Agrobacterium-mediated genetic transformation of plants: biology and biotechnology

Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at understanding and improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains, plasmids and technologies currently used for the successful transformation of numerous plant species. Unlike the role of bacterial proteins, the role of host factors in the transformation process has remained obscure for nearly a century of Agrobacterium research, and only recently have we begun to understand how Agrobacterium hijacks host factors and cellular processes during the transformation process. The identification of such factors and studies of these processes hold great promise for the future of plant biotechnology and plant genetic engineering, as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants.     

Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression

Agrobacterium tumefaciens is a plant pathogen that incites crown gall tumors by transferring to and expressing a portion of a resident plasmid in plant cells. Currently, little is known about the host response to Agrobacterium infection. Using suppressive subtractive hybridization and DNA macroarrays, we identified numerous plant genes that are differentially expressed during early stages of Agrobacterium-mediated transformation. Expression profiling indicates that Agrobacterium infection induces plant genes necessary for the transformation process while simultaneously repressing host defense response genes, thus indicating successful utilization of existing host cellular machinery for genetic transformation purposes. A comparison of plant responses to different strains of Agrobacterium indicates that transfer of both T-DNA and Vir proteins modulates the expression of host genes during the transformation process.     

Agrobacterium-mediated genetic transformation of plants: The role of host

Agrobacterium-mediated genetic transformation is the most widely used technology to obtain overexpression of recombinant proteins in plants. Molecular events that occur within Agrobacterium during interactions with host plants have been studied extensively, and now we have a reasonable understanding the key factors involved in the regulation of T-DNA nuclear import and genomic integration. By contrast, very little is known about the events that take place in the host cells during genetic transformation by Agrobacterium. Here, we describe the plant-related factors including genotype, genes, proteins, competency of target tissues and phenolic compounds that participate in Agrobacterium-mediated genetic transformation and discuss their possible roles in this process. Because Agrobacterium probably adapts existing cellular processes for its life cycle, identifying the processes in host cells during Agrobacterium infection might contribute to better understanding of basic biological processes as cell communication, intracellular transport and DNA repair and recombination as well as to expanding the host range of Agrobacterium as a genetic engineering tool.     

Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.     

Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool.

Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this "natural genetic engineer" for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.     

The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation

To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus. As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.     

Agrobacterium-mediated gene delivery and the biology of hostrange limitations

Insertion of foreign DNA into Ti plasmid-derived vectors in Agrobacterium tumefaciens is currently the most frequently used strategy for generating transgenic plants in a wide variety of species. Limitations of the host range of Agrobacterium restrict its usefulness in many cases, particularly when dealing with monocotyledonous plants. The objective of this presentation is to briefly discuss the efficiency of the transformation process utilized by Agrobacterium tumefaciens , potential barriers to efficient transformation by Agrobacterium that result in limitation of its useful host range, and how an understanding of the successful Agrobacterium /plant cell interaction might lead to advances in a variety of DNA delivery methodologies.     

VirD4-independent transformation by CloDF13 evidences an unknown factor required for the genetic colonization of plants via Agrobacterium

Agrobacterium uses a mechanism similar to conjugation for trans-kingdom transfer of its oncogenic T-DNA. A defined VirB/VirD4 Type IV secretion system is responsible for such a genetic transfer. In addition, certain virulence proteins as VirE2 can be mobilized into host cells by the same apparatus. VirE2 is essential to achieve plant but not yeast transformation. We found that the limited host range plasmid CloDF13 can be recruited by the virulence apparatus of Agrobacterium for transfer to eukaryotic hosts. As expected the VirB transport complex was required for such trans-kingdom DNA transfer. However, unexpectedly, the coupling factor VirD4 turned out to be necessary for transfer to plants but not for transport into yeast. The CloDF13 encoded coupling factor (Mob) was essential for transfer to both plants and yeast though. This is interpreted by the different specificities of Mob and VirD4. Hence, Mob being required for the transport of the CloDF13 transferred DNA (to both plants and yeast) and VirD4 being required for transport of virulence proteins such as VirE2. Nevertheless, the presence of the VirE2 protein in the host plant was not sufficient to restore the deficiency for VirD4 in the transforming bacteria. We propose that Mob functions encoded by the plasmid CloDF13 are sufficient for DNA mobilization to eukaryotic cells but that the VirD4-mediated pathway is essential to achieve DNA nuclear establishment specifically in plants. This suggests that other Agrobacterium virulence proteins besides VirE2 are translocated and essential for plant transformation.     

Molecular biology of Ri-plasmid—A review

Agrobacterium rhizogenes transfers a segment of its plasmid to the plant genome. The transferred DNA contains genes which are involved in the synthesis of plant hormones. These genes express in the plant cell and give rise to rooty-tumors at the infection site. Transgenic plants can be readily regenerated from the rooty-tumors and the transferred DNA is transmitted to progeny plants. High regeneration potential and sustained maintenance of transferred DNA makes the bacterium a suitable vector for plant genetic engineering. DNA sequences homologous to the transferred DNA ofAgrobacterium rhizogenes were detected in some untransformed plant species suggesting a past infection byAgrobacterium rhizogenes during evolution of some genera, notably Nicotiana.     

Agrobacterium T-DNA integration: molecules and models

Genetic transformation mediated by Agrobacterium involves the transfer of a DNA molecule (T-DNA) from the bacterium to the eukaryotic host cell, and its integration into the host genome. Whereas extensive work has revealed the biological mechanisms governing the production, Agrobacterium-to-plant cell transport and nuclear import of the Agrobacterium T-DNA, the integration step remains largely unexplored, although several different T-DNA integration mechanisms have been suggested. Recent genetic and functional studies have revealed the importance of host proteins involved in DNA repair and maintenance for T-DNA integration. In this article, we review our understanding of the specific function of these proteins and propose a detailed model for integration.     

Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro

Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.     

Transfer of T-DNA from Agrobacterium to the plant cell.

Agrobacterium tumefaciens is the causative agent of crown gall, a disease of dicotyledonous plants characterized by a tumorous phenotype. Earlier in this century, scientific interest in A. tumefaciens was based on the possibility that the study of plant tumors might reveal mechanisms that were also operating in animal neoplasia. In the recent past, the tumorous growth was shown to result from the expression of genes coded for by a DNA segment of bacterial origin that was transferred and became stably integrated into the plant genome. This initial molecular characterization of the infection process suggested that Agrobacterium might be used to deliver genetic material into plants. The potential to genetically engineer plants generated renewed interest in the study of A. tumefaciens. In this review, we concentrate on the most recent advances in the study of Agrobacterium-mediated gene transfer, its relationship to conjugation, DNA processing and transport, and nuclear targeting. In the following discussion, references for earlier work can be found in more comprehensive reviews (Hooykaas and Schilperoort, 1992; Zambryski, 1992; Hooykaas and Beijersbergen, 1994).     

DNA replication and cell cycle in plants: learning from geminiviruses

Plant cell growth and development depend on continuous cell proliferation which is restricted to small regions of the plant called meristems. Infection by geminiviruses, small DNA viruses whose replicative cycle relies on host cell factors, is excluded from those proliferating areas. Since most of the replicative factors are present, almost exclusively, in proliferating cells, geminivirus infection is believed to induce a cellular state permissive for viral DNA replication, e.g. S-phase or, at least, some specific S-phase functions. The molecular basis for this effect seems to be the interference that certain geminivirus proteins exert on the retinoblastoma-related (RBR) pathway, which analogously to that of animal cells, regulates plant cell cycle activation and G(1)-S transition. In some cases, geminiviruses induce cell proliferation and abnormal growth. Mechanisms other than sequestering plant RBR probably contribute to the multiple effects of geminivirus proteins on cellular gene expression, cell growth control and cellular DNA replication. Current efforts to understand the coupling of geminivirus DNA replication to cell cycle and growth control as well as the directions in which future research is aiming are reviewed.     

Epigenetic control of Agrobacterium T-DNA integration

To genetically transform plants, Agrobacterium transfers its T-DNA into the host cell and integrates it into the plant genome, resulting in neoplastic growths. Over the past 2 decades, a great deal has been learned about the molecular mechanism by which Agrobacterium produces T-DNA and transports it into the host nucleus. However, T-DNA integration, which is the limiting, hence, the most critical step of the transformation process, largely remains an enigma. Increasing evidence suggests that Agrobacterium utilizes the host DNA repair machinery to facilitate T-DNA integration. Meanwhile, it is well known that chromatin modifications, including the phosphorylation of histone H2AX, play an important role in DNA repair. Thus, by implication, such epigenetic codes in chromatin may also have a considerable impact on T-DNA integration, although the direct evidence to demonstrate this hypothesis is still lacking. In this review, we summarize the recent advances in our understanding of Agrobacterium T-DNA integration and discuss the potential link between this process and the epigenetic information in the host chromatin. This article is part of a Special Issue entitled: Epigenetic Control of cellular and developmental processes in plants.     

On tracks and locomotives: the long route of DNA to the nucleus

Molecular motors have prominent functions in organelle transport, cytoskeletal organization, division and motility. The dyneins are one of the three families of cytoskeleton-based molecular motors and they travel along the cytoplasmic microtubule network towards the minus end of the microtubule. This directed movement is used by DNA viruses to deliver their infectious genome and proteins to the host cell nucleus. In recent studies, it has been hypothesized that Agrobacterium species use a similar pathway to deliver their infectious unit--a large complex between single-stranded DNA and proteins--to the host cell nucleus and that a karyophilic protein carrier that can deliver synthetic DNA to the nucleus is also driven by a dynein motor. These studies shed light on the mechanism of Agrobacterium-mediated genetic transformation and could lead to new methods for the efficient transfection of synthetic DNA.     

Identification of Yeast Factors Involved in the Replication of Mungbean Yellow Mosaic India Virus Using Yeast Temperature-Sensitive Mutants

正Dear Editor,The geminiviruses are small single-stranded plant DNA viruses belonging to the family Geminiviridae, which cause serious diseases in many economically important     

农杆菌T-复合物的形成与转运研究进展

在简要介绍农杆菌T-DNA转运全过程的基础上,结合作者近年的工作,重点对T-复合物的形成和T-复合物在农杆菌细胞内的转运机理的最新进展进行归纳和评述．农杆菌能够将其Ti质粒上的一段DNA以单链DNA-蛋白质复合物(简称T-复合物)的形式,通过其细胞两端的四型分泌系统(typeⅣ secretion system,T4SS)转运到宿主植物中,并使宿主发生遗传转化,因而农杆菌介导的T-DNA转运技术已成为应用最广泛的植物转基因技术,同时,由于转运T-复合物的T4SS也是某些质粒接合转移和许多病源微生物分泌致病效应蛋白的通道,因此,农杆菌T-DNA转运机理的研究受到了广泛的重视和关注,使得这方面的研究进展非常迅速．     

Odyssey of agrobacterium T-DNA

Agrobacterium tumefaciens, a plant pathogen, is characterized by the unique feature of interkingdom DNA transfer. This soil bacterium is able to transfer a fragment of its DNA, called T-DNA (transferred DNA), to the plant cell where T-DNA is integrated into the plant genome leading to "genetic colonization" of the host. The fate of T-DNA, its processing, transfer and integration, resembles the journey of Odysseus, although our hero returns from its long trip in a slightly modified form.