まれるものの、これまで日本国内で分離されたオオムギ縞萎縮ウイルスとは異なる系統である事実を示した。

第三章では、JK05株のRNA1とRNA2に対する全長cDNAをT7プロモーターの直下流に配置し、pBR322由来のプラスミドにクローニングし、全長cDNAクローンpBY1とpBY2を構築した。ウイルス感染の検出には、RNA1にコードされた外被タンパク質CPおよびゲノム結合タンパク質VPg、RNA2にコードされたP1タンパク質をそれぞれGST融合タンパク質として大腸菌内で発現し、ウサギに免疫して抗血清を作製した。pBY1およびpBY2由来in vitro転写産物をオオムギ葉肉プロトプラストに接種したところ、RNA1のみでCPとVPgが検出され、RNA1が自律複製することが明らかになった。in vitro転写産物をオオムギ幼苗に接種したところ、RNA1単独では感染せず、RNA1とRNA2を同時に接種した場合にのみ全身感染性を示した。全身感染個体からウイルス粒子を精製し、抽出RNAからRT-PCR増幅して直接塩基配列を決定した結果、複製ウイルスRNAの塩基配列は鋳型cDNAの塩基配列と完全に一致することが確認された。以上の結果より、オオムギ縞萎縮ウイルスの感染性全長cDNAクローンの構築が成功し、本ウイルスの逆遺伝学が確立した。

第四章では、RNA2にコードされるP1およびP2タンパク質の全身感染性における機能について検討した。P1のみ、P2のみ、およびP12融合タンパク質のみをそれぞれ発現する変異型RNA2 cDNAクローンを構築し、先ず無細胞タンパク合成系を用いて目的タンパク質の翻訳を確認した後、オオムギ葉肉プロトプラストとオオムギ幼苗に対して接種試験を行った。その結果、P1は全身感染性に必須であり、P2は細胞間移行と全身感染性に機能することが明らかとなった。

第五章では、欧州産オオムギ縞萎縮ウイルス分離株の研究から、本ウイルス病原性の重要な決定因子と推察されるVPgタンパク質の132番目のアミノ酸に注目し、その部位のアミノ酸のウイルス複製と全身感染性における機能を検討した。欧州産オオムギ縞萎縮ウイルス分離株はVPg132にLysを持ち、そのrym4抵抗性打破変異株ではAsnかHisを持つ。一方、本研究で用いたJK05株は同じ部位にHisを持つ。そこでVPg132のアミノ酸をLys、Tyr、AsnおよびAlaに置換した変異型RNA1 cDNAクローンを作出し、rym4抵抗性を持つオオムギ品種由来の葉肉プロトプラストと幼苗に対して接種試験を行った。その結果、どの変異体もrym4抵抗性を細胞レベルで打破することができず、病原性にはVPg132以外のアミノ酸も関与することが明らかとなった。

第六章では、オオムギ縞萎縮ウイルスに対する6種の抵抗性遺伝子rym1~6を一つずつ持つオオムギ品種を用い、野生型in vitro転写RNA1およびRNA2を用いて、葉肉プロトプラストと幼苗に接種試験を行った。その結果、JK05株RNAはrym3遺伝子を持つ品種では細胞レベルで複製したが個体レベルでは感染しなかった。rym1、2、4、5および6を持つ品種は細胞レベルでも抵抗性を示し、宿主抵抗性が細胞レベルか移行レベルかを識別可能となった。

以上、本研究ではオオムギの重要病原ウイルスであるオオムギ縞萎縮ウイルスの逆遺伝学を世界で初めて確立し、ウイルスの複製、病原性および宿主抵抗性を遺伝子レベルで解析することを可能としたものであり、学術上、応用上、今後の研究に貢献するところは極めて大きい。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。