Agrobacterium tumefaciens#Biotechnological uses

The classification of Agrobacterium tumefaciens and related species, collectively the Agrobacterium tumefaciens species complex, has greatly outpaced the change in terminology employed by plant scientists.

Before 1980 the division of Agrobacterium largely reflected disease symptomology and host range. A. radiobacter is defined as the "avirulent" species, A. tumefaciens the one causing crown gall, A. rhizogenes causing hairy root disease, and A. rubi causijng cane gall.

With the discovery of the Ti plasmid it was realized that symptomology mostly depend on the particular version of the plasmid carried, not anything that resembles a biological species concept. By 2000, the "biovar" concept, using growth and metabolic characteristics, had divided Agrobacterium into three biovars later shown to be mostly congruent with genetic differentiation. Biovar 1 would remain in Agrobacterium, biovar 2 to Rhizobium rhizogenes, and biovar 3 to Allorhizobium vitis. By 2014 there is very little, if any, confusion for what Agrobacterium in the strict sense would refer to.{{cite journal |vauthors = Ramírez-Bahena MH, Vial L, Lassalle F, Diel B, Chapulliot D, Daubin V, Nesme X, Muller D | year = 2014 | title = Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid | journal = Mol Phylogenet Evol | volume = 73 | pages = 202–207 | pmid = 24440816 | doi = 10.1016/j.ympev.2014.01.005| bibcode = 2014MolPE..73..202R }}

However, another issue remains with the classification inside of biovar 1, specifically inside the Agrobacterium tumefaciens species complex, where biological species remain hard to differentiate without DNA sequencing. Researchers largely still stuck to the old nomenclature based on symptomology, save for a few who take the time to delimit the "genomovars" or "genomospecies" inside of this complex.{{cite journal |last1=Vargas Ribera |first1=PR |last2=Kim |first2=N |last3=Venbrux |first3=M |last4=Álvarez-Pérez |first4=S |last5=Rediers |first5=H |title=Evaluation of sequence-based tools to gather more insight into the positioning of rhizogenic agrobacteria within the Agrobacterium tumefaciens species complex. |journal=PLOS ONE |date=2024 |volume=19 |issue=11 |pages=e0302954 |doi=10.1371/journal.pone.0302954 |doi-access=free |pmid=39561304|pmc=11575935 |bibcode=2024PLoSO..1902954V }} To add to the confusion, the Approved Lists of 1980 changed the type strain of A. tumefaciens without explanation to "B6", a strain now properly classified as Agrobacterium radiobacter (genomovar 4), causing misled researchers to propose the synonimization of the two. The original type strain of A. tumefaciens, reinstated in 2023, belongs to genomovar 1.

Another strain of "A. tumefaciens" commonly used in early research was C58, which belongs to genomovar 8. For a review of the currently-known structure of the species complex, see Vargas Ribera et al. (2024), which also lists names that have been separately proposed for the genomovars.

This article cites a great number of sources that do not distinguish among the genomovars. Most text in this article should be treated as describing the species complex as a whole.

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.{{cite journal | vauthors = Gordon JE, Christie PJ | title = The Agrobacterium Ti Plasmids | journal =Microbiology Spectrum| volume = 2 | issue = 6 | date = December 2014 | pmid = 25593788 | pmc = 4292801 | doi = 10.1128/microbiolspec.PLAS-0010-2013 }} 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 N-(3-oxo-octanoyl)-L-homoserine lactone (3OC8HSL) or the Agrobacterium autoinducer.{{Cite journal |last1=Oger |first1=Philippe |last2=Farrand |first2=Stephen K. |date=2002 |title=Two Opines Control Conjugal Transfer of an Agrobacterium Plasmid by Regulating Expression of Separate Copies of the Quorum-Sensing Activator Gene traR |journal=Journal of Bacteriology |language=en |volume=184 |issue=4 |pages=1121–1131 |doi=10.1128/jb.184.4.1121-1131.2002 |issn=0021-9193 |pmc=134798 |pmid=11807073}} This activates the transcription factor TraR, positively regulating the transcription of genes required for conjugation.{{Cite journal |last1=Zhang |first1=Hai-Bao |last2=Wang |first2=Lian-Hui |last3=Zhang |first3=Lian-Hui |date=2002 |title=Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens |journal=Proceedings of the National Academy of Sciences |language=en |volume=99 |issue=7 |pages=4638–4643 |doi=10.1073/pnas.022056699 |doi-access=free |issn=0027-8424 |pmc=123700 |pmid=11930013|bibcode=2002PNAS...99.4638Z }}

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.{{cite journal |last=Gelvin |first=Stanton B |date=March 2003 |title=Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool |url= |journal=Microbiology and Molecular Biology Reviews |volume=67 |issue=1 |pages=16–37, table of contents |doi=10.1128/mmbr.67.1.16-37.2003 |pmc=150518 |pmid=12626681}}

At least 25 vir genes on the Ti plasmid are necessary for tumor induction.{{Cite journal |last=Winans |first=Stephen C. |date=1992 |title=Two-way chemical signaling in Agrobacterium-plant interactions |journal=Microbiological Reviews |language=en |volume=56 |issue=1 |pages=12–31 |doi=10.1128/mr.56.1.12-31.1992 |issn=0146-0749 |pmc=372852 |pmid=1579105}} 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.

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.{{cn|date=February 2023}}

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.{{cn|date=February 2023}}

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).{{cite journal |vauthors=Barate PL, Kumar RR, Waghmare SG, Pawar KR, Tabe RH |year=2018 |title=Effect of different parameters on Agrobacterium mediated transformation in Glycine max |url=https://www.researchgate.net/publication/323177967 |journal=International Journal of Advanced Biological Research |volume=8 |issue=1 |pages=99–105}}

Possible plant compounds that initiate Agrobacterium to infect plant cells:{{Cite patent|country=US|number=6483013|title=Method for agrobacterium mediated transformation of cotton|status=patent|pubdate=2002-11-19|assign1=Bayer BioScience N.V. (BE)}}

• Acetosyringone and other phenolic compounds
• alpha-Hydroxyacetosyringone
• Catechol
• Ferulic acid
• Gallic acid
• p-Hydroxybenzoic acid
• Protocatechuic acid
• Pyrogallic acid
• Resorcylic acid
• Sinapinic acid
• Syringic acid
• Vanillin

To transfer T-DNA into a 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 thought 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.{{Cite journal |last1=Beltran |first1=Leticia C. |last2=Cvirkaite-Krupovic |first2=Virginija |last3=Miller |first3=Jessalyn |last4=Wang |first4=Fengbin |last5=Kreutzberger |first5=Mark A. B. |last6=Patkowski |first6=Jonasz B. |last7=Costa |first7=Tiago R. D. |last8=Schouten |first8=Stefan |last9=Levental |first9=Ilya |last10=Conticello |first10=Vincent P. |last11=Egelman |first11=Edward H. |last12=Krupovic |first12=Mart |date=2023-02-07 |title=Archaeal DNA-import apparatus is homologous to bacterial conjugation machinery |journal=Nature Communications |volume=14 |issue=1 |pages=666 |doi=10.1038/s41467-023-36349-8 |issn=2041-1723 |pmc=9905601 |pmid=36750723|bibcode=2023NatCo..14..666B }}

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.

[[File:Transfection by Agrobacterium.svg|thumb|300px|right|

{{#invoke:list|ordered|list_style_type=upper-alpha

|Agrobacterium cell

|Agrobacterium chromosome

|Ti Plasmid (a. T-DNA, b. vir genes, c. replication origin, d. opines catabolism)

|Plant cell

|Plant mitochondria

|Plant chloroplast

|Plant nucleus

}}

{{#invoke:list|ordered|

| VirA recognition

| VirA phosphorylates VirG

| VirG causes transcription of Vir genes

| Vir genes cut out T-DNA and form nucleoprotein complex ("T-complex")

| T-complex enters plant cytoplasm through T-pilus

| T-DNA enters into plant nucleus through nuclear pore

| T-DNA achieves integration

}}

]]

The T-DNA must be cut out of the circular plasmid. This is typically done by the Vir genes within the helper plasmid.{{cite web |last=Kroemer |first=Tyasning |title=A Guide to T-DNA Binary Vectors in Plant Transformation |url=https://goldbio.com/articles/article/a-guide-to-agrobacterium-t-dna-binary-vectors#_Toc61524553 |access-date=January 9, 2024 |website=GoldBio}} 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.

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.{{cite journal | vauthors = Zupan J, Muth TR, Draper O, Zambryski P | title = The transfer of DNA from agrobacterium tumefaciens into plants: a feast of fundamental insights | journal =The Plant Journal| volume = 23 | issue = 1 | pages = 11–28 | date = July 2000 | pmid = 10929098 | doi = 10.1046/j.1365-313x.2000.00808.x }} 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.{{Cite journal |last1=Escobar |first1=M. A. |last2=Civerolo |first2=E. L. |last3=Polito |first3=V. S. |last4=Pinney |first4=K. A. |last5=Dandekar |first5=A. M. |date=January 2003 |title=Characterization of oncogene-silenced transgenic plants: implications for Agrobacterium biology and post-transcriptional gene silencing |journal=Molecular Plant Pathology |language=en |volume=4 |issue=1 |pages=57–65 |doi=10.1046/j.1364-3703.2003.00148.x |issn=1464-6722|doi-access=free |pmid=20569363 |bibcode=2003MolPP...4...57E }}

Two nopaline type Ti plasmids, pTi-SAKURA and pTiC58, were fully sequenced. "A. fabrum" C58,{{efn|The sequenced genomes of strain C58 is not sufficiently similar to the type strain (either the old B6 and the new A1) of Agrobacterium tumefaciens to be considered the same species. The name "Agrobacterium fabrum" {{au|Lassalle et al. 2011}} was proposed to contain this strain but the name was not validated.{{cite web |title=Species: Agrobacterium fabrum |url=https://lpsn.dsmz.de/species/agrobacterium-fabrum |website=lpsn.dsmz.de |language=en}}}} the first fully sequenced pathovar, was first isolated from a cherry tree crown gall. The genome was simultaneously sequenced by Goodner et al.{{cite journal |last1=Goodner |first1=Brad |last2=Hinkle |first2=Gregory |last3=Gattung |first3=Stacie |last4=Miller |first4=Nancy |last5=Blanchard |first5=Mary |last6=Qurollo |first6=Barbara |last7=Goldman |first7=Barry S. |last8=Cao |first8=Yongwei |last9=Askenazi |first9=Manor |last10=Halling |first10=Conrad |last11=Mullin |first11=Lori |last12=Houmiel |first12=Kathryn |last13=Gordon |first13=Jeffrey |last14=Vaudin |first14=Mark |last15=Iartchouk |first15=Oleg | s2cid = 86255214 | display-authors = 6 | title = Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58 | journal =Science| volume = 294 | issue = 5550 | pages = 2323–2328 | date = December 2001 | pmid = 11743194 | doi = 10.1126/science.1066803 | bibcode = 2001Sci...294.2323G |issn=0036-8075}} and Wood et al.{{cite journal |last1=Wood |first1=Derek W. |last2=Setubal |first2=Joao C. |last3=Kaul |first3=Rajinder |last4=Monks |first4=Dave E. |last5=Kitajima |first5=Joao P. |last6=Okura |first6=Vagner K. |last7=Zhou |first7=Yang |last8=Chen |first8=Lishan |last9=Wood |first9=Gwendolyn E. |last10=Almeida |first10=Nalvo F. |last11=Woo |first11=Lisa |last12=Chen |first12=Yuching |last13=Paulsen |first13=Ian T. |last14=Eisen |first14=Jonathan A. |last15=Karp |first15=Peter D. | s2cid = 2761564 | display-authors = 6 | title = The genome of the natural genetic engineer Agrobacterium tumefaciens C58 | journal =Science| volume = 294 | issue = 5550 | pages = 2317–23 | date = December 2001 | pmid = 11743193 | doi = 10.1126/science.1066804 | citeseerx = 10.1.1.7.9501 | bibcode = 2001Sci...294.2317W |issn=0036-8075}} in 2001. The genome of strain C58consists 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,{{efn|"At plasmid" or "pAt" when talking about related plasmids}} once dubbed the "cryptic" plasmid.

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.{{cite journal | vauthors = Vaudequin-Dransart V, Petit A, Chilton WS, Dessaux Y | year = 1998 | title = The cryptic plasmid of Agrobacterium tumefaciens cointegrates with the Ti plasmid and cooperates for opine degradation | journal =Molecular Plant-Microbe Interactions | volume = 11 | issue = 7| pages = 583–591 | doi=10.1094/mpmi.1998.11.7.583| doi-access = free | bibcode = 1998MPMI...11..583V }} If the Ti plasmid is removed, the tumor growth that is the means of classifying this species of bacteria does not occur.

{{See also|Agroinfiltration}}

Image:Transformation with Agrobacterium.JPGs]]

The Asilomar Conference in 1975 established widespread agreement that recombinant techniques were insufficiently understood and needed to be tightly controlled.{{Cite journal |last1=Berg |first1=Paul |last2=Baltimore |first2=David |last3=Brenner |first3=Sydney |last4=Roblin |first4=Richard O. |last5=Singer |first5=Maxine F. |date=1975-06-06 |title=Asilomar Conference on Recombinant DNA Molecules |url=https://www.science.org/doi/10.1126/science.1056638 |journal=Science |language=en |volume=188 |issue=4192 |pages=991–994 |doi=10.1126/science.1056638 |pmid=1056638 |bibcode=1975Sci...188..991B |issn=0036-8075|url-access=subscription }}{{cite book | author= ((National Research Council)) | author2= ((Board on Agriculture and Natural Resources)) | author3= ((Committee on Genetically Modified Pest-Protected Plants)) | title=Genetically Modified Pest-Protected Plants: Science and Regulation | publisher=National Academies Press | publication-place=Washington, D.C. | year=2000 | isbn=978-0-309-06930-4 | oclc=894124744 | pages=xxiii, 263 | doi= 10.17226/9795| pmid= 25032472 }} The DNA transmission capabilities of Agrobacterium have been vastly explored in biotechnology as a means of inserting foreign genes into plants. Shortly after the Asilomar Conference, Marc Van Montagu and Jeff Schell 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.{{cite book |last1=Schell |first1=J. |last2=Van Montagu |first2=M. |editor1-last=Hollaender |editor1-first=Alexander |editor2-last=Burris |editor2-first=R. H. |editor3-last=Day |editor3-first=P. R. |editor4-last=Hardy |editor4-first=R. W. F. |chapter = The Ti-Plasmid of Agrobacterium Tumefaciens, A Natural Vector for the Introduction of NIF Genes in Plants? | title = Genetic Engineering for Nitrogen Fixation | series = Basic Life Sciences | volume = 9 | pages = 159–79 | year = 1977 | pmid = 336023 | doi = 10.1007/978-1-4684-0880-5_12 | isbn = 978-1-4684-0882-9 }} The plasmid T-DNA that is transferred to the plant is an ideal vehicle for genetic engineering.{{cite journal | vauthors = Zambryski P, Joos H, Genetello C, Leemans J, Montagu MV, Schell J | title = Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity | journal = The EMBO Journal| volume = 2 | issue = 12 | pages = 2143–50 | year = 1983 | pmid = 16453482 | pmc = 555426 | doi = 10.1002/j.1460-2075.1983.tb01715.x }} 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. This process has been performed using the 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.{{cite journal | vauthors = Root M | year = 1988 | title = Glow in the dark biotechnology | journal =BioScience| volume = 38 | issue = 11| pages = 745–747 | doi=10.2307/1310781| jstor = 1310781 | doi-access = free }} 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, T-DNA has also been transferred to human cells, demonstrating the diversity of insertion application.{{cite journal | vauthors = Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V | title = Genetic transformation of HeLa cells by Agrobacterium| journal =Proceedings of the National Academy of Sciences of the United States of America| volume = 98 | issue = 4 | pages = 1871–6 | date = February 2001 | pmid = 11172043 | pmc = 29349 | doi = 10.1073/pnas.041327598 | bibcode = 2001PNAS...98.1871K | doi-access = free }}

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.{{citation needed|date=December 2019}} This makes Agrobacterium an important topic of medical research, as well.{{citation needed|date=December 2019}}

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.{{cite journal | vauthors = Demanèche S, Kay E, Gourbière F, Simonet P | title = Natural transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in soil | journal =Applied and Environmental Microbiology| volume = 67 | issue = 6 | pages = 2617–21 | date = June 2001 | pmid = 11375171 | pmc = 92915 | doi = 10.1128/AEM.67.6.2617-2621.2001 | bibcode = 2001ApEnM..67.2617D }}

File:A tumefaciens disease cycle.jpg]]

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.{{cite journal| vauthors = Schroth MN, Weinhold AR, Mccain AH |date=March 1971|title=Biology and Control of Agrobacterium tumefaciens|journal=Hilgardia|volume=40|issue=15|pages=537–552|doi=10.3733/hilg.v40n15p537|doi-access=free}} 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.{{Cite book |last=Agrios |first=George N. |title=Plant pathology |publisher=Elsevier Academic Press |year=2005 |isbn=9780120445653 |edition=5th |location=Amsterdam |language=en |doi=10.1016/C2009-0-02037-6 |oclc=55488155}}

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{{Cite web |last1=Bilderback |first1=Ted |last2=Bir |first2=R. E. |last3=Ranney |first3=T. G. |date=June 30, 2014 |title=Grafting and Budding Nursery Crop Plants |url=https://content.ces.ncsu.edu/grafting-and-budding-nursery-crop-plants |access-date=December 12, 2017 |website=NC State Extension Publications |language=en-US}} 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. 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.{{Cite web |last1=Koetter |first1=Rebecca |last2=Grabowski |first2=Michelle |title=Crown gall |url=https://www.extension.umn.edu/garden/yard-garden/trees-shrubs/crown-gall/ |archive-url=https://web.archive.org/web/20171016014523/https://www.extension.umn.edu/garden/yard-garden/trees-shrubs/crown-gall/ |archive-date=October 16, 2017 |access-date=October 15, 2017 |publisher=University of Minnesota Extension |language=en}}

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 a strain of Rhizobium rhizogenes{{cite web |title=Taxonomy browser (Rhizobium rhizogenes K84) |url=https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=311403 |website=www.ncbi.nlm.nih.gov}} (formerly classified under A. radiobacter, but later reclassified) 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, a deletion mutant strain based on K84, known as K1026, was created. This strain is just as successful in controlling crown gall as K84 without the caveat of resistance gene transfer.{{Cite journal |vauthors=Ryder MH, Jones DA |date=October 1, 1991 |title=Biological Control of Crown Gall Using Using Agrobacterium Strains K84 and K1026 |journal=Functional Plant Biology |volume=18 |issue=5 |pages=571–579 |doi=10.1071/pp9910571|bibcode=1991FunPB..18..571R }}{{cite journal |last1=Kim |first1=Jung-Gun |last2=Park |first2=Byoung Keun |last3=Kim |first3=Sung-Uk |last4=Choi |first4=Doil |last5=Nahm |first5=Baek Hie |last6=Moon |first6=Jae Sun |last7=Reader |first7=John S. |last8=Farrand |first8=Stephen K. |last9=Hwang |first9=Ingyu |title=Bases of biocontrol: Sequence predicts synthesis and mode of action of agrocin 84, the Trojan Horse antibiotic that controls crown gall |journal=Proceedings of the National Academy of Sciences |date=6 June 2006 |volume=103 |issue=23 |pages=8846–8851 |doi=10.1073/pnas.0602965103|doi-access=free |pmid=16731618 |pmc=1482666 |bibcode=2006PNAS..103.8846K }}

File:Crown Gall of Sunflower.jpg

Host, environment, and pathogen are extremely important concepts in regards to plant pathology. Agrobacteria have the widest host range of any plant pathogen,{{Cite web |last=Ellis |first=Michael A. |date=Apr 15, 2016 |title=Bacterial Crown Gall of Fruit Crops |url=https://ohioline.osu.edu/factsheet/plpath-fru-19 |access-date=October 20, 2017 |website=Ohioline |publisher=Ohio State University Extension |language=en}} 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.{{Cite web |date=October 19, 2017 |title=Crown Gall – A Growing Concern in Vineyards |url=https://extension.psu.edu/crown-gall-a-growing-concern-in-vineyards |url-status=dead |archive-url=https://web.archive.org/web/20171020200755/https://extension.psu.edu/crown-gall-a-growing-concern-in-vineyards |archive-date=October 20, 2017 |access-date=October 20, 2017 |website=Penn State Extension |language=en}} 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.{{cite journal | vauthors = Karimi M, Van Montagu M, Gheysen G | title = Nematodes as vectors to introduce Agrobacterium into plant roots | journal =Molecular Plant Pathology | volume = 1 | issue = 6 | pages = 383–7 | date = November 2000 | pmid = 20572986 | doi = 10.1046/j.1364-3703.2000.00043.x | s2cid = 35932276 | doi-access = free | bibcode = 2000MolPP...1..383K }} Finally, temperature is a factor when considering A. tumefaciens infection. The optimal temperature for crown gall formation due to this bacterium is {{ Convert |22|C}} because of the thermosensitivity of T-DNA transfer. Tumor formation is significantly reduced at higher temperature conditions.{{Cite journal| vauthors = Dillen W, De Clereq J, Kapila J, Van Montagu ZM, Angenon G |date=1997-12-01|title=The effect of temperature on Agrobacterium tumefaciens-mediated gene transfer to plants|journal=The Plant Journal|volume=12|issue=6|pages=1459–1463|doi=10.1046/j.1365-313x.1997.12061459.x |doi-access=free}}

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• {{cite book | vauthors = Dickinson M | date = 2003 | title = Molecular Plant Pathology | publisher = BIOS Scientific Publishers }}
• {{cite journal | vauthors = Lai EM, Kado CI | title = The T-pilus of Agrobacterium tumefaciens | journal =Trends in Microbiology| volume = 8 | issue = 8 | pages = 361–9 | date = August 2000 | pmid = 10920395 | doi = 10.1016/s0966-842x(00)01802-3 }}
• {{cite journal | vauthors = Ward DV, Zupan JR, Zambryski PC | title = Agrobacterium VirE2 gets the VIP1 treatment in plant nuclear import | journal =Trends in Plant Science| volume = 7 | issue = 1 | pages = 1–3 | date = January 2002 | pmid = 11804814 | doi = 10.1016/s1360-1385(01)02175-6 | bibcode = 2002TPS.....7....1W }}
• {{cite journal| vauthors = Webster J, Thomson J |s2cid=180063|title=Genetic Analysis of an Agrobacterium tumefaciens strain producing an agrocin active against biotype 3 Pathogen|journal=Molecular and General Genetics|date=1988|volume=214|issue=1|pages=142–147|doi=10.1007/BF00340192}}

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• [https://archive.today/20140223073629/http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=ntat01 "Agrobacterium fabrum" C58 Genome Page] — as sequenced by Cereon Genomics/University of Richmond

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Category:Gram-negative bacteria

Category:Bacterial plant pathogens and diseases

Category:Bacterial grape diseases

Category:Bacteria described in 1907

Category:Rhizobiaceae