称 号 及 び氏 名

博士（応用生命科学）

学位授与の日付

2021 年 3 月 31 日

論

Epidermal cell-patterning genes of a stem parasitic plant

文

名

Sabrina Sultana

Cuscuta campestris, are involved in the holdfast development（茎寄生植物アメリカネナシカズラの表皮細胞パターン形成
遺伝子群は付着器の発生に関与する）
論文審査委員

主査
副査
副査

青木 考
小泉 望
東條 元昭

Cuscuta campestris, a stem parasitic plant, is a holoparasite that does not perform
photosynthesis and takes up water and nutrient from host plant. Upon contacting host stem
surface, C. campestris coils around the stem, and initiates parasitic processes. The parasitic
processes begin with tight adhesion to the stem surface of host plant. C. campestris forms a
specialized disc-like adhesive structure called holdfast which helps parasitic stem to stay incontact with the host stem during haustorium penetration. Development of holdfast is always
associated with the outgrowth of papillar epidermal cells. The morphology of epidermal cells
in the holdfast is very much similar to leaf trichome and root hair of dicotyledonous plants.
However, regulatory network underlying the development of holdfast has not been elucidated
yet. In this study, we assessed the roles of epidermal cell-patterning genes on the developmentof holdfast.
Chapter 1: Holdfast cell outgrowth and the expression of epidermal cell-patterning genes
occurs in the early stage of attachment of parasitization
Holdfast is a pad-like attachment structure that develops on the stem surface,contacting to host. In this study, the time of initiation of the holdfast developments wasdetermined. Development of the pad-like structure of holdfast and a group of cells on thecontacting surface showed outgrowth toward the host surface between 24 and 36 hours afterattachment (haa) in our growth condition. The analysis of transcriptome data of parasitic
interface between Cuscuta japonica and Glycine max confirmed that a group of genes thatshowed high expression levels in the early parasitic stages such as 24 and 48 haa contained
epidermal

cell-patterning

genesof C.

japonica, suchas

homologsof JACKDAW(JKD), GLABRA2 (GL2) and ROOT HAIR DEFECTIVE 3(RHD3). To examine whether theepidermal cell-patterning genes of Cuscuta campestris are involved in the outgrowth ofholdfast epidermal cells or not, the expression profiles of the genes in the parasitic interface at0, 12, 24, 36, and 48 haa was analyzed. CcJKD showed a rapid increase after attachment,reached the maximum level at 12 haa, and then decreased. C. campestris
GLABRA3(GL3), TRANSPARENTTESTAGLABRA1(TTG1), WEREWOLF(WER), and GL2 reached the maximum levels at 24 haa, and then decreased. The expression
profile of C. campestris

CAPRICE (CPC) did not show significant change during

the period. These results demonstrated that most of the epidermal cell-patterning genes showed
high expression transiently before the onset of the outgrowth of holdfast epidermal cells,
except CcCPC.

Chapter 2: Interaction pattern of transcription factors encoded by epidermal cellpatterning genes
GL3, TTG1, WER, CPC and GL2 have been localized to epidermal cells previously
whereas JKD was localized in quiescent center and cortex. To test whether C. campestris
homologs show tissue-specific expression patterns consistent with previous results in or not, insitu hybridization to localize transcripts of CcGL2 and CcJKD was performed. At 36 haa, when
the outgrowth of holdfast epidermal cells was ongoing, unexpectedly CcGL2 and CcJKD weredetected in the epidermal cells of the holdfast. These results suggested that CcJKD and CcGL2
were expressed in the same cells in the epidermis of the holdfast when the holdfast epidermalcells exhibit outgrowth. To test whether CcGL3, CcTTG1, CcWER and CcCPC expressed inparasitic interface are functionally equivalent to those expressed in leaf or root epidermal cellsof other plants, the formation of Myb-bHLH-WD40 complex was assessed. By using yeast twohybrid (Y2H) assay, pair-wise interactions between CcGL3 and CcTTG1, CcGL3 and CcCPC,CcTTG1 and CcCPC, and CcWER and CcCPC were detected. CcWER did not interact withCcTTG1. These results indicate that CcCPC-CcGL3-CcTTG1 complexes can be formed in theparasitic interface. The interaction of CcJKD with CcTTG1 and with CcCPC was also
confirmed by Y2H assay. In this experimental method CcGL2 interacts with CcTTG1 and withCcCPC. Interaction of CcJKD with CcGL2 and CcGL2 with CcWER were assessed by protein
complex immunoprecipitation assay, because CcJKD, CcGL2 and CcWER fused to binding
domain exhibited autoactivation of reporter genes. A proof-of-concept experiment by using
AD-HA-CcTTG1 and BD-Myc-CcCPC showed that the assay can capture interaction between
proteins successfully. Pairs of proteins which are AD-HA-CcGL2 and BD-Myc-CcJKD wereco-expressed in the same yeast cell, and AD-HA-CcGL2 by anti-HA-tag mAB-magnetic
agarose. BD-Myc-CcJKD were absent in the respective CcGL2-captured fraction.
Chapter 3: CcJKD regulates the epidermal cell-patterning genes and holdfast celloutgrowth but does not control establishment of successful parasitization
The unexpected CcJKD mRNA expression in the same epidermis of the holdfast
with CcGL2 raises curiosity about their protein interaction profile, done by Y2H and
immunoprecipitation assay. The formation of the Myb-bHLH-WD40 complex in
the Cuscuta holdfast epidermis like Arabidopsis root epidermis provides possibility of similar
regulatory interaction in root hair trichome formation. On the other hand, the cloned
transcription factors' unusual interacting ability opens the possibility of different additional
functions of those proteins. Previous transcriptome analyses revealed that Cuscuta
2campestris genome lacs homologs of GL1 or TRIPTYCHON (TRY) are involved in thetrichome formation regulatory network. JKD is the upstream regulator of the Arabidopsis
thaliana root epidermis network, and CcJKD showed few conserved findings and new. It wasa logical, sequential approach to find out the role of CcJKD.

To investigate the roleof CcJKD expressed in the epidermal cells, CcJKD was silenced utilizing a host induced genesilencing (HIGS) approach. Both at 24 and 36 haa, on the second host A. thaliana, expression
levels of the target CcJKD were decreased compared to those parasitized on wild type N.
tabacum, indicating the silencing effect on the target gene. Expression levelsof CcGL3, CcTTG1, CcWER, and CcGL2 were also decreased except for CcCPC. Theseresults suggested that CcJKD controls expression of CcGL3, CcTTG1, CcWER, and CcGL2,either directly or indirectly, but not CcCPC. Cell morphology in the epidermal layer of theholdfast on the second host was observed. Cell length were significantly shorter in CcJKDsilenced C. campestris than in the non-silenced C. campestris, whereas cell width was not
significantly different. We further evaluated the effect of CcJKD-silencing on the establishmentof parasitism by estimating the percentage of parasitic connection. Percentage of theestablishment of parasitism by CcJKD-silenced C. campestris was not significantly different
from that by non-silenced C. campestris. This indicates that silencing of CcJKD represses theprocesses leading to outgrowth of holdfast epidermal cells, but the extent of repressive effectwas not enough to inhibit the establishment of parasitism.
From current experimental model, epidermal cell patterning genes are found to beinvolved in the holdfast developments in early stage of parasitization. Moreover, CcJKD acts
as the regulator of this model regulatory network since expression of CcJKD in the same
epidermal cell with other epidermal cell patterning genes in the early parasitic stage upregulates
CcWER, CcTTG1, CcGL3 and also activates downstream gene CcGL2 and results in epidermal
cell protrusion towards host. As CcCPC expression were not affected by CcJKD expression, inthe non-parasitic stage relative abundance of CcCPC protein became higher than the abundanceand CcJKD and CcWER proteins that leads to the increase the abundance of CcCPC-CcGL3CcTTG1 complex, which is a repressor complex of CcGL2 leads to repress outgrowth ofholdfast epidermal cells. This model helps partially explaining the regulatory events of holdfast
development in the early stage of parasitization and in future the ongoing CcGL2 silencing
method will helps to identify the down stream genes of CcGL2 which may involve in theestablishment of successful parasitization.
3

審査結果の要旨
寄生植物は、宿主植物から水や養分を吸収して自身が生長と繁殖をする植物である。宿主範囲が広い寄生植物も多く、農業的に重要な作物に大規模な寄生被害をもたらすこともあり、世界的に大きな問題となっている。寄生植物は、水分や養分を吸収するために宿主植物の維管束組織に自身の維管束組織を接続するが、この組織接続に先駆けて宿主植物表面への付着を行わなければならない。付着する際に、寄生植物は宿主植物に接触した部位の表皮に付着器を形成する。付着器は、宿主に接触している部分の細胞の変形、ならびに粘着物質の分泌、という二つの機能によって特徴づけられる。中でも接触部分での細胞の変形に関しては未知な点が多い。同じ特性は寄生植物のみならず蔓植物やよじ登り植物においても見られ、表皮にパッドやフックを持った付着器を形成するが、いずれの植物においても付着器形成の分子機構は明らかにされていない。そこで本研究では、寄生植物の付着器形成段階における表皮形態形成遺伝子群の解析を実施し、付着器に特徴的な表皮細胞形態がどのようにして形成されるかについて解明を試みた。
本研究では茎寄生植物アメリカネナシカズラ (Cuscuta campestris)と宿主植物シロイヌナズナ（Arabidopsis thaliana）からなる寄生系を用いた。アメリカネナシカズラの付着器では、表皮細胞が宿主植物方向に突出する特徴を持つ。第１章ではまず、アメリカネナシカズラがシロイヌナズナに接触した後、24 時間から 36 時間の間に付着器表皮細胞の突出伸長が開始されることを明らかにした。付着器形成開始の前後の時間帯において、モデル植物でトライコームと根毛における表皮細胞パターン形成への関与が知られている遺伝子群の相同遺伝子、すなわち JACKDAW (CcJKD)、 GLABRA3 (CcGL3)、TRANSPARENTTESTA GLABRA1 (CcTTG1)、WEREWOLF (CcWER)、CAPRICE（CcCPC）、GLABRA2 (CcGL2)の発現パターンを調べてみると、CcJKD は接触後 12 時間で、CcGL3、CcTTG1、CcWER、CcGL2 は接触後 24 時間で発現が上昇していた。CcCPC だけは発現レベルが変動しなかった。さらに in situ ハイブリダイゼーション法により、CcJKD と CcGL2 が付着器表皮細胞で発現していることが明らかにされた。これらのことから、CcCPC を除く表皮細胞パターン形成遺伝子群の付着器形成への関与が示唆された。
第２章では、表皮細胞パターン形成遺伝子群がコードするタンパク質間の相互作用が、酵母ツーハイブリッド法と免疫共沈降法を用いて調べられた。この結果、付着器においてCcGL3-CcTTG1-CcCPC 複合体が形成されること、CcTTG1 と CcCPC は直接相互作用をすること、ならびに CcJKD が CcTTG1、CcCPC と相互作用することが明らかにされ、付着器表皮細胞内に表皮細胞パターン形成の中心となる三つの転写因子タンパク質からなる複合体が存在することが示唆された。
第３章では、付着器表皮形態形成において CcJKD が他の遺伝子の上流に位置すると仮定し、Host-induced gene silencing 法で CcJKD の発現抑制を試み、その寄生への影響を評価した。CcJKD をサイレンシングすると CcGL3、CcTTG1、CcWER、CcGL2 の発現レベルも低下した。
CcCPCの発現には影響が見られなかった。
よってCcJKDはCcGL3、CcTTG1、CcWER、CcGL2 の上流に位置し、直接または間接的に発現を制御していると考えられた。
CcJKD のサイレンシングは、付着器表皮細胞の突出伸長を抑制したため、CcJKD、CcGL3、CcTTG1、CcWER、CcGL2 は付着器表皮細胞伸長を正に制御していると考えられた。しかしながら寄生率そのものは CcJKD のサイレンシングによって低下しなかったため、突出伸長抑制は寄生抑制のために十分ではないことが示された。
本研究では寄生植物の付着器表皮細胞形態が、表皮細胞パターン形成遺伝子群によって制御されることを初めて明らかにした。表皮細胞パターン形成遺伝子群がコードするタンパク質間の相互作用パターンは、モデル植物のトライコームや根毛内における相互作用パターンと共通しているものの、付着器表皮に特異的な相互作用パターンも認められた。本研究における表皮付着分子機構の成果は、植物生理・分子生物学ならびに植物保護学的な観点から高く評価できる。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。