Rhizobium radiobacter (syn. Agrobacterium radiobacter) is a saprophytic,
gram negative rod belonging to alpha proteobacteria, family
Rhizobiaceae. Agrobacterium and Rhizobium are highly related and their
species are interwoven. Agrobacterium species have been reclassified in
the genus Rhizobium, based on 16S rRNA homology amalgamating the
pathogenic, symbiotic nitrogen-fixing and non-pathogenic species in a
generic circumscription of Rhizobium (Young et al., 2001). Agrobacterium
tumefaciens (syn. Agrobacterium radiobacter) was conserved as the type
species of Agrobacterium, but the epithet radiobacter took precedence as
Rhizobium radiobacter in the revised genus. Agrobacterium (Rhizobium)
radiobacter does not contain the Ti plasmid associated with tumour
induction in plants (Sawada et al., 1993). Rhizobium radiobacter is a fast
growing, keto-lactose negative, reported as a plant growth promoting
bacteria and to infect soybean and has been isolated from its root nodules
(Ansari et al 2014). Plant-pathogenic, soil inhabitant R. radiobacter is
not characterized as a true human pathogen but as an opportunistic human
pathogen. Human infections caused by R. radiobacter that are reported
include catheter-related bacteraemia, endocarditis, continuous ambulatory
peritoneal dialysis peritonitis, urinary tract infections, pneumonia and
chronic endopthalmitis.
Agrobacterium tumefaciens (lat. für „Tumor machendes Ackerbakterium“; neuerdings auch bezeichnet als Rhizobium radiobacter) ist ein pflanzenpathogenes, gramnegatives Bodenbakterium. Es gehört zur Alpha-Klasse der Proteobakterien.
A. tumefaciens ist ein Modellorganismus und verfügt über die Fähigkeit, DNA in pflanzliche Zellen zu übertragen. Dieser Vorgang wurde erstmals durch Jozef Schell und Marc Van Montagu im Jahr 1983 beschrieben.
Durch A. tumefaciens hervorgerufene Gallen an den Wurzeln der Pekannuss (Carya illinoinensis).
Durch A. tumefaciens hervorgerufene Galle an einem Zweig der Forsythie (Forsythia × intermedia)
Unter natürlichen Bedingungen kann das Bakterium nur verletzte, zweikeimblättrige Pflanzen infizieren. Die Infektion erfolgt meist an der Stängelbasis, es können aber auch andere oberirdische Pflanzenteile infiziert werden. Die Erkennung zwischen Bakterien- und Pflanzenzellen erfolgt mit Hilfe bestimmter Signalmoleküle (Pektine und Glucane) auf deren Oberfläche. Unter Laborbedingungen ist auch die Infektion von Monokotylen möglich.
Die Wucherungen, die A. tumefaciens durch Eingriff in den pflanzlichen Hormonhaushalt auslöst, haben bei Nutzpflanzen oft bestimmte Namen. Beim Befall der Weinrebe spricht man von Maukekrankheit, bei der Zuckerrübe von Wurzelkropf. Bei anderen Pflanzen wie etwa Meerrettich und Rhabarber werden die Tumoren auch als crown-galls (Wurzelhalsgallentumor) bezeichnet.
Pathogene Agrobakterien tragen stets ein Plasmid. Dieses Ti-Plasmid (Ti steht für tumor inducing – Tumor induzierend) enthält Gene, die für die spätere DNA-Übertragung in die Pflanzenzelle notwendig sind. Die Transkription dieser sogenannten „Virulenz-“ oder vir-Gene wird durch sekundäre Pflanzenstoffe (1), die aus verwundeten Pflanzenteilen austreten, aktiviert (2). Man spricht auf Seiten des Bakteriums auch von positiver Chemotaxis, da sich die Bakterien gezielt zur Quelle des Botenstoffs hin bewegen.
Mögliche Pflanzenstoffe, die Agrobacterium zu einer Infektion veranlassen:
Das aktivierte vir-Genom des Bakteriums (3) befindet sich auf seinem Ti-Plasmid und codiert für Proteine (4), die in die Pflanze eindringen (5) und in der Pflanze Fremdgene installieren können. Mit Hilfe der vir-Genprodukte kann ein anderer Teil der DNA des Ti-Plasmides als so genannte „Transfer- oder t-DNA“ in die Pflanzenzelle übertragen werden. Dabei wird die t-DNA als einzelsträngiges DNA-Molekül mit Hilfe des bakteriellen Typ IV Sekretionssystems in die pflanzliche Wirtszelle eingeschleust. Diese t-DNA ist an ihrem 5'-Ende kovalent an das bakterielle VirD2-Protein gebunden, welches durch ein bestimmtes Motiv dazu beiträgt, dass die DNA in den pflanzlichen Zellkern transportiert wird (6, Kernlokalisierungssignal). Des Weiteren wird die t-DNA von VirE2-Proteinen gebunden und bildet nun den t-Komplex. Aus Arabidopsis thaliana ist bekannt, dass VirE2 mit dem pflanzlichen VIP1-Protein oder seinen homologen interagieren kann, nachdem dieses durch die pflanzliche MAP-Kinase 3 phosphoryliert wurde. Das aktivierte VIP1-Protein fungiert nun als Adaptor der pflanzlichen Kernimportmaschinerie (insbesondere Importin alpha) und dem bakteriellen VirE2-Protein und trägt nun ebenfalls zum Kerntransport des t-Komplexes bei. Im Zellkern muss der t-Strang von den VirE2 und VIP1-Proteinen befreit werden. Dies geschieht durch das bakterielle VirF-Protein, welches die Proteine dem Proteasom-vermittelten Abbau zuführt. Nun kann die t-DNA ins Wirtsgenom integrieren. In eukaryotischen Zellen ist DNA nur hier vor Abbau geschützt und kann repliziert und transkribiert werden.
Im Zellkern angekommen wird die DNA in das pflanzliche Genom integriert (7). Der Ort der Insertion im Wirtsgenom ist rein zufällig. Promotoren, die von der Pflanzenzelle erkannt werden, sorgen dafür, dass die Gene in der Pflanze auch aktiv sind. Die übertragenen Gene besitzen also eine typisch eukaryotische Struktur, obwohl sie aus einem Bakterium stammen. Die übertragenen Gene lösen Tumoren aus und veranlassen die pflanzliche Zelle, Opine (Octopin und Nopalin) zu produzieren, die den Bakterien als Nahrung dienen, für die Pflanze aber wertlos sind. Der Name Agrobacterium tumefaciens leitet sich von der tumorinduzierenden Eigenschaft ab.
Die Aktivierung der vir-Gene kann dazu benutzt werden, gewünschte Gene in Pflanzen einzubringen, so genannte Transformation durch Agrobakterien. Agrobakterien können Pflanzenzellen also gezielt genetisch verändern. Wegen dieser Eigenschaft gilt es seit Beginn der Gentechnik als wichtiger Vektor, um Gene auf Pflanzen zu übertragen. Unter Laborbedingungen gelingt dies bei vielen Pflanzen und sogar einigen Pilzen. Hierfür werden die Gene der t-DNA, die normalerweise für den Opin-Metabolismus und die Tumor-Induktion notwendig sind, durch andere ersetzt. Transformationsziele sind neben zweikeimblättrigen Pflanzen wie Kartoffeln, Raps, Soja und Tabak auch einkeimblättrige Pflanzen aus der Familie der Süßgräser. In dieser Familie stehen die meisten der wichtigsten Getreidepflanzen, wie Mais, Weizen, Roggen, Reis und Gerste. Erfolgreiche Transformationen wurden für alle der genannten Monokotylen beschrieben.[2][3][4][5][6][7][8][9][10]
Das Genom von A. tumefaciens wurde 2001 sequenziert.
Ein weiterer bekannter Vertreter der Gattung ist Agrobacterium rhizogenes, der Pflanzen hauptsächlich an den Wurzeln infiziert und zu massiven Wucherungen der Wurzelhaare führt. Das Äquivalent des Ti-Plasmides ist hier das Ri-Plasmid.
Agrobacterium tumefaciens (lat. für „Tumor machendes Ackerbakterium“; neuerdings auch bezeichnet als Rhizobium radiobacter) ist ein pflanzenpathogenes, gramnegatives Bodenbakterium. Es gehört zur Alpha-Klasse der Proteobakterien.
A. tumefaciens ist ein Modellorganismus und verfügt über die Fähigkeit, DNA in pflanzliche Zellen zu übertragen. Dieser Vorgang wurde erstmals durch Jozef Schell und Marc Van Montagu im Jahr 1983 beschrieben.
Ang Agrobacterium tumefaciens (binago ang siyentipikong panglan sa Rhizobium radiobacter, kasinghulugan ng Agrobacterium radiobacter)[2][3][4] ay ang sanhi ng sakit na crown gall (o ang pagkabuo ng tumor) sa higit isang daan at apatnapung espesye ng eudicots. Ito ay bakteryum sa lupa na hugis-baras at negatibo ang Gram. Ang mga sintomas ay dulot ng pagpasok ng maliit na piraso ng DNA (kilala bilang T-DNA, para sa transfer DNA, na iba sa tRNA na naglilipat ng asidong amino sa pagbuo ng protina, na nakalilitong tinatawag na transfer RNA), mula sa isang plasmid, paloob ng selula ng halaman, kung saan ito ay ihinahalo ng medyo walang pili o random sa loob ng genome ng halaman.
Ang A. tumafaciens ay isang alphaproteobacteria ng pamilya Rhizobiacae, na kinabibilangan ng bakterya sa loob ng mga butil na taga-ayos ng nitroheno. Di tulad ng ibang bakterya na tumutulong sa halaman, ang espesye ng Agrobacterium ay nakakapagpabunga ng tumor sa halaman, at ito’y hindi nakatutulong sa mga halaman Dahil maraming uri ng halaman ang apektado ng Agrobacterium, ito ay isang nakababahalang problema sa industriya ng agrikultura.
Sa ekonomiya, ang A. tumefaciens ay isang malaking salot ng mga nogales, ubas, prutas na bato, mga puno ng nuwes, matamis na aselga, horseradish, at ruwibarbo.
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(tulong) Ang Agrobacterium tumefaciens (binago ang siyentipikong panglan sa Rhizobium radiobacter, kasinghulugan ng Agrobacterium radiobacter) ay ang sanhi ng sakit na crown gall (o ang pagkabuo ng tumor) sa higit isang daan at apatnapung espesye ng eudicots. Ito ay bakteryum sa lupa na hugis-baras at negatibo ang Gram. Ang mga sintomas ay dulot ng pagpasok ng maliit na piraso ng DNA (kilala bilang T-DNA, para sa transfer DNA, na iba sa tRNA na naglilipat ng asidong amino sa pagbuo ng protina, na nakalilitong tinatawag na transfer RNA), mula sa isang plasmid, paloob ng selula ng halaman, kung saan ito ay ihinahalo ng medyo walang pili o random sa loob ng genome ng halaman.
Ang A. tumafaciens ay isang alphaproteobacteria ng pamilya Rhizobiacae, na kinabibilangan ng bakterya sa loob ng mga butil na taga-ayos ng nitroheno. Di tulad ng ibang bakterya na tumutulong sa halaman, ang espesye ng Agrobacterium ay nakakapagpabunga ng tumor sa halaman, at ito’y hindi nakatutulong sa mga halaman Dahil maraming uri ng halaman ang apektado ng Agrobacterium, ito ay isang nakababahalang problema sa industriya ng agrikultura.
Sa ekonomiya, ang A. tumefaciens ay isang malaking salot ng mga nogales, ubas, prutas na bato, mga puno ng nuwes, matamis na aselga, horseradish, at ruwibarbo.
Agrobacterium radiobacter (more commonly known as Agrobacterium tumefaciens)[6][3][7] is the causal agent of crown gall disease (the formation of tumours) in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium.[1] Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA, for 'transfer DNA', not to be confused with tRNA that transfers amino acids during protein synthesis), from a plasmid into the plant cell,[8] which is incorporated at a semi-random location into the plant genome. Plant genomes can be engineered by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors.
Agrobacterium tumefaciens is an Alphaproteobacterium of the family Rhizobiaceae, which includes the nitrogen-fixing legume symbionts. Unlike the nitrogen-fixing symbionts, tumor-producing Agrobacterium species are pathogenic and do not benefit the plant. The wide variety of plants affected by Agrobacterium makes it of great concern to the agriculture industry.[9]
Economically, A. tumefaciens is a serious pathogen of walnuts, grape vines, stone fruits, nut trees, sugar beets, horse radish, and rhubarb, and the persistent nature of the tumors or galls caused by the disease make it particularly harmful for perennial crops.[10]
Agrobacterium tumefaciens grows optimally at 28 °C (82 °F). The doubling time can range from 2.5–4h depending on the media, culture format, and level of aeration.[11] At temperatures above 30 °C (86 °F), A. tumefaciens begins to experience heat shock which is likely to result in errors in cell division.[11]
To be virulent, the bacterium contains a tumour-inducing plasmid (Ti plasmid or pTi) 200 kbp long, which contains the T-DNA and all the genes necessary to transfer it to the plant cell.[12] Many strains of A. tumefaciens do not contain a pTi.
Since the Ti plasmid is essential to cause disease, prepenetration events in the rhizosphere occur to promote bacterial conjugation - exchange of plasmids amongst bacteria. In the presence of opines, A. tumefaciens produces a diffusible conjugation signal called 30C8HSL or the Agrobacterium autoinducer. This activates the transcription factor TraR, positively regulating the transcription of genes required for conjugation.
Agrobacterium tumefaciens infects the plant through its Ti plasmid. The Ti plasmid integrates a segment of its DNA, known as T-DNA, into the chromosomal DNA of its host plant cells. A. tumefaciens has flagella that allow it to swim through the soil towards photoassimilates that accumulate in the rhizosphere around roots. Some strains may chemotactically move towards chemical exudates from plants, such as acetosyringone and sugars, which indicate the presence of a wound in the plant through which the bacteria may enter. Phenolic compounds are recognised by the VirA protein, a transmembrane protein encoded in the virA gene on the Ti plasmid. Sugars are recognised by the chvE protein, a chromosomal gene-encoded protein located in the periplasmic space.[13]
At least 25 vir genes on the Ti plasmid are necessary for tumor induction. In addition to their perception role, virA and chvE induce other vir genes. The VirA protein has autokinase activity: it phosphorylates itself on a histidine residue. Then the VirA protein phosphorylates the VirG protein on its aspartate residue. The virG protein is a cytoplasmic protein produced from the virG Ti plasmid gene. It is a transcription factor, inducing the transcription of the vir operons. The ChvE protein regulates the second mechanism of the vir genes' activation. It increases VirA protein sensitivity to phenolic compounds.[13]
Attachment is a two-step process. Following an initial weak and reversible attachment, the bacteria synthesize cellulose fibrils that anchor them to the wounded plant cell to which they were attracted. Four main genes are involved in this process: chvA, chvB, pscA, and att. The products of the first three genes apparently are involved in the actual synthesis of the cellulose fibrils. These fibrils also anchor the bacteria to each other, helping to form a microcolony.
VirC, the most important virulent protein, is a necessary step in the recombination of illegitimate recolonization. It selects the section of the DNA in the host plant that will be replaced and it cuts into this strand of DNA.
After production of cellulose fibrils, a calcium-dependent outer membrane protein called rhicadhesin is produced, which also aids in sticking the bacteria to the cell wall. Homologues of this protein can be found in other rhizobia. Currently, there are several reports on standardisation of protocol for the Agrobacterium-mediated transformation. The effect of different parameters such as infection time, acetosyringone, DTT, and cysteine have been studied in soybean (Glycine max).[14]
Possible plant compounds that initiate Agrobacterium to infect plant cells:[15]
To transfer the T-DNA into the plant cell, A. tumefaciens uses a type IV secretion mechanism, involving the production of a T-pilus. When acetosyringone and other substances are detected, a signal transduction event activates the expression of 11 genes within the VirB operon which are responsible for the formation of the T-pilus.
The pro-pilin is formed first. This is a polypeptide of 121 amino acids which requires processing by the removal of 47 residues to form a T-pilus subunit. The subunit was though to be circularized by the formation of a peptide bond between the two ends of the polypeptide. However, high-resolution structure of the T-pilus revealed no cyclization of the pilin, with the overall organization of the pilin subunits being highly similar to those of other conjugative pili, such as F-pilus.[16]
Products of the other VirB genes are used to transfer the subunits across the plasma membrane. Yeast two-hybrid studies provide evidence that VirB6, VirB7, VirB8, VirB9 and VirB10 may all encode components of the transporter. An ATPase for the active transport of the subunits would also be required.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS). The structure of the T-pilus showed that the central channel of the pilus is too narrow to allow the transfer of the folded VirD2, suggesting that VirD2 must be partially unfolded during the conjugation process.[16]
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLSs, located on the VirE2 and VirD2, are recognised by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. VIP1 also appears to be an important protein in the process, possibly acting as an adapter to bring the VirE2 to the importin. Once inside the nucleus, VIP2 may target the T-DNA to areas of chromatin that are being actively transcribed, so that the T-DNA can integrate into the host genome.
To cause gall formation, the T-DNA encodes genes for the production of auxin or indole-3-acetic acid via the IAM pathway. This biosynthetic pathway is not used in many plants for the production of auxin, so it means the plant has no molecular means of regulating it and auxin will be produced constitutively. Genes for the production of cytokinins are also expressed. This stimulates cell proliferation and gall formation.
The T-DNA contains genes for encoding enzymes that cause the plant to create specialized amino acid derivatives which the bacteria can metabolize, called opines.[17] Opines are a class of chemicals that serve as a source of nitrogen for A. tumefaciens, but not for most other organisms. The specific type of opine produced by A. tumefaciens C58 infected plants is nopaline (Escobar et al., 2003).
Two nopaline type Ti plasmids, pTi-SAKURA and pTiC58, were fully sequenced. "A. fabrum" C58, the first fully sequenced pathovar, was first isolated from a cherry tree crown gall. The genome was simultaneously sequenced by Goodner et al.[18] and Wood et al.[19] in 2001. The genome of A. tumefaciens C58 consists of a circular chromosome, two plasmids, and a linear chromosome. The presence of a covalently bonded circular chromosome is common to Bacteria, with few exceptions. However, the presence of both a single circular chromosome and single linear chromosome is unique to a group in this genus. The two plasmids are pTiC58, responsible for the processes involved in virulence, and pAtC58,[b] once dubbed the "cryptic" plasmid.[18][19]
The pAtC58 plasmid has been shown to be involved in the metabolism of opines and to conjugate with other bacteria in the absence of the pTiC58 plasmid.[20] If the Ti plasmid is removed, the tumor growth that is the means of classifying this species of bacteria does not occur.
The Asilomar Conference (Berg et al. 1975) established widespread agreement that recombinant techniques were insufficiently understood and needed to be tightly controlled.[21] The DNA transmission capabilities of Agrobacterium have been vastly explored in biotechnology as a means of inserting foreign genes into plants. Shortly after Asilomar, Marc Van Montagu and Jeff Schell, (University of Ghent and Plant Genetic Systems, Belgium) discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter the bacterium into an efficient delivery system for genetic engineering in plants.[22] The plasmid T-DNA that is transferred to the plant is an ideal vehicle for genetic engineering.[23] This is done by cloning a desired gene sequence into T-DNA binary vectors that will be used to deliver a sequence of interest into eukaryotic cells. Soon it was widely agreed that Asilomar like protections were needed in plant technologies as well.[21] This process has been performed using firefly luciferase gene to produce glowing plants. This luminescence has been a useful device in the study of plant chloroplast function and as a reporter gene.[24] It is also possible to transform Arabidopsis thaliana by dipping flowers into a broth of Agrobacterium: the seed produced will be transgenic. Under laboratory conditions, the T-DNA has also been transferred to human cells, demonstrating the diversity of insertion application.[25]
The mechanism by which Agrobacterium inserts materials into the host cell is by a type IV secretion system which is very similar to mechanisms used by pathogens to insert materials (usually proteins) into human cells by type III secretion. It also employs a type of signaling conserved in many Gram-negative bacteria called quorum sensing. This makes Agrobacterium an important topic of medical research, as well.
Natural genetic transformation in bacteria is a sexual process involving the transfer of DNA from one cell to another through the intervening medium, and the integration of the donor sequence into the recipient genome by homologous recombination. A. tumefaciens can undergo natural transformation in soil without any specific physical or chemical treatment.[26]
Agrobacterium tumefaciens overwinters in infested soils. Agrobacterium species live predominantly saprophytic lifestyles, so its common even for plant-parasitic species of this genus to survive in the soil for lengthy periods of time, even without host plant presence.[27] When there is a host plant present, however, the bacteria enter the plant tissue via recent wounds or natural openings of roots or stems near the ground. These wounds may be caused by cultural practices, grafting, insects, etc. Once the bacteria have entered the plant, they occur intercellularly and stimulate surrounding tissue to proliferate due to cell transformation. Agrobacterium performs this control by inserting the plasmid T-DNA into the plant's genome. See above for more details about the process of plasmid DNA insertion into the host genome. Excess growth of the plant tissue leads to gall formation on the stem and roots. These tumors exert significant pressure on the surrounding plant tissue, which causes this tissue to become crushed and/or distorted. The crushed vessels lead to reduced water flow in the xylem. Young tumors are soft and therefore vulnerable to secondary invasion by insects and saprophytic microorganisms. This secondary invasion causes the breakdown of the peripheral cell layers as well as tumor discoloration due to decay. Breakdown of the soft tissue leads to release of the Agrobacterium tumefaciens into the soil allowing it to restart the disease process with a new host plant.[28]
Crown gall disease caused by Agrobacterium tumefaciens can be controlled by using various methods. The best way to control this disease is to take preventative measures, such as sterilizing pruning tools so as to avoid infecting new plants. Performing mandatory inspections of nursery stock and rejecting infected plants as well as not planting susceptible plants in infected fields are also valuable practices. Avoiding wounding the crowns/roots of the plants during cultivation is important for preventing disease. In horticultural techniques in which multiple plants are joined to grow as one, such as budding and grafting[29] these techniques lead to plant wounds. Wounds are the primary location of bacterial entry into the host plant. Therefore, it is advisable to perform these techniques during times of the year when Agrobacteria are not active. Control of root-chewing insects is also helpful to reduce levels of infection, since these insects cause wounds (aka bacterial entryways) in the plant roots.[28] It is recommended that infected plant material be burned rather than placed in a compost pile due to the bacteria's ability to live in the soil for many years.[30]
Biological control methods are also utilized in managing this disease. During the 1970s and 1980s, a common practice for treating germinated seeds, seedlings, and rootstock was to soak them in a suspension of K84. K84 is composed of A. radiobacter, which is a species related to A. tumefaciens but is not pathogenic. K84 produces a bacteriocin (agrocin 84) which is an antibiotic specific against related bacteria, including A. tumefaciens. This method, which was successful at controlling the disease on a commercial scale, had the risk of K84 transferring its resistance gene to the pathogenic Agrobacteria. Thus, in the 1990s, the use of a genetically engineering strain of K84, known as K-1026, was created. This strain is just as successful in controlling crown gall as K84 without the caveat of resistance gene transfer.[31]
Host, environment, and pathogen are extremely important concepts in regards to plant pathology. Agrobacteria have the widest host range of any plant pathogen,[32] so the main factor to take into consideration in the case of crown gall is environment. There are various conditions and factors that make for a conducive environment for A. tumefaciens when infecting its various hosts. The bacterium can't penetrate the host plant without an entry point such as a wound. Factors leading to wounds in plants include cultural practices, grafting, freezing injury, growth cracks, soil insects, and other animals in the environment causing damage to the plant. Consequently, in exceptionally harsh winters, it is common to have an increased incidence of crown gall due to the weather-related damage.[33] Along with this, there are methods of mediating infection of the host plant. For example, nematodes can act as a vector to introduce Agrobacterium into plant roots. More specifically, the root parasitic nematodes damage the plant cell, creating a wound for the bacteria to enter through.[34] Finally, temperature is a factor when considering A. tumefaciens infection. The optimal temperature for crown gall formation due to this bacterium is 22 °C (72 °F) because of the thermosensitivity of T-DNA transfer. Tumor formation is significantly reduced at higher temperature conditions.[35]
Agrobacterium radiobacter (more commonly known as Agrobacterium tumefaciens) is the causal agent of crown gall disease (the formation of tumours) in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA, for 'transfer DNA', not to be confused with tRNA that transfers amino acids during protein synthesis), from a plasmid into the plant cell, which is incorporated at a semi-random location into the plant genome. Plant genomes can be engineered by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors.
Agrobacterium tumefaciens is an Alphaproteobacterium of the family Rhizobiaceae, which includes the nitrogen-fixing legume symbionts. Unlike the nitrogen-fixing symbionts, tumor-producing Agrobacterium species are pathogenic and do not benefit the plant. The wide variety of plants affected by Agrobacterium makes it of great concern to the agriculture industry.
Economically, A. tumefaciens is a serious pathogen of walnuts, grape vines, stone fruits, nut trees, sugar beets, horse radish, and rhubarb, and the persistent nature of the tumors or galls caused by the disease make it particularly harmful for perennial crops.
Agrobacterium tumefaciens grows optimally at 28 °C (82 °F). The doubling time can range from 2.5–4h depending on the media, culture format, and level of aeration. At temperatures above 30 °C (86 °F), A. tumefaciens begins to experience heat shock which is likely to result in errors in cell division.
Agrobacterium tumefaciens estas planto-malsaniga, gramnegativa grundobakterio[1]. Ĝi apartenas al Alfa-klaso de la proteobakterioj.
A. tumefaciens estas model-organismo kaj posedas kapablon transporti DNA en plantan ĉelon. Tiun okazon priskribis unuafoje Jozef Schell.
En la naturo, ĝi malsanigas nur vunditajn dukotiledonajn plantojn kaj estigas ĉe ili trokreskon sur la radikoj. Ĝia genomo estis sekvencite en 2001.
Agrobacterium tumefaciens estas planto-malsaniga, gramnegativa grundobakterio. Ĝi apartenas al Alfa-klaso de la proteobakterioj.
A. tumefaciens estas model-organismo kaj posedas kapablon transporti DNA en plantan ĉelon. Tiun okazon priskribis unuafoje Jozef Schell.
En la naturo, ĝi malsanigas nur vunditajn dukotiledonajn plantojn kaj estigas ĉe ili trokreskon sur la radikoj. Ĝia genomo estis sekvencite en 2001.
Agrobacterium tumefaciens - actualmente denominado Rhizobium radiobacter - es una bacteria que causa en las plantas dicotiledóneas unos tumores conocidos como "agallas" o "tumores del cuello", que crecen en la zona donde se unen la raíz y el tallo (cuello).
Agrobacterium es una proteobacteria alpha de la familia Rhizobiaceae, la cual también incluye a las fijadoras de nitrógeno que viven en simbiosis con las legumbres. A diferencia de éstas, Agrobacterium es un parásito y causa grave daño a la planta afectada.
Es de notar que Agrobacterium no es ni el único ni el más común causante de tumores en las plantas. Muchos pueden ser causados por insectos o larvas que segregan ciertas sustancias que producen el mismo efecto aparente.
La bacteria se guía por sustancias que la planta expulsa (fenoles; entre los más conocidos se encuentran la acetosiringona, la vainillina y el ácido p-hidroxibenzoico) a través de heridas pequeñas, por las que se introduce. Se ubica entonces en los espacios intercelulares y desde allí transfiere a las células de la planta un fragmento de su material genético, un plásmido (ADN circular extracromosómico bacteriano) para transferir un segmento de ADN conocido como T-DNA o ADN-T, que se integra en alguna zona del genoma de la planta (el T-DNA no debe confundirse con el tRNA, la molécula adaptadora que acarrea los aminoácidos hacia el ribosoma y está involucrada en la síntesis de proteínas).
Este T-DNA inyectado por el Agrobacterium contiene genes que provocan la producción de reguladores del crecimiento vegetal, de lo cual resulta el desarrollo del tumor. El T-DNA también contiene genes codificadores de enzimas que causan que la planta produzca aminoácidos especializados llamados opinas. Estas son una fuente de energía específica para A. tumefaciens, pero no para otros organismos. Existen gran variedad de opinas, y cada cepa de A. tumefaciens suele dar lugar a un único tipo de opina. Por ejemplo, la cepa C58 produce nopalina como opina.
La capacidad de transmisión de ADN por el Agrobacterium está siendo extensamente explotada en biotecnología, como medio de insertar genes foráneos dentro de las plantas y desarrollar organismos modificados genéticamente. Se trata entonces de un vehículo óptimo para operaciones de ingeniería genética. Entre los transgénicos creados por este método están las plantas productoras del insecticida Bt, una toxina originalmente producida por el Bacillus thuringiensis.
Los laboratorios del proyecto australiano CAMBIA obtuvieron relevancia al desarrollar una versión de Agrobacterium tumefaciens modificada mediante tecnología de código abierto.
Agrobacterium tumefaciens - actualmente denominado Rhizobium radiobacter - es una bacteria que causa en las plantas dicotiledóneas unos tumores conocidos como "agallas" o "tumores del cuello", que crecen en la zona donde se unen la raíz y el tallo (cuello).
Agrobacterium es una proteobacteria alpha de la familia Rhizobiaceae, la cual también incluye a las fijadoras de nitrógeno que viven en simbiosis con las legumbres. A diferencia de éstas, Agrobacterium es un parásito y causa grave daño a la planta afectada.
Es de notar que Agrobacterium no es ni el único ni el más común causante de tumores en las plantas. Muchos pueden ser causados por insectos o larvas que segregan ciertas sustancias que producen el mismo efecto aparente.
Rhizobium radiobacter[1] ou Agrobacterium tumefaciens est une bactérie à coloration de Gram négative trouvée dans les sols. C'est un pathogène des végétaux responsable d'une maladie appelée galle du collet (ou en anglais : crown-gall). A. tumefaciens a été identifiée à partir de galles des marguerites en 1907[2].
Le mécanisme de formation des galles s'apparente à la conjugaison, il est dû à un plasmide bactérien, fragment d'ADN circulaire, appelé plasmide Ti qui rend les bactéries virulentes. Un fragment d'ADN du plasmide Ti, l'ADN-T est transféré de la bactérie vers la plante, puis intégré dans le génome végétal où il induit la formations des galles caractérisés par la multiplication anarchique des cellules végétales. Agrobacterium tumefaciens infecte essentiellement les dicotylédones à la faveur d'une blessure.
Cette observation constitue le fondement d'une des techniques les plus utilisées en ingénierie génétique des végétaux pour produire des organismes génétiquement modifiés.
La classification de cette bactérie, appartenant à un groupe présentant des particularités complexes, a été reconsidérée plusieurs fois. D'abord baptisée Bacterium tumefaciens par ses découvreurs de 1907[2], elle a été ensuite introduite dans le genre Agrobacterium, créé en 1942. À partir de 1993, des études phylogénétiques de l'ADN de différentes souches d'Agrobacterium et de Rhizobium aboutissent à la considération que les deux genres sont entrelacés et ne peuvent être distingués[3], formant un groupe d'espèces proches. En 2001, une autre étude considérait qu'Agrobacterium tumefaciens ne devait pas être considérée comme une espèce à part entière, mais comme une forme pathogène (parce que contenant certains plasmides) d'Agrobacterium radiobacter, décrite 1902 par Beijerinck et van Delden dans un contexte de symbiose racinaire permettant la fixation de l'azote, et seule espèce non phytopathogène du genre. Elle rejetait par conséquent la dénomination d'Agrobacterium tumefaciens, proposant de retenir radiobacter en tant qu'épithète d'origine, et Rhizobium pour le genre[4]. Ces conclusions taxonomiques ont été contestées en 2003[5], sur la base de différences moléculaires entre les "biovars", groupes identiques génétiquement mais présentant des caractéristiques biochimiques ou physiologiques différentes[6]. La problématique porte à la fois sur le statut des biovars, le rôle d'éléments génétiques transmissibles pour la classification, et le fait d'admettre ou non une nomenclature basée sur des propriétés particulières ("special purpose nomenclature")[7].
Des études plus récentes tendent vers la préservation du genre Agrobacterium[8]. Les progrès de l'analyse génomique ont abouti à améliorer en 2018-2019 l'analyse du génome de Agrobacterium radiobacter de 2014[9], qui aurait été réalisée sur des échantillons contaminés, et de considérer A. tumefaciens comme une sous-espèce d'Agrobacterium radiobacter. Les deux sous-espèces seraient appelées respectivement Agrobacterium radiobacter subsp. radiobacter, et Agrobacterium radiobacter subsp. tumefaciens[10].
"Agrobacterium" est formé de "bacterium", latin scientifique issu du grec, βακτηρία, baktêria, bâton, pour leur forme au microscope, de bâtonnet, et agro-, relatif aux champs, la bactérie vivant dans le sol. "Tumefaciens" signifie qui produit des tumeurs (de tumor, en latin, gonflement), pour désigner les galles qu'elle produit.
"Rhizobium" vient du grec ῥίζα, riza (« racine »), et de βίος, bíos (« vie »), qui renvoie à l'association symbiotique caractéristique de ce genre.
"Radiobacter" est construit à partir de radio-, lié aux rayons, et "bacter" pour bactérie (cf. ci-dessus).
Le génome d' Agrobacterium tumefaciens (souche de référence C58, séquencée en 2001) se compose de deux chromosomes, l'un linéaire de 2,8 Mb contenant une origine de réplication plasmidique et l'autre circulaire de 2 Mb portant une origine de réplication de type cori. A. tumefaciens C58 porte également deux plasmides AtC58 et TiC58 (le plasmide Ti) respectivement de 540 Kb et 214 Kb[11].
La bactérie Agrobacterium tumefaciens infecte les végétaux (essentiellement des dicotylédones) à la faveur d'une blessure. Des composés phénoliques produits par la plante - généralement bactériostatiques ou antibiotiques - attirent au contraire Agrobacterium vers le site de la blessure. La bactérie s'attache aux cellules végétales. Sous l'action de ces composés phénoliques, Agrobacterium met en place un système de transfert d'un fragment de son ADN, vers la cellule blessée. Cet ADN, dit ADN-T (ou T-DNA en anglais), est porté par le plasmide Ti (tumor-inducing) et s'intègre au génome nucléaire de la cellule végétale. Les gènes portés par l'ADN-T s'expriment dans le végétal et conduisent:
Les opines sont spécifiquement utilisées par les agrobactéries qui ont induit la formation de la tumeur. Cette spécificité est liée au fait que les gènes déterminant l'utilisation des opines sont portés par le plasmide Ti. De plus, certaines opines induisent le transfert du plasmide Ti d'une agrobactérie vers une autre par conjugaison. Les opines sont donc des médiateurs chimiques clefs de l'interaction Agrobacterium - plante, dont la présence dans la tumeur favorise la croissance des pathogènes et concourt à leur dissémination.
L'interaction Agrobacterium - plante peut donc être vue comme une manipulation génétique naturelle au cours de laquelle la bactérie détourne à son profit l'activité métabolique de la plante.
La bactérie perçoit les signaux phénoliques (qui sont émis par la plante) grâce à une protéine transmembranaire codée par le gène virA (1). La protéine VirA, possède une activité kinase, s'autophosphoryle et transfère le phosphate à une autre protéine, cytosolique cette fois et codée par virG (2). Cette dernière active la transcription des gènes de virulence (3).
La plante blessée émet également des signaux glucidiques, qui sont captés par une protéine codée par chvE. Celle-ci active VirA et la rend réceptive à des concentrations phénoliques faibles. L'augmentation du pH du milieu est captée par ChvI, qui active également les gènes de virulence.
Les gènes « vir » synthétisent plusieurs protéines dont une, VirD2, reconnaît les séquences double brin spécifiques des bordures droite et gauche de l'ADN-T. VirD1 ouvre les doubles brins, qui sont restreints (coupés) par VirD2; l'ADN-T est donc excisé du plasmide (4). La protéine VirD2 reste fixée sur la bordure droite de l'ADN-T, ce qui l'oriente pour sortir de la bactérie. Cette sécrétion s'effectue par un pilus protéique reliant la bactérie à la cellule végétale (5). Cet appareil de sécrétion (dit système de sécrétion de type IV) est essentiellement codé par les gènes de l'opéron virB, et par virD4 Il a été proposé que le complexe nucléoprotéique VirD2-ADN-T soit recruté par la protéine de couplage VirD4 pour traverser la membrane interne puis serait prix en charge par le complexe composé des produits des gènes virB [12]. Avant ou après son entrée dans la cellule (selon les auteurs), l'ADN-T est recouvert de plusieurs monomères de la protéine VirE2. Celle-ci est également d'origine bactérienne, codée par le gène virE2, et permet de protéger l'ADN-T monocaténaire de la dégradation dans la cellule végétale. VirD2 et VirE2 possèdent des séquences signal de localisation nucléaire dites NLS (pour Nuclear Localisation Signal) qui sont reconnues par la cellule végétale, permettant d'adresser le complexe nucléoprotéique ADN-T/VirE2/VirD2 vers le noyau de la cellule végétale (6). Ce complexe pénètre donc dans le noyau, où il s'intègre à l'ADN végétal. Les ADN polymérases du végétal l'utilisent comme matrice, et le transforment en ADN double brin (7).
Les gènes codés par l'ADN-T ne sont généralement pas exprimés dans une cellule procaryote, donc chez Agrobacterium.
Le génie génétique végétal repose en grande partie sur l'utilisation d' Agrobacterium comme vecteur naturel de gènes. La méthode est appelée "Agrobacterium tumefaciens-Mediated Transformation" (ATMT). Les biotechnologistes utilisent le plus souvent des plasmides proches du plasmide Ti, dit plasmides désarmés, car leur ADN-T ne porte plus les gènes responsables du pouvoir pathogène. Sur ces plasmides désarmés, les gènes tumoraux sont en effet remplacés par un (ou plusieurs) gène d’intérêt agronomique (par exemple le gène codant la toxine de Bacillus thuringiensis dit gène Bt, ou des gènes de résistance à l'herbicide non sélectif glyphosate) et par un ou plusieurs gènes marqueurs permettant de sélectionner les cellules transformées, puis de les multiplier sur des milieux de culture, in vitro. Des plantes entières sont ensuite régénérées par des techniques classiques de culture in vitro, faisant intervenir les hormones végétales auxine et cytokinine.
Une étude réalisée en 2003 montre que Agrobacterium tumefaciens présente les caractéristiques d'un acidocalcisome dans sa structure, organite que l'on pensait n'exister que chez les archées et les eucaryotes. Cette découverte est la première à montrer une caractéristique commune à tous les règnes du vivant.
Parmi les genres de plantes qui peuvent être touchées par Agrobacterium tumefaciens, toutes des dicotylédones, on peut mentionner[13]:
Rhizobium radiobacter ou Agrobacterium tumefaciens est une bactérie à coloration de Gram négative trouvée dans les sols. C'est un pathogène des végétaux responsable d'une maladie appelée galle du collet (ou en anglais : crown-gall). A. tumefaciens a été identifiée à partir de galles des marguerites en 1907.
Le mécanisme de formation des galles s'apparente à la conjugaison, il est dû à un plasmide bactérien, fragment d'ADN circulaire, appelé plasmide Ti qui rend les bactéries virulentes. Un fragment d'ADN du plasmide Ti, l'ADN-T est transféré de la bactérie vers la plante, puis intégré dans le génome végétal où il induit la formations des galles caractérisés par la multiplication anarchique des cellules végétales. Agrobacterium tumefaciens infecte essentiellement les dicotylédones à la faveur d'une blessure.
Cette observation constitue le fondement d'une des techniques les plus utilisées en ingénierie génétique des végétaux pour produire des organismes génétiquement modifiés.
Agrobacterium tumefaciens (á que recentemente se lle cambiou o nome a Rhizobium radiobacter)[2][3] é unha especie bacteriana que causa a formación de tumores nunhas 140 especies de plantas eudicotiledóneas. Ten forma de bacilo, é gramnegativa e vive no solo.[1] A doenza orixínase na planta pola inserción no xenoma da planta dun pequeno segmento de ADN (chamado ADN-T, ou ADN de transferencia), procedente dun plásmido da bacteria,[4] chamado plásmido Ti, que se incorpora de xeito semialeatorio no xenoma da planta.
A. tumefaciens é unha alfaproteobacteria da familia Rhizobiaceae, na que se inclúen tamén os simbiontes de legumes fixadores de nitróxeno. A diferenza dos simbiontes fixadores de nitróxeno, as especies de Agrobacterium que producen tumores son patóxenas e non benefician en absoluto á planta. A ampla variedade de plantas afectadas por Agrobacterium fai que esta bacteria sexa unha importante preocupación para a agricultura.[5] Economicamente, A. tumefaciens é un importante patóxeno das nogueiras, vides, Prunus, remolachas, ravos picantes (Armoracia rusticana), e ruibarbos.
Para ser virulenta, esta bacteria debe conter un plásmido indutor de tumores (plásmido Ti ou pTi), de 200 kb, que contén o ADN-T (o ADN que se transfire) e todos os xenes necesarios para transferilo á célula da planta. Moitas cepas de A. tumefaciens non conteñen o pTi.
Como o plásmido Ti é esencial para causar enfermidades na planta, os eventos de prepenetración que teñen lugar an rizosfera promoven a conxugación bacteriana, na que as bacterias intercambian plásmidos entre elas. En presenza de substancias chamadas opinas, A. tumefaciens produce un sinal de conxugación difusible chamado 30C8HSL ou autoindutor de Agrobacterium. Este activa o factor de transcrición TraR, regulando positivamente a transcrición dos xenes necesarios para a conxugación.
A. tumefaciens infecta a planta por medio dos seus plásmidos Ti. O plásmido Ti integra un segmento do seu ADN, chamado ADN-T, no ADN dun cromosoma da célula hóspede da planta. A. tumefaciens ten flaxelos que lle permiten nadar polo solo dirixíndose a onde hai moléculas producidas na fotosíntese (fotoasimilados) que se acumulan na rizosfera arredor das raíces. Algunhas cepas poden moverse quimiotacticamente cara aos exsudados químicos das plantas, como a acetosiringona e azucres. A acetosiringona é recoñecida pola proteína VirA, unha proteína transmembrana codificada no xene virA do plásmido Ti. Os azucres son recoñecidos pola proteína chvE, unha proteína codificada no cromosoma da bacteria presente no espazo periplásmico.[6]
Para a indución de tumores cómpren polo menos 25 xenes vir do plásmido Ti. Ademais do seu papel de percepción de compostos, virA e chvE inducen outros xenes vir. A proteína virA ten actividade de autoquinase, xa que se autofosforila nun residuo de histidina. Despois, a proteína virA fosforila a proteína virG no seu residuo aspartato. A proteína virG é unha proteína citoplasmática producida a partir do xene virG do plásmido Ti. É un factor de transcrición, que induce a transcrición dos operóns vir. A proteína chvE regula o segundo mecanismo da activación dos xenes vir. Incrementa a sensibilidade da proteína VirA a compostos fenólicos.[6]
A adhesión á planta é un proceso que ten lugar en dúas etapas. Despois dunha adhesión inicial feble e reversible, a bacteria sintetiza fibrilas de celulosa que ancoran a bacteria á planta con feridas á cal foi atraída. Neste proceso están implicados catro xenes principais, que son: chvA, chvB, pscA, e att. Os produtos dos primeiros tres xenes están implicados aparentemente na propia síntese das fibrilas de celulosa. Estas fibrilas tamén serven para ancorar as bacterias unhas a outras, o que facilita a formación dunha microcolonia.
O xene VirC é o xene de virulencia máis importante, e a súa intervención é un paso necesario na recombinación. Selecciona a sección do ADN na planta hóspede que será substituída, e corta nesa febra do ADN.
Despois da produción de fibrilas de celulosa, prodúcese unha proteína da membrana externa dependente do calcio chamada ricadhesina, que tamén contribúe a adherir a bacteria á parede celular. Poden atoparse homólogos desta proteína noutros rizobios.
Posibles compostos da planta que inician Agrobacterium para infectar células da planta son:[7]
Para transferir o ADN-T á célula da planta, A. tumefaciens utiliza un mecanismo de secreción de tipo IV[8], no que se ten que orixinar un pilus T. Cando se detecta a acetosiringona e outras substancias, desencadéase unha transdución de sinais que activa a expresión de 11 xenes situados no operón VirB, os cales son responsables da formación do pilus T.
Primeiramente, fórmase a pro-pilina, que é un polipéptido de 121 aminoácidos que debe ser procesado eliminándolle 47 residuos para así formar unha subunidade de pilus T. A subunidade circularízase ao formarse un enlace peptídico entre os dous extremos do polipéptido.
Os produtos dos outros xenes VirB utilízanse para transferir as subunidades a través da membrana plasmática. Estudos feitos en sistemas de dobre híbrido en lévedos proporcionaron probas de que VirB6, VirB7, VirB8, VirB9 e VirB10 poden codificar compoñentes do transportador. Cómpre tamén a actuación dunha ATPase para o transporte activo das subunidades.
O ADN-T debe ser cortado e escindido do plásmido circular. O complexo VirD1/D2 fai un corte no ADN nas secuencias dos extremos esquerdo e dereito do ADN-T. A proteína VirD2 únese covalentemente ao extremo 5'. A VirD2 contén un motivo que fai que o complexo nucleoproteico se una ao sistema de secreción de tipo IV (T4SS).
No citoplasma da célula receptora, o complexo ADN-T queda cuberto por proteínas VirE2, as cales son exportadas a través do sistema T4SS independentemente do complexo ADN-T. Uns sinais de localización nuclear (NLS), localizados no VirE2 e VirD2, son recoñecidos pola proteína importina alfa, a cal despois se asocia coa importina beta e co complexo do poro nuclear para transferir o ADN-T ao interior do núcleo. O VIP1 tamén parece ser unha proteína importante no proceso, que posiblemente actúa como un adaptador para traer a VirE2 á importina. Unha vez dentro do núcleo, a VIP2 pode levar o ADN-T a áreas da cromatina que están sendo transcritas activamente, para que así o ADN-T poida integrarse no xenoma do hóspede.
Para causar a formación dun tumor na planta, o ADN-T codifica xenes para a produción das hormonas auxinaa (xeralmente ácido indol-3-acético) por medio da vía IAM. Esta vía biosintética non é a utilizada por moitas plantas para a produción de auxina, polo que isto significa que a planta non ten ningunha maneira molecular de regulala e a auxina prodúcese de forma continua (constitutivamente). Tamén se expresan os xenes para a produción de citocininas. Isto estimula a proliferación celular e a formación do tumor.
O ADN-T contén xenes que codifican encimas que causa que a planta produza derivados de aminoácidos especializados que a bacteria pode metabolizar, chamadas opinas.[9] As opinas son unha clase de compostos químicos que serven como fonte de nitróxeno para A. tumefaciens, pero non para a maioría dos demais organismos. O tipo específico de opina producido polas plantas infectadas por A. tumefaciens C58 é a nopalina.[10]
Secuenciáronse completamente dous plásmidos Ti de tipo nopalina, que son: pTi-SAKURA e pTiC58. A. tumefaciens C58 foi o primeiro patovar secuenciado totalmente, e foi illado por primeira vez dos tumores das cereixeiras. O xenoma foi secuenciado simultaneamente por Goodner et al.[11] e Wood et al.[12] en 2001. O xenoma de A. tumefaciens C58 consta dun cromosoma circular, dous plásmidos, e un cromosoma linear. A presenza dun cromosoma circular pechado por enlaces covalentes é o normal nas bacterias, con poucas excepcións. Porén, a presenza á vez dun cromosoma circular e outro liñal é exclusiva dun grupo deste xénero. Os dous plásmidos son pTiC58, responsable dos procesos implicados na virulencia, e o pAtC58, denominado o plásmido "críptico".[11][12]
O plásmido pAtC58 está implicado no metabolismo das opinas e na conxugación con outras bacterias en ausencia do plásmido pTiC58.[13] Se o plásmido pTi é eliminado, non se produce o crecemento do tumor, que é a característica para clasificar esta especie de bacterias.
A capacidade de transmisión de ADN de Agrobacterium foi exhaustivamente explorado en biotecnoloxía como un medio para inserir xenes alleos en plantas. Marc Van Montagu e Jeff Schell, (da Universidade de Gante e Plant Genetic Systems, Bélxica) descubriron o mecaismo de transferencia entre Agrobacterium e plantas, o cal deu lugar ao desenvolvemento de métodos para alterar a bacteria xerando un sistema de traspaso do xene para modificar plantas por enxeñaría xenética.[14] O ADN-T de plásmidos que se transfire á planta é un vehículo ideal para o seu uso en enxeñaría xenética.[15] Isto faise clonando a secuencia do xene desexado no ADN-T que se inserirá no ADN do hóspede. Este proceso realizouse usando o xene da luciferase do vagalume para producir un resplandor na planta. Esta produción de luminescencia foi unha ferramenta útil no estudo da función do cloroplasto da planta e como xene reporteiro.[16] É tamén posible transformar a planta Arabidopsis thaliana mergullando as súas flores nun caldo de Agrobacterium; deste xeito, as sementes producidas nas flores serán transxénicas. Baixo as condicións de laboratorio, o ADN-T foi tamén transferido a células humanas, demostrando a versatilidade das aplicacións da inserción.[17]
O mecanismo polo cal Agrobacterium insire materiais na célula hóspede por un sistema de secreción de tipo IV é moi similar aos mecanismos usados polos patóxenos para inserir materiais (xeralmente proteínas) en células humanas por secreción de tipo III. Tamén emprega un tipo de sinalización conservado en moitas bacterias gramnegativas chamado percepción do quórum. Isto fai que Agrobacterium sexa tamén un importante obxecto de estudo na investigación médica.
Webster, Thomson, Jocelyn, Jennifer (1988). "Genetic Analysis of an Agrobacterium Tumefaciens strain producing an agrocin active against biotype 3 Pathogen". Molecular and General Genetics 214 (1): 142-147. doi:10.1007/BF00340192.
Agrobacterium tumefaciens (á que recentemente se lle cambiou o nome a Rhizobium radiobacter) é unha especie bacteriana que causa a formación de tumores nunhas 140 especies de plantas eudicotiledóneas. Ten forma de bacilo, é gramnegativa e vive no solo. A doenza orixínase na planta pola inserción no xenoma da planta dun pequeno segmento de ADN (chamado ADN-T, ou ADN de transferencia), procedente dun plásmido da bacteria, chamado plásmido Ti, que se incorpora de xeito semialeatorio no xenoma da planta.
A. tumefaciens é unha alfaproteobacteria da familia Rhizobiaceae, na que se inclúen tamén os simbiontes de legumes fixadores de nitróxeno. A diferenza dos simbiontes fixadores de nitróxeno, as especies de Agrobacterium que producen tumores son patóxenas e non benefician en absoluto á planta. A ampla variedade de plantas afectadas por Agrobacterium fai que esta bacteria sexa unha importante preocupación para a agricultura. Economicamente, A. tumefaciens é un importante patóxeno das nogueiras, vides, Prunus, remolachas, ravos picantes (Armoracia rusticana), e ruibarbos.
Agrobacterium tumefaciens adalah bakteri patogen pada tanaman yang banyak digunakan untuk memasukkan gen asing ke dalam sel tanaman untuk menghasilkan suatu tanaman transgenik.[1] Bakteri ini dipopulerkan oleh Mary-Dell Chilton sejak tahun 1977 melalui penelitiannya. Secara alami, A. tumefaciens dapat menginfeksi tanaman dikotiledon melalui bagian tanaman yang terluka sehingga menyebabkan tumor mahkota empedu (crown gall tumor).[1] Bakteri yang tergolong ke dalam gram negatif ini memiliki sebuah plasmid besar yang disebut plasmid-Ti yang berisi gen penyandi faktor virulensi penyebab infeksi bakteri ini pada tanaman.[2] Untuk memulai pembentukan tumor, A. tumefaciens harus menempel terlebih dahulu pada permukaan sel inang dengan memanfaatkan polisakarida asam yang akan digunakan untuk mengkoloniasi/menguasai sel tanaman.[1] Selain tanaman dikotiledon, tanaman monokotiledon seperti jagung, gandum, dan tebu telah digunakan untuk memasukkan sel asing ke dalam genom tanaman.[1] Agrobacterium tumefaciens adalah bakteri patogen pada tanaman yang banyak digunakan untuk memasukkan gen asing ke dalam sel tanaman untuk menghasilkan suatu tanaman transgenik.[1]
Agrobacterium tumefaciens adalah bakteri patogen pada tanaman yang banyak digunakan untuk memasukkan gen asing ke dalam sel tanaman untuk menghasilkan suatu tanaman transgenik. Bakteri ini dipopulerkan oleh Mary-Dell Chilton sejak tahun 1977 melalui penelitiannya. Secara alami, A. tumefaciens dapat menginfeksi tanaman dikotiledon melalui bagian tanaman yang terluka sehingga menyebabkan tumor mahkota empedu (crown gall tumor). Bakteri yang tergolong ke dalam gram negatif ini memiliki sebuah plasmid besar yang disebut plasmid-Ti yang berisi gen penyandi faktor virulensi penyebab infeksi bakteri ini pada tanaman. Untuk memulai pembentukan tumor, A. tumefaciens harus menempel terlebih dahulu pada permukaan sel inang dengan memanfaatkan polisakarida asam yang akan digunakan untuk mengkoloniasi/menguasai sel tanaman. Selain tanaman dikotiledon, tanaman monokotiledon seperti jagung, gandum, dan tebu telah digunakan untuk memasukkan sel asing ke dalam genom tanaman. Agrobacterium tumefaciens adalah bakteri patogen pada tanaman yang banyak digunakan untuk memasukkan gen asing ke dalam sel tanaman untuk menghasilkan suatu tanaman transgenik.
L'Agrobacterium tumefaciens (nome scientifico aggiornato Rhizobium radiobacter[1]) è un batterio gram negativo, di forma bastoncellare, capace di infettare le piante attraverso la trasmissione di un segmento di DNA, definito T-DNA, che penetra all'interno delle cellule vegetali integrandosi nel loro genoma. E' un proteobatterio della famiglia delle Rhizobiaceae, la stessa cui appartengono molti batteri azotofissatori simbionti delle piante, ma a differenza di questi ultimi, tuttavia, si comporta da parassita; in questi termini, ha una notevole importanza in campo economico, in grado di arrecare danni a coltivazioni quali quelle della vite. È capace di infettare principalmente le dicotiledoni e causare in esse una crescita paragonabile a quella tumorale, una patologia nota con il nome di galla del colletto.
Dal punto di vista genetico, la principale peculiarità del genoma di A. tumefaciens è quella di possedere un plasmide, chiamato plasmide Ti (Tumor Inducing), di circa 180 kb (migliaia di coppie di basi), che codifica il T-DNA e tutti i geni necessari a trasferirlo nella cellula vegetale. La presenza del plasmide è necessaria perché avvenga l'infezione: cellule batteriche prive del plasmide non danno origine alla patologia. Il batterio è quindi detto oncogeno o virulento.
Poiché la presenza del plasmide è necessaria per l'infezione, prima dell'infezione stessa avvengono normalmente, nella rizosfera della pianta, numerosi eventi di coniugazione. Tali eventi sono mediati dalla presenza di opine: questi fattori stimolano la produzione, da parte dei batteri stessi, di una molecola segnale chiamata 30C8HSL (o autoinduttore) la quale attiva a sua volta il fattore di trascrizione TraR, che regola la trascrizione dei geni responsabili della coniugazione.
Il T-Dna è una piccola porzione di DNA che comprende 8 geni, contenuta all'interno del plasmide Ti, che penetra all'interno della cellula vegetale per dare il via all'infezione. Sebbene il T-DNA da solo non sia in grado di provocare la malattia (in quanto sono necessari anche i geni vir presenti sul plasmide, oltre che alcuni geni del cromosoma batterico), esso codifica per i geni responsabili della formazione del tumore nella pianta, la cosiddetta galla. Il T-DNA codifica inoltre per alcuni ormoni vegetali, come l'auxina e la citochinina, oltre che per le opine.
L'infezione di una pianta da parte di A. tumefaciens è un processo che consta di diversi eventi consecutivi: inizialmente avviene l'attacco dei batteri ai tessuti della pianta, generalmente a livello delle radici, attraverso delle lesioni preesistenti; successivamente avviene il trasferimento di materiale genetico dalle cellule batteriche a quelle vegetali, con modalità fondamentalmente simili a quelle della coniugazione batterica. Una volta che il DNA batterico (il T-DNA) si è integrato nel genoma della cellula della pianta, questa viene spinta a produrre enzimi ed ormoni che aiutano la crescita e la proliferazione del batterio, provocando al contempo la formazione della escrescenza tumorale nella pianta infetta.
Le cellule di A. tumefaciens possiedono dei flagelli, tramite i quali possono muoversi attraverso il suolo; questi batteri vengono solitamente attratti da segnali chimici rilasciati dai tessuti vegetali in caso di lesione e si radunano perciò in prossimità di ferite nei tessuti vegetali esterni delle piante (un processo noto come chemiotassi).
A questo punto avviene l'attacco vero e proprio: questo è un processo in due fasi, la prima delle quali vede la sintesi, da parte dei batteri, di sottili fibrille di cellulosa, che ancorano i batteri stessi alla parete della pianta ferita. Queste fibrille legano inoltre i batteri tra loro, contribuendo a formare una colonia; i geni coinvolti nella sintesi delle fibrille sono quattro, localizzati sul cromosoma batterico: chvA, chvB, pscA e att.
Il processo di trasmissione del T-DNA all'interno della cellula vegetale richiede la formazione di un pilus, detto Pilus-T (una struttura filamentosa proteica con funzione di ancoraggio, simile al pilus F che si forma durante la coniugazione batterica); tale meccanismo è definito "meccanismo di secrezione di tipo IV". L'intero processo di trasferimento genetico è sotto il controllo di un gruppo di geni, localizzati sul plasmide batterico (ma non a livello del T-DNA) chiamati complessivamente vir, ed identificati con una lettera (VirA, VirB, VirG, VirC, VirD, VirE). L'attivazione dei vari geni è interdipendente, e l'evento iniziale è il legame di composti fenolici, rilasciati dalle cellule vegetali nelle zone sottoposte a lesione, al sensore a due componenti VirA/VirG; questo legame attiva un processo di trasduzione del segnale che porta a sua volta all'attivazione dell'operone VirB, responsabile della produzione del pilus-T. La prima proteina ad essere sintetizzata dall'operone è la pro-pilina, un polipeptide di 121 amminoacidi, che viene processato tramite eventi post-traduzionali fino ad essere trasformato in pilina, la subunità fondamentale del pilus. Il processamento comprende l'eliminazione di 47 amminoacidi, ed il ripiegamento della molecola in una struttura circolare, tramite un legame peptidico tra le estremità del polipeptide.
Il processo di trasferimento del T-DNA all'interno della cellula vegetale può essere diviso in due fasi: la prima consiste nel taglio del T-DNA stesso dal plasmide Ti. Questo evento è catalizzato dai prodotti genici del complesso VirD1/D2; VirD1 riconosce le LB e RB del T-DNA e taglia il singolo filamento a livello della RB al 5' del T-DNA, quindi si lega VirD2 che taglia LB al 3'. Il 3' è libero e parte la sintesi del DNA. Il T-DNA viene poi legato da proteina quali VirE e VirD2. VirD2 è inoltre necessaria in quanto viene riconosciuta dal complesso trasportatore del pilus.
La seconda fase consiste nel trasporto del T-DNA all'interno del nucleo della cellula vegetale: questo processo richiede ancora la presenza di VirE e VirD2, che, legati al DNA stesso, vengono riconosciuti dalla proteina importina all'interno della cellula vegetale stessa. Questa conduce il T-DNA all'interno del nucleo cellulare, dove altre proteine, chiamate VIP1 e VIP2 permetto l'integrazione nel genoma ospite. A questo punto il T-DNA viene attivamente trascritto e tradotto, dando origine alla patologia vegetale vera e propria.
Il batterio Agrobacterium tumefaciens è utilizzato in laboratorio per ottenere l'impianto di DNA ricombinante all'interno di piante dicotiledoni (mentre per le monocotiledoni si utilizzano bombardamenti con particelle di tungsteno).
L'Agrobacterium tumefaciens (nome scientifico aggiornato Rhizobium radiobacter) è un batterio gram negativo, di forma bastoncellare, capace di infettare le piante attraverso la trasmissione di un segmento di DNA, definito T-DNA, che penetra all'interno delle cellule vegetali integrandosi nel loro genoma. E' un proteobatterio della famiglia delle Rhizobiaceae, la stessa cui appartengono molti batteri azotofissatori simbionti delle piante, ma a differenza di questi ultimi, tuttavia, si comporta da parassita; in questi termini, ha una notevole importanza in campo economico, in grado di arrecare danni a coltivazioni quali quelle della vite. È capace di infettare principalmente le dicotiledoni e causare in esse una crescita paragonabile a quella tumorale, una patologia nota con il nome di galla del colletto.
근두암종병(根頭癌腫病, crown gall)은 배, 사과, 장미 등에 주로 생기는 병이다. 뿌리혹병이라고도 한다. 원인이 되는 병원균은 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens)다. 감염된 식물들은 증식하지 못하고 심해지면 죽는다.[1]
증상으로는 뿌리줄기와 잎 등에 혹이 생기며 이상증식한다.
첫 번째로 토양에 의해 전염된다. 두 번째로는 세포 분열로 증식하며 뿌리혹을 섭취한 곤충에 의해 전염되기도 한다.