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標題: 以蛋白質交互作用分析在農桿菌感染過程中AtRTNLB1-8與AtRab8B之功能
Protein interaction study of AtRTNLB1-8 and AtRab8B functions during Agrobacterium infection process
作者: 黃凡真
Huang, Fan-Chen
關鍵字: Arabidopsis
出版社: 生命科學系所
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Pseudomonas Type III effector AvrPto suppresses the programmed cell dea
摘要: 農桿菌(Agrobacterium tumefaciens)是一種可跨”界”(kingdom)轉移自身遺傳物質至真核生物中之植物病原菌,其基因體組成除了一線狀及環狀染色體外,尚有pAtC58與pTiC58兩個質體。Ti質體(tumor-inducing plasmid)上含有一段T-DNA (transfer DNA),可藉由第四型分泌系統(type IV secretion system,T4SS)輸出進入植物細胞中。T4SS是由農桿菌的致病蛋白質(virulence protein,Vir)VirB1-11及VirD4所組成,因此又稱為VirB/D4輸出系統。另外,在Ti質體上的致病基因表現區(vir region)中,包含了感染植物過程所需的致病基因,分別參與了植物訊息的辨認、T-DNA的產生、轉移、進入植物細胞及到達植物細胞核的過程,使得T-DNA可成功地嵌入植物染色體中及表現。而在T-DNA由農桿菌轉移至植物細胞的過程中,T4SS扮演了非常重要的角色,而其中的T線毛構造雖有研究指出並非T-DNA轉移的必要條件,但組成T線毛(T-pilus)的VirB2、VirB5與VirB7若缺乏,則造成農桿菌喪失致病力。而在農桿菌感染植物的過程中,植物中的蛋白質可被農桿菌利用,目前已知阿拉伯芥中的AtRTNLB1、2、4及AtRab8B皆可與農桿菌的VirB2相互結合,故本研究利用雙分子螢光互補(bimolecular fluorescence complementation,BiFC)實驗檢測蛋白質間的交互關係,檢測結果顯示AtRTNLB1、2、4與AtRab8B可於原生質體中相互結合,可知此四個蛋白質應可在植物細胞形成一複合體,在植物細胞中共同作用。並且已知在大量表現AtRTNLB1、2或4轉殖株中其被農桿菌感染的效率顯著提升。因此進一步以反轉錄聚合酶連鎖反應(reverse transcription polymerase chain reaction,RT-PCR)檢測病原菌相關基因(pathogen-related genes 1-5,PR1-5)與植物防禦基因(plant defensin 1.2,PDF1.2) 在上述轉殖株中的基因表現量有無改變。在大量表現AtRTNLB1、2或4之轉殖株中,PR1-5及PDF1.2基因的表現量整體有下降的趨勢。另外,為了檢測AtRTNLB家族其他成員是否亦參與農桿菌的感染過程,因此利用酵母菌雙雜合實驗檢測AtRTNLB3、5-8與農桿菌致病蛋白質或AtRab8B的交互作用,結果顯示只有AtRTNLB8可與VirB2或AtRab8B相互結合,因此推測AtRTNLB8較可能參與農菌感染植物的過程。此外針對AtRTNLB8、9、10及13突變株進行農桿菌感染分析實驗,實驗結果顯示上述突變株其短暫表現T-DNA的能力或被野生種農桿菌感染後產生腫瘤的效率,與野生株相較降低的幅度僅限於10-30%,亦或無明顯差異;可知當突變株中只有AtRTNLB8、9、10或13基因功能缺失時,可能不會對農桿菌感染此植株能力造成顯著影響。
Agrobacterium tumefaciens is a plant pathogen that can transfer its own genetic material into eukaryotic cells. The A. tumefaciens genome consists of a linear and a circular chromosomes, and two plasmids, pAtC58 and pTiC58. The tumor-inducing (Ti) plasmid contains a specific DNA region, transfer DNA (T-DNA), which can be export and transfer into plant cells by a type IV secretion system (T4SS). The A. tumefaciens T4SS, also called the VirB/D4 transport system, is composed of VirB1-11 and VirD4 proteins. The Ti plasmid also contains several virulence (vir) genes that are responsible for T-DNA processing, transferring from bacteria into plant cells, nuclear targeting, and finally integration into plant chromosome. During A. tumefaciens infections, the T4SS mediates T-DNA and Vir proteins export. Although the T-pilus has been reported that might not be necessary for T-DNA transfers, the components of T-pilus, VirB2, VirB5, and VirB7, are essential for A. tumefaciens virulence. A previous study showed that Arabidopsis proteins, AtRTNLB1, 2, 4, and AtRab8B, interact with VirB2 in yeast and in vitro. In order to understand further the interactions between AtRTNLB1, 2, 4, and AtRab8B in plant cells, bimolecular fluorescence complementation (BiFC) tests were utilized and demonstrated that AtRTNLB1, 2, 4, and AtRab8B may form a protein complex in plant protoplasts. The over-expression AtRTNLB1, 2 or 4 transgenic plants were hyper-susceptible to Agrobacterium-mediated plant transformation. We therefore performed RT-PCR to determine RNA accumulation levels of the pathogen-related gene 1-5 (PR1-5) and plant defesin gene 1.2 (PDF1.2) in both over-expression transgenic plants and wild-type plants. Most of the PR1-5 and PDF1.2 genes RNA accumulated more in over-expression AtRTNLB1, 2 or 4 transgenic lines than in wild-type plants. Additionally, in order to determine if other members of AtRTNLB participate A. tumefaciens transformation process, we cloned AtRTNLB3, and 5-8 genes from Arabidopsis and performed yeast-two hybrid assays between RTNLB3, 5-8 and several Vir proteins. So far, only AtRTNLB8 interacted with either VirB2 or AtRab8B, suggesting that AtRTNLB8 may be involved in Agrobacterium transformation process. We also examined Agrobacterium infection abilities of the Arabidopsis rtnlb8, rtnlb9, rtnlb10, and rtnlb13 T-DNA insertion mutants The transient and stable transformation efficiencies of some of these mutants showed only slightly 10-30% reductions in comparison to wild-type plants. These results suggest when either the AtRTNLB8-10, or 13 gene expression was affected due to the T-DNA insertion, the Agrobacterium-mediated plant transformation process might no significantly affected in these mutants.
其他識別: U0005-0901201218341600
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