农杆菌T4SS的结构及其底物转运机理的最新进展

细菌Ⅳ型分泌系统(type Ⅳ secretion system,T4SS)不仅是农杆菌转运T-DNA的通道,也是多种质粒接合的通道,还能转运多种底物分子到不同的靶细胞(包括细菌和真核细胞),因此,T4SS的结构及其底物转运机理的研究受到了人们的极大关注,近几年来,这方面的研究进展迅速。农杆菌转运T-DNA的T4SS又叫Vir B/Vir D4系统,是革兰氏阴性菌中最典型的T4SS之一,有关细菌Ⅳ型分泌系统的研究均以农杆菌的Vir B/Vir D4系统为参考模型。现将综述这几年来在农杆菌T4SS结构研究方面取得的最新进展,并介绍目前对T4SS底物转运机理的一些最新认识。     

冠瘿瘤基因在植物体中的表达

1　实验目的利用质粒转移技术将有用 (目的 )基因整合到植物基因组 DNA中 ,创造出植物遗传新类型。本实验用根癌农杆菌介导 ,将菌体中的冠瘿瘤基因 (T- DNA)导入桑、紫景天中 ,使其形成植物瘤。通过本实验操作 ,可使学生了解植物转基因技术的基本原理 ,提高对生物课程学习的兴趣具有重要意义。2　实验原理植物经人工刺伤并感染农杆菌后 ,因伤流中含有一定量的酚类、氨基酸、糖等有机物 ,这类物质能诱导农杆菌合成参与 T- DNA剪切、加工、转移的酶系 ,并将 T-DNA整合到宿主细胞的基因组 DNA中。由于 T- DNA中有编码生长素 (tmsl、tm…     

T-DNA转移研究进展

植物遗传转化技术近年在农作物性状改良、植物生物反应器利用以及基因功能鉴定等方面得到了广泛的应用.T-DNA转移是植物细胞农杆菌介导遗传转化整合和表达外源基因的基础.农杆菌Ti质粒vir基因编码蛋白、农杆菌一些染色体基因编码蛋白及植物细胞一些基因编码蛋白或因子均参与T-DNA转移.转移过程包括农杆菌对植物细胞的识别、附着,细菌对植物信号物质的感受,细菌vir基因的诱导表达,T复合体的形成,跨膜运输,进核运输和整合等一序列过程.植物细胞因子与农杆菌T-DNA转移相关蛋白的相互作用最近被认为在T-DNA转移过程中起重要作用.     

影响农杆菌介导遗传转化的植物因子研究进展

农杆菌介导法是植物遗传转化中最常用的一种方法.越来越多的研究表明,植物遗传因子是决定农杆菌遗传转化效率的重要因素,它们至少影响了转化过程的如下5个方面:1)受伤植物释放的酚类物质和糖分子等介导农杆菌的趋化运动和毒性基因vir的诱导表达;2)农杆菌吸附到植物表面;3)T-DNA和毒性蛋白通过由VirB和virD4蛋白组成的Ⅳ型分泌系统从细菌转移到植物细胞质;4)T-复合体利用细胞质ACTIN骨架和输入蛋白进行核定位和核输入;5)T-DNA利用植物的修复装置整合进宿主基因组.就以上5个方面涉及的植物因子研究进展予以综述.     

Ti质粒基因在单子叶植物中的表达

<正> 根癌农杆菌感染双子叶植物时,能侵入双子叶植物的细胞壁,并通过一种未知机制将其Ti质粒DNA导入植物细胞内。导入的Ti质粒DNA(T-DNA)能整合到植物细胞的核基因组中,并被转录。通过遗传操作插入Ti质粒T区的任何DNA片段似乎都能随根癌农杆菌一起转移到植物细胞内。因此,根癌农杆菌作为载体,已广泛用于高等植物外源遗传物质的导入研究。它最终有可能给作物的基因组加入一些有益的遗传成分。遗憾的是,把Ti质粒用作转     

植物DNA断裂修复基因对农杆菌T-DNA整合的作用

转基因植物在作物新品种培育和生物制药中已发挥了巨大作用。农杆菌介导的遗传转化是广泛用于基因组分析的强大工具,也是获得转基因植物的主导技术。农杆菌介导的基因转移是极其复杂的生物学过程,需要许多农杆菌和植物的遗传因子协同参与完成。经过20多年的研究,人们对T-DNA产生和转运的分子机制以及农杆菌与寄主植物的互作已有所了解。T-DNA整合是农杆菌介导转化过程中最为关键的一步,但对于其整合机制所知仍有限。越来越多的证据表明,寄主植物细胞的DNA断裂修复基因对农杆菌T-DNA整合具有重要作用。该文首先介绍T-DNA转移的大致过程,重点讨论DNA断裂损伤修复相关基因对T-DNA整合的作用,为通过DNA损伤修复基因的遗传操纵来提高农杆菌介导植物遗传转化的效率提供参考。     

AcvB基因的发现及表达

被根癌农杆菌感染的植物 ,其部分细胞发生了变异 ,最终形成植物癌。究其原因是由于农杆菌中的 T-DNA转移至植物细胞所致。T- DNA剪切、加工、转移受Ti- DNA中的多个操纵子的共同调控。最近 ,小岛研究室用转座子标记法对根癌农杆菌 (A- 2 0 8株 ) T- DNA转移机能进行了研究 ,发现了 3个位于根癌农杆菌染色体上的 Acv B、chv A、ch VB基因 ,它们在 T- DNA转移过程中担负重要角色。1　 Acv B基因的发现用大肠肝菌 (PJB4 J1)与农杆菌 (A- 2 0 8)在 2 8℃条件下共培养 ,在含卡那霉素 (Kmr)固体培养基上选取单菌落 ,取其菌体分别接…     

我国葡萄根癌农杆菌Ti质粒的特征

以窄宿主葡萄农杆菌Ag162Ti质粒的T-DNA区tmr、tmsl和ocs基因座位以及T_A-DNA和T_B-DNA片段为探针,对12株我国分离的不同生物型、质粒类型和寄主范围的葡萄根癌农杆菌的引质粒转移DNA(T-DNA)进行Southern杂交分析。在9株生物3型octoplne Ti质粒菌株中,与上述探针均同源。其中窄宿主葡萄根癌农杆菌菌株杂交片段彼此较一致。广宿主葡萄根癌农杆菌菌株的杂交片段彼此差异较大。1株无致瘤能力的生物1型菌株与5个探针均不杂交。1株生物3型nopaline Ti质粒菌株及1株诱导冠瘿瘤中只合成精氨酸的菌株,杂交带的变化也大。由此可见葡萄农杆菌在生物进化过程中其转移DNA呈多态性,成为农杆菌中特殊类群。本分析对葡萄根癌农杆菌致病菌株的鉴定亦有帮助。     

农杆菌介导的单子叶植物遗传转化研究进展

根癌农杆菌(Agrobacterium tumefaciens)Ti质粒转化系统的建立使植物遗传工程进入了一个飞速发展的时期。近年来,发根农杆菌(A.rhizogenes)Ri质粒毛根转化系统的研究十分迅速,展示了美好的前景,农杆菌介导的植物遗传转化已成为目前研究和应用最广泛的系统。但是,农杆菌的宿主范围一般仅限于双子叶植物和一些裸子植物,这就直接防碍着这种比较完善的基因转移技术在单子叶植物,尤其是禾谷类作物转化的应用。本文介绍了农杆菌介导的单子叶植物遗传转化的进展;对扩大农杆菌宿主范围、实现对单子叶植物转化的途径进行了探讨。 (一)农杆菌介导的单子叶植物转化的方法 目前建立的单子叶植物基因转移系统有:(1)农杆菌载体系统;(2)外源DNA     

农杆菌接种法作为一种简便的植物病毒载体的侵染方法

烟草花叶病毒(TMV)表达载体30B是一个目前广泛应用的植物病毒表达载体,但用其生产外源蛋白时,必须先将它体外转录成RNA,才能被用来接种宿主植物。由于RNA体外转录费用昂贵、操作复杂,因此限制了30B表达载体的进一步应用。针对这一不足,我们用农杆菌接种法(agroinoculation)接种该病毒载体,即将30B cDNA置于花椰菜花叶病毒(CaMV)的35S启动子和终止子之间,再将整个表达框架插入到农杆菌T-DNA的左边界和右边界之内,构建成质粒p35S-30B,将转入该质粒的农杆菌注射到植物的叶片中,30B cDNA随T-DNA进入植物细胞后,被转录成可自我复制的RNA形式,进而发生系统侵染。为了检测此接种方式的可行性,绿色荧光蛋白(GFP)报告基因被克隆到p35S-30B中,构建成p35s-30B：：GFP,用含有该质粒的农杆菌进行注射操作。证实该病毒载体可通过简便的农杆菌接种法侵染Nicotiana benthamiana,在被接种植物的系统叶中,GFP的表达量可占植物总可溶蛋白的5．2％。     

Several components of SKP1/Cullin/F-box E3 ubiquitin ligase complex and associated factors play a role in Agrobacterium-mediated plant transformation

? Successful genetic transformation of plants by Agrobacterium tumefaciens requires the import of bacterial T-DNA and virulence proteins into the plant cell that eventually form a complex (T-complex). The essential components of the T-complex include the single stranded T-DNA, bacterial virulence proteins (VirD2, VirE2, VirE3 and VirF) and associated host proteins that facilitate the transfer and integration of T-DNA. The removal of the proteins from the T-complex is likely achieved by targeted proteolysis mediated by VirF and the plant ubiquitin proteasome complex. ? We evaluated the involvement of the host SKP1/culin/F-box (SCF)-E3 ligase complex and its role in plant transformation. Gene silencing, mutant screening and gene expression studies suggested that the Arabidopsis homologs of yeast SKP1 (suppressor of kinetochore protein 1) protein, ASK1 and ASK2, are required for Agrobacterium-mediated plant transformation. ? We identified the role for SGT1b (suppressor of the G2 allele of SKP1), an accessory protein that associates with SCF-complex, in plant transformation. We also report the differential expression of many genes that encode F-box motif containing SKP1-interacting proteins (SKIP) upon Agrobacterium infection. ? We speculate that these SKIP genes could encode the plant specific F-box proteins that target the T-complex associated proteins for polyubiquitination and subsequent degradation by the 26S proteasome.     

VirE2: a unique ssDNA-compacting molecular machine

The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.     

Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant

VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.     

Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions

Agrobacterium tumefaciens translocates T-DNA through a polar VirB/D4 type IV secretion (T4S) system. VirC1, a factor required for efficient T-DNA transfer, bears a deviant Walker A and other sequence motifs characteristic of ParA and MinD ATPases. Here, we show that VirC1 promotes conjugative T-DNA transfer by stimulating generation of multiple copies per cell of the T-DNA substrate (T-complex) through pairwise interactions with the processing factors VirD2 relaxase, VirC2, and VirD1. VirC1 also associates with the polar membrane and recruits T-complexes to cell poles, the site of VirB/D4 T4S machine assembly. VirC1 Walker A mutations abrogate T-complex generation and polar recruitment, whereas the native protein recruits T-complexes to cell poles independently of other polar processing factors (VirC2, VirD1) or T4S components (VirD4 substrate receptor, VirB channel subunits). We propose that A. tumefaciens has appropriated a progenitor ParA/MinD-like ATPase to promote conjugative DNA transfer by: (i) nucleating relaxosome assembly at oriT-like T-DNA border sequences and (ii) spatially positioning the transfer intermediate at the cell pole to coordinate substrate-T4S channel docking.     

Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems

The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals. In Gram-negative bacteria, the conjugation machines are composed of a cell envelope-spanning secretion channel and an extracellular pilus. These dynamic structures (i) direct formation of stable contacts-the mating junction-between donor and recipient cell membranes, (ii) transmit single-stranded DNA as a nucleoprotein particle, as well as protein substrates, across donor and recipient cell membranes, and (iii) mediate disassembly of the mating junction following substrate transfer. This review summarizes recent progress in our understanding of the mechanistic details of DNA trafficking with a focus on the paradigmatic Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation systems.     

农杆菌-植物间基因转移的分子基础

植物病原细菌多以Ⅲ型分泌系统运送毒性因子或无毒基因产物到植物细胞,但根癌农杆菌利用Ⅳ型分泌系统转移致瘤基因片断T-DNA到植物细胞核,并整合到植物基因组,使植物产生肿瘤,作者将介绍vir基因的诱导、T-DNA的加工、T-DNA的转移,以及T-复合体运输的装备等方面的最新研究进展,以探讨农杆菌-植物间基因转移的分子基础,研究该系统转移基因的分子基础将有利于开发和改良植物遗传工程的载体工具;另外,农杆菌-植物作为一种模式植物病害系统,其研究也为植物-病原菌的基础理论研究提供参考。由于有些人体病原细菌也采用Ⅳ型分泌系统运送毒性因子到人体细胞,研究农杆菌-植物间的基因转移系统也有利于医学研究。     

Will you let me use your nucleus? How Agrobacterium gets its T-DNA expressed in the host plant cell

Agrobacterium is the only known bacterium capable of natural DNA transfer into a eukaryotic host. The genes transferred to host plants are contained on a T-DNA (transferred DNA) molecule, the transfer of which begins with its translocation, along with several effector proteins, from the bacterial cell to the host-cell cytoplasm. In the host cytoplasm, the T-complex is formed from a single-stranded copy of the T-DNA (T-strand) associated with several bacterial and host proteins and it is imported into the host nucleus via interactions with the host nuclear import machinery. Once inside the nucleus, the T-complex is most likely directed to the host genome by associating with histones. Finally, the chromatin-associated T-complex is uncoated from its escorting proteins prior to the conversion of the T-strand to a double-stranded form and its integration into the host genome.     

The emerging structure of the Agrobacterium T-DNA transfer complex

Single-stranded DNA-protein complex (T-complex) is proposed to mediate T-DNA transfer from Agrobacterium to plant cells. A novel model for transfer is presented which incorporates features of both bacterial conjugation and viral infection. Specific protein components of the T-complex, its ultrastructure and possible functions in the plant cell are discussed.     

Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation

Agrobacterium tumefaciens uses a type IV secretion system (T4SS) to transfer T-DNA and virulence proteins to plants. The T4SS is composed of two major structural components: the T-pilus and a membrane-associated complex that is responsible for translocating substrates across both bacterial membranes. VirB2 protein is the major component of the T-pilus. We used the C-terminal-processed portion of VirB2 protein as a bait to screen an Arabidopsis thaliana cDNA library for proteins that interact with VirB2 in yeast. We identified three related plant proteins, VirB2-interacting protein (BTI) 1 (BTI1), BTI2, and BTI3 with unknown functions, and a membrane-associated GTPase, AtRAB8. The three BTI proteins also interacted with VirB2 in vitro. Preincubation of Agrobacterium with GST-BTI1 protein decreased the transformation efficiency of Arabidopsis suspension cells by Agrobacterium. Transgenic BTI and AtRAB8 antisense and RNA interference Arabidopsis plants are less susceptible to transformation by Agrobacterium than are wild-type plants. The level of BTI1 protein is transiently increased immediately after Agrobacterium infection. In addition, overexpression of BTI1 protein in transgenic Arabidopsis results in plants that are hypersusceptible to Agrobacterium-mediated transformation. Confocal microscopic data indicate that GFP-BTI proteins preferentially localize to the periphery of root cells in transgenic Arabidopsis plants, suggesting that BTI proteins may contact the Agrobacterium T-pilus. We propose that the three BTI proteins and AtRAB8 are involved in the initial interaction of Agrobacterium with plant cells.     

Structural and dynamic properties of bacterial type IV secretion systems (review)

The type IV secretion systems (T4SS) are widely distributed among the gram-negative and -positive bacteria. These systems mediate the transfer of DNA and protein substrates across the cell envelope to bacterial or eukaryotic cells generally through a process requiring direct cell-to-cell contact. Bacteria have evolved T4SS for survival during establishment of pathogenic or symbiotic relationships with eukaryotic hosts. The Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation machines serve as models for detailed mechanistic studies aimed at elucidating the nature of translocation signals, machine assembly pathways and architectures, and the dynamics of substrate translocation. The A. tumefaciens VirB/D4 T4SS are polar-localized organelles composed of a secretion channel and an extracellular T pilus. These T4SS are assembled from 11 or more subunits. whose membrane topologies, intersubunit contacts and, in some cases, 3-dimensional structures are known. Recently, powerful in vivo assays have identified C-terminal translocation signals, defined for the first time the translocation route for a DNA substrate through a type IV secretion channel, and supplied evidence that ATP energy consumption contributes to a late stage of machine morphogenesis. Together, these recent findings describe the mechanics of type IV secretion in unprecedented detail.