Sabitree, Shahi

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Okayama University Scientific Achievement Repository

氏    名 SHAHI SABITREE 授与した学位 博 士 専攻分野の名称 農 学 学位授与番号 博甲第 ６４１９ 号 学位授与の日付 ２０２１年 ３月２５日 学位授与の要件 環境生命科学研究科 農生命科学専攻  （学位規則第４条第１項該当） 学位論文の題目 Experimental introduction of mycoviruses into phytopathogenic filamentous fungi and their biologic characterization （菌類ウイルスの植物病原糸状菌への導入と生物学的性状解析） 論文審査委員 教授 鈴木 信弘 教授 ガリス イバン 准教授 近藤 秀樹 学位論文内容の要旨 Fungal viruses have been the subjects of great interest from phytopathological and virological viewpoints. Fungal viruses, having the capabilities of reducing the virulence of their host phytopathogenic fungi, could be exploited as biological control agents. Furthermore, studies on mycoviruses have greatly advanced our knowledge about virus diversity and evolution. However, it is generally difficult to experimentally introduce mycoviruses into fungi, which impede the exploration of virus/host interactions. In this study, a few methods for the experimental introduction of fungal viruses into filamentous fungi were examined toward their biological characterization.  Among tested viruses are two major viruses of filamentous ascomycetes Cryphonectria spp., Cryphonectria parasitica mitovirus 1 (CpMV1), and Cryphonectria nitschkei chrysovirus 1 (CnCV1). The capsidless mitovirus CpMV1 of the chestnut blight fungus Cryphonectria parasitica was the first mitochondrially-replicating virus that encodes only an RNA-dependent RNA polymerase. Here, we developed a protoplast fusion-based protocol for horizontal transmission of CpMV1. This method allowed an expansion of the host range to many other C. parasitica strains. Species within and outside the family Cryphonectriacea like C. radicalis and Valsa ceratosperma, respectively, also supported the replication of CpMV1 at a level comparable to that in the natural host. However, no stable maintenance of CpMV1 was observed in Helminthosporium victoriae. Polymerase chain reaction-based haplotyping of virus-infected fungal strains confirmed the recipient mitochondrial genetic background. Phenotypic comparison between CpMV1-free and -infected isogenic strains revealed no overt effects of the virus on colony morphology in the newly established host fungi. Nevertheless, virulence assay with the standard strain of C. parasitica EP155 showed the hypovirulence induction by CpMV1 on living chestnut trees. Accumulation levels in antiviral RNA silencing-proficient and -deficient strains of EP155 are comparable, suggesting the avoidance of antiviral RNA silencing by CpMV1, consistent with its mitochondrial replication.  Cryphonectria nitschkei chrysovirus 1 (CnCV1) was described earlier from an ascomycete fungus Cryphonectria nitschkei strain OB5/11, collected in Japan. The partial sequence for CnCV1 was reported a decade ago. Complete sequencing of the four genomic double-stranded RNA segments revealed molecular features similar to but distinct from previously reported members of the family Chrysoviridae. The unique features of this virus include the presence of a mini-cistron preceding the major large open reading frame in each genomic segment. The presence of CAA repeats in the 5’-untranslated regions and conserved terminal sequences are among the common features that CnCV1 shared with its previously described counterparts. Virion transfection, protoplast fusion, and hyphal fusion using ZnCl2 (inhibitor of vegetative incompatibility reactions) were compared for CnCV1 transfer. CnCV1 could laterally be transferred to C. nitschkei and to its relatives like C. radicalis, C. carpinicola, and C. naterciae via coculturing, virion transfection, and protoplast fusion, but neither to any other fungal species nor to any members within the genus Cryphonectria. This is suggestive of a very narrow host range for this virus. Phenotypic comparison of a few sets of CnCV1-infected and -free isogenic strains showed symptomless infection in new hosts. While all three methods could transfer the virus horizontally to other fungal strains or species, protoplast fusion technique turned out to be the most efficient method among all. This is the first study to establish a virion transfection protocol for a chrysovirus and shows an asymptomatic infection of Cryphonectria spp. by CnCV1. Collectively, this study establishes protoplast fusion as a versatile and universal method for experimentally introducing diverse types of fungal viruses into different filamentous fungi including phytopathogens. This research also provides a solid foundation for further exploration of virus/fungus interactions.  論文審査結果の要旨 菌類ウイルス（マイコウイルス）は植物病理学的およびウイルス学的に注目されている。菌類ウイルスが植物病原糸状菌の病原力を衰退させるのであれば，潜在的ヴァイロコントロール因子となり得る。また，マイコウイルスの研究は，ウイルスの多様性・進化の理解に大きく貢献している。しかし，菌類ウイルスを接種することは一般的に難しく，それらを生物学に解析するには困難をともなう。 本研究では，複製様式。ゲノム型が大きく異なる多様なウイルスを用いて，主に植物病原糸状菌に実験的に導入し，生物学的解析を試みた。して菌類での混合感染は，特に高頻度に観察される。クリ胴枯病菌のミトコンドリアで複製するキャプシドレスウイルス，Cryphonectria parasitica mitovirus 1 (CpMV1)に対しては，プロトプラスト融合法による接種法を確立した。そして，CpMV1がクリ胴枯病菌(Cryphonectria parasitica)の他系統，別種のC. radicalis, Valsa ceratospermaなどでも複製可能であることを証明した。これらの新規宿主が受容菌のミトコンドリア遺伝子型を持つことも確認した。興味深いことに，ウイルス感染によって培地上でのコロニー形態に大きな影響を受けないが，クリ樹を用いた病原性試験によりクリ胴枯病菌の病原力が衰退することを証明した。一方，細胞質基質で複製する球形２本鎖RNAウイルス，Cryphonectria nitschkei chrysovirus 1 (CnCV1)の導入には，粒子トランスフェクション，ZnCl2を用いた菌糸融合，プロトプラスト融合が利用可能であるが，プロトプラスト融合が最も効率的であることが判明した。CnCV1は宿主範囲が狭く，C. radicalis, C. carpinicola, C. naterciae 等一部のCryphonectria属の種でのみ複製し，無病徴感染した。以上のように，菌糸融合はウイルスの複製部位，粒子形成能の有無，ゲノム型に拘らず，利用可能であることが明らかとなり，今後のマイコウイルス・宿主相互作用研究に貢献すると考えられる。 SHAHI 女史は，博士号に価する上記のように十分な研究成果を挙げ，またその過程で十分な研鑽を積んだことを学位論文審査員として認める。 

菌類ウイルスの植物病原糸状菌への導入と生物学的性状解析

https://ousar.lib.okayama-u.ac.jp/files/public/6/62169/20210614135656676089/K0006419_abstract_review.pdf

菌類ウイルスの植物病原糸状菌への導入と生物学的性状解析

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