称号及び氏名

博士（応用生命科学）

学位授与の日付

２０２１年５月３１日

論Trans-species long-distance movement of small RNAs in the文名Bera Subhankar

parasitic complexes（寄生複合体における低分子 RNA の種間長距離移行）
論文審査委員

主査

青 木

考副査

横井修司

副査

高野順平

論文要旨
Stem holoparasitic plants Cuscuta attack domesticated and non-domesticated plants andcause serious damage to commercial crops. Cuscuta withdraws water and nutrients from hosts. In
addition to water and nutrients, vascular connections allow Cuscuta to exchange larger molecules
including various types of RNAs with the host. The exchange of mRNAs has been reported
previously between Cuscuta and host plants. The movement of the endogenous mRNAs via thephloem within a single plant regulates developmental processes. The trans-species movement ofmRNAs strongly suggests that RNA-based control is involved in the establishment of the parasiticcomplex. In addition to the mRNAs, the exchange of small RNAs (sRNAs) between pathogenicorganisms and host plants has previously been found. The pathogenic fungus control host plant’s
immunity by exporting sRNAs. Conversely, host plants export microRNAs (miRNAs) to control
the fungal hyphae, by silencing the genes expression essential for fungal virulence. In the Cuscuta
campestris-Arabidopsis thaliana parasitic complex, C. campestris miRNAs were induced in thehaustorial interface and repress the expression of Arabidopsis genes involved in the defense
response and vascular development. These studies suggest that sRNA-based mechanisms areinvolved in the establishment of mutual regulation at the interfaces between host plants and otherorganisms. However, it has not yet been elucidated whether sRNAs derived from donor plants inparasitic complexes were translocated to distant tissues in the recipient plants or if they showedeffects after the establishment of a stable parasitic connection. In this study, we analyzed theaccumulation of sRNAs of two host-parasitic plant complexes and tried to find out the common
phenomenon involved in sRNA accumulation. To obtain the transcriptome of sRNA, RNAs wereextracted from the parasitic stems of C. campestris-A. thaliana parasitic complex, and Cuscuta
japonica-Glycine max parasitic complex. RNAs were also extracted from the non-parasitic stems.
sRNA-seq reads obtained from a single sequencing library were mapped to the genome databasesof both the parasite and host to identify mobile sRNAs. The analyses of sRNA accumulation oftwo parasitic complexes along with trans-species sRNA movement and biogenesis were describedin four chapters.

Chapter 1: Analyses of small RNAs in Cuscuta campestris-Arabidopsis thaliana parasitic
complex
The analyses of sRNA-seq data of different tissues of C. campestris-A. thaliana parasitic
complex reveals the accumulation of sRNAs in parasitic conditions. In A. thaliana, sRNAaccumulation was increased in 103 genes and decreased in 119 genes. Gene ontology (GO)analyses of the genes onto which sRNA accumulation were increased showed that majority of thegenes were involved in ‘glucosinolate biosynthesis’, ‘response to stress’ and ‘symplast’. ThesRNA reads associated with genes, where reads per million (RPM) values increased more than
2-fold in parasitic condition, revealed that 22-nt reads were enriched. On the contrary, sRNA reads
associated with genes that decreased more than 2-fold in parasitized stems of A. thaliana wereenriched with 21-nt reads. In C. campestris, sRNA accumulation was increased in 458 genes anddecreased in 445 genes in the parasitic condition. GO terms of genes onto which sRNA
accumulation were increased were enriched with ‘response to cadmium ion’ and ‘response to metalion’. In the parasite, no significant enrichment was observed for any length of sRNA reads in C.
campestris. Percentage of 21-nt reads significantly decreased that associated with genes that
increased more than 2-fold in C. campestris.

Chapter 2: Analyses of small RNAs in Cuscuta japonica-Glycine max parasitic complex
In C. japonica-G. max parasitic complex, the genes which were responsible for thegeneration of sRNAs in parasitic condition were analyzed. In G. max, 666 genes of which sRNAaccumulation was increased where 365 genes of which sRNA accumulation was decreased inparasitic condition. GO terms of genes onto which sRNA accumulation were increased suggest theinvolvement of two biological processes: hormone-mediated response such as ‘auxin binding’,‘regulation of gibberellic acid-mediated signaling pathway’, ‘cellular response to hormone
stimulus’ and hormone-mediated signaling pathway and associated with cellular connection suchas ‘phloem transport’, ‘plasmodesma’, and ‘cell-cell junction’. In C. japonica, sRNA
accumulation was increased in 197 genes, whereas decreased in 367 genes, in the parasiticcondition. GO analyses reveal that genes associated with lignin accumulation such as ‘lignin

biosynthetic process’, ‘lignin metabolic process regulated’ was controlled by sRNA accumulationin parasitic conditions. We mention that the whole loci analyses of the C. japonica genome havenot to be done due to the unavailability of the full genome of C. japonica. In parasites (C.
campestris and C. japonica), genes associated with ‘response to cadmium ion’ and ‘response tometal ion’ were commonly involved in sRNA accumulation in parasitic condition. In hosts, (A.
thaliana and G. max), genes associated with ‘stress response’ were commonly involved in sRNA
accumulation.

Chapter 3: Trans-species long-distance movement of small RNAs in the parasitic complexes
The screening of trans-species mobile sRNAs resulted in the selection of 20 sRNAs of C.
campestris, 10 sRNAs of A. thaliana in C. campestris-A. thaliana parasitic complex, whereas 5
sRNAs of C. japonica and 20 sRNAs of G. max in C. japonica-G. max parasitic complex. Fromthe predicted mobile sRNAs, we prioritized 7 of C. campestris sRNA (CcsRNA) and 6 of A.
thaliana sRNA (AtsRNA) to confirm the long-distance movement. We confirmed 4 of the CcsRNA(CcsRNA0641, CcsRNA0897, CcsRNA4295 and CcsRNA5627) and 2 of the AtsRNAs(AtsRNA3031 and AtsRNA4348) were mobile to the apical region of the recipient plant.
CcsRNA0641 was also detected in the distant oegans of A. thaliana such as leaf, stem and root inparasitic condition. The GO analysis of predicted target genes of the mobile sRNAs did not show
any enrichment of specific GO terms. The decrease in the accumulation of predicted target genes(AtNPC5 and AtMo25 which were targets of CcsRNA4295, and AtSKD1 which was a target ofCcsRNA5672) strengthened the hypothesis of trans-species sRNA-mediated repression in theparasitic condition.

Chapter 4: Biogenesis of sRNA
To clarify the regulation of sRNA biogenesis in the parasitic complex, we measured theexpression levels of sRNA biogenesis genes. Expression levels of A. thaliana SUPPRESSOR OF
GENE SILENCING 3 (AtSGS3) and RNA-DEPENDENT RNA POLYMERASE 6 (AtRDR6), whichplay crucial roles in the tasiRNA production pathway, were significantly upregulated in theparasitized stems. The enrichment of 21-nt and 22-nt sRNAs of A. thaliana may correspond to theupregulation of AtSGS3 and AtRDR6, which support the possibility of secondary siRNAproduction in the parasitic condition. In C. campestris, DICER-LIKE 1 (CcDCL1), CcDCL3, andCcRDR6 were significantly downregulated. The decrease in the proportion of 21-nt reads in theparasitic stems of C. campestris may be associated with the downregulation of CcDCL1 andCcRDR6. Secondary siRNAs production triggered by trans-species mobile sRNAs was notdetected in the parasitic condition. By using A. thaliana sgs3 and rdr6 mutants, trans-species
mobile sRNAs from C. campestris moved long distances in the host plant, suggesting that
long-distance movement of trans-species mobile sRNAs occurs without siRNA production.

In summary, the results of my study indicate that donor-derived sRNAs move long
distances in recipient plants. In hosts, secondary siRNA producing pathway not only plays acrucial role in the accumulation of secondary sRNA but also control the mobility ofparasite-derived sRNA. Comprehensive profiling of the trans-species sRNAs and their target
genes will provide deeper insights into the RNA-mediated interactions between parasitic and hostplants.

審査結果の要旨
低分子 RNA（small RNA、以下 sRNA）は、塩基配列特異的な mRNA の分解、翻訳抑制、ならびに転写抑制などを引き起こすことで、生体機能の調節に様々な役割を果たしている。
sRNA は細胞から細胞へ移行することが知られており、移行による遺伝子機能の調節が植物と微生物の間で見られている。ある種の植物病原菌と宿主植物の間では、お互いに隣接する相手の細胞へ sRNA を送り込み、植物側の防御反応や病原菌側の病原性を弱めている。
近年、sRNA の相互交換は多細胞性の異種植物個体間でも起こることが、寄生植物と宿主植物の研究から明らかになった。寄生植物は、寄生時に吸器とよばれる特殊な器官で sRNAを発現し、これらが吸器周辺の宿主植物細胞へ移行する。そして移行した sRNA は、宿主内で二次的な sRNA の生産を促し宿主植物の防御反応を弱める、という分子機構の存在が解明されている。一方で、寄生植物は吸器が形成される寄生部位から遠く離れた宿主植物の器官においても、特定の遺伝子群の発現レベルを低下させる。この現象に対しては、sRNAが器官間長距離移行することの関与が示唆されていた。しかしながら寄生植物と宿主植物という異種個体間で sRNA が移行するか、また移行した植物内で sRNA が長距離移行して機能する事が可能かどうかは未解明であった。
そこで申請者は本研究において、二つの異なる寄生植物‐宿主植物複合体を用いて、異種植物間で移行する sRNA の存在ならびに長距離移行性を検証し、それらの sRNA が寄生時にいかにして生成され、移行先でどのような機能を果たすのかを明確にするための解析を実施した。
第１章では、寄生植物 Cuscuta campestris と宿主植物 Arabidopsis thaliana を用いて、smallRNA sequencing（sRNA-seq）解析での結果に基づいて、それぞれの植物内での sRNA プロフィールが明らかにされた。A. thaliana において sRNA が増加している座位では、22 塩基長の sRNA が主要な生成物となっていた。
第２章では、宿主植物 Cuscuta japonica と寄生植物 Glycine max を用いて、sRNA プロフィールが明らかにされた。C. japonica において sRNA が増加している座位では、22 塩基長の sRNA が主要な生成物となっていた。第１章と第２章での結果を比較し、寄生によってsRNA 量増加がおこる座位のうち、二つの寄生系で共通しているのは、寄生植物側、宿主植物側で共にストレス応答や細胞間連絡形成に関与する遺伝子群であった。
第３章では、C. campestris‐A. thaliana 寄生系を用いて異種間移行する sRNA の選抜と、移行性の実証が行われた。sRNA-seq のデータ解析により候補化された sRNA の異種間移行性を、sRNA 配列特異的ステムループ RT-PCR 法で実験的に検証し、C. campestris から A.
thaliana に移行する 4 つの sRNA、ならびに A. thaliana から C. campestris に移行する 2 つのsRNA が同定された。さらにこれらの sRNA の標的遺伝子を予測し qRT-PCR による発現量の測定を行ったところ、A. thaliana の 3 つの標的遺伝子が被寄生状態において抑制されていることが確認され、移行性 sRNA による抑制効果が示唆された。
第４章では、C. campestris‐A. thaliana 寄生系における sRNA 生合成機構の研究がなされた。sRNA 生合成系遺伝子発現を解析したところ、A. thaliana では SGS3 と RDR6 の発現が被寄生時に上昇していた。このことは A. thaliana での 22 塩基長 sRNA の増加と整合し、二次的 siRNA 生合成の活性化が示唆された。そこで異種間移行性 sRNA が移行先で二次的siRNA 生合成を誘導するかどうかを sRNA-seq データから解析したところ、この可能性は否定された。さらに、A. thaliana の sgs3 ならびに rdr6 変異体を宿主として、C. campestris由来の異種間移行性 sRNA が移行先植物内で長距離移行するために二次的 siRNA 生合成が必要かどうかを検証した。両変異体内で C. campestris 由来の sRNA は茎頂で検出され、移行先植物内での長距離移行に二次的 siRNA 生合成は必要ではないことが示された。
申請者は本研究で、寄生植物と宿主植物の複合体における異種個体間で sRNA の双方向移行があること、移行性 sRNA は移行先で長距離移行し、標的遺伝子の発現を抑制する事が可能であることを示した。ならびに移行先植物内での長距離移行に二次的 siRNA 生合成は必要がなく、異種間移行した sRNA が直接長距離移行することが示された。本研究は個体間での新規な RNA の機能様式を解明した独創性の高いもので、細胞分子生物学や植物生理学的な成果として高く評価できる。ならびに RNA を活用した植物保護や新育種技術への展開も期待される。よって本論文の審査ならびに最終試験の結果と併せて博士（応用生命科学）の学位を授与することを適当と認める。