Editing Agrobacterium tumefaciens

{{Short description|Bacterium, genetic engineering tool}}
{{cs1 config|name-list-style=vanc}}
{{hatnote|Between 1980 and 2022, ''Agrobacterium tumefaciens'' had a type strain (B6) now classified as ''[[Agrobacterium radiobacter]]''. This article cites a great number of sources from this period. Statements backed by these sources should be treated as possibly describing ''A. radiobacter'' or a different species.}}
{{Speciesbox
| image = Agrobacterium-tumefaciens.png
| image_caption = ''Agrobacterium tumefaciens'' attaching itself to a carrot cell
| genus = Agrobacterium
| species = tumefaciens
| authority = (Smith and Townsend 1907) Conn 1942 (Approved Lists 1980)
| type_strain = ATCC 4720<ref>{{cite journal |vauthors = Velazquez E, Flores-Felix JD, Sanchez-Juanes F, Igual JM, Peix A | title = Strain ATCC 4720<sup>T</sup> is the authentic type strain of ''Agrobacterium tumefaciens'', which is not a later heterotypic synonym of ''Agrobacterium radiobacter'' | journal =[[Int J Syst Evol Microbiol]]| year = 2020 | volume = 70 | issue = 9 | pages = 5172–5176 | pmid = 32915125 | doi = 10.1099/ijsem.0.004443| doi-access = free }}</ref><ref name=jo127>{{cite journal |last1=Arahal |first1=David R. |last2=Bull |first2=Carolee T. |last3=Busse |first3=Hans-Jürgen |last4=Christensen |first4=Henrik |last5=Chuvochina |first5=Maria |last6=Dedysh |first6=Svetlana N. |last7=Fournier |first7=Pierre-Edouard |last8=Konstantinidis |first8=Konstantinos T. |last9=Parker |first9=Charles T. |last10=Rossello-Mora |first10=Ramon |last11=Ventosa |first11=Antonio |last12=Göker |first12=Markus |title=Judicial Opinions 123–127 |journal=International Journal of Systematic and Evolutionary Microbiology |date=27 April 2023 |volume=72 |issue=12 |doi=10.1099/ijsem.0.005708|pmid=36748499 |hdl=10261/295959 |hdl-access=free }} [N.B.: Judicial Opinion 127 assigns the strain ATCC 4720 as the type strain of Agrobacterium tumefaciens.]<!-- the NB is from NCBI, which is in the public domain.--></ref>{{efn|also known as: CCM:1000, CCUG:3555, CFBP:2412, CIP:104335, DSM:30150, ICMP:5793, BCCM/LMG:182, NCIMB:8150, NCPPB:2992, IAM:14141, IAM:1524, JCM:21034, personal::A1<ref name=ncbi/>}}
| synonyms =
'''Homotypic synonyms'''
* ''Bacterium tumefaciens'' <small>Smith and Townsend 1907</small><ref name=Smith1907>{{cite journal | vauthors = Smith EF, Townsend CO | title = A Plant-Tumor of Bacterial Origin | journal =[[Science (journal)|Science]]| volume = 25 | issue = 643 | pages = 671–3 | date = April 1907 | pmid = 17746161 | doi = 10.1126/science.25.643.671 | url = https://zenodo.org/record/1447982 | bibcode = 1907Sci....25..671S }}</ref>
* ''Pseudomonas tumefaciens'' <small>(Smith and Townsend 1907) Duggar 1909</small>
* ''Phytomonas tumefaciens'' <small>(Smith and Townsend 1907) Bergey ''et al''. 1923</small>
* ''Polymonas tumefaciens'' <small>(Smith and Townsend 1900) Lieske 1928</small>

'''Heterotypic synonyms'''
* ''Agrobacterium fabacearum'' <small>Delamuta et al 2020<ref>{{Cite journal |last1=Delamuta |first1=Jakeline Renata Marçon |last2=Scherer |first2=Anderson José |last3=Ribeiro |first3=Renan Augusto |last4=Hungria |first4=Mariangela |date=2020 |title=Genetic diversity of Agrobacterium species isolated from nodules of common bean and soybean in Brazil, Mexico, Ecuador and Mozambique, and description of the new species Agrobacterium fabacearum sp. nov. |url=https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijsem.0.004278 |journal=International Journal of Systematic and Evolutionary Microbiology |volume=70 |issue=7 |pages=4233–4244 |doi=10.1099/ijsem.0.004278 |pmid=32568030 |issn=1466-5034}}</ref></small> (by ANI)<ref name=ncbi/>

''[[Agrobacterium radiobacter]]'' <small>(Beijerinck and van Delden 1902) Conn 1942 (Approved Lists 1980)</small> is NOT a synonym.<ref name=jo127/> The two used to be synonimized<ref>{{cite journal |vauthors=Tindall BJ |collaboration=Judicial Commission | title = Judicial Opinion No. 94: ''Agrobacterium radiobacter'' (Beijerinck and van Delden 1902) Conn 1942 has priority over ''Agrobacterium tumefaciens'' (Smith and Townsend 1907) Conn 1942 when the two are treated as members of the same species based on the principle of priority and Rule 23a, Note 1 as applied to the corresponding specific epithets | journal =[[Int J Syst Evol Microbiol]]| year = 2014 | volume = 64 | issue = Pt 10 | pages = 3590–3592 | doi = 10.1099/ijs.0.069203-0| pmid = 25288664 | doi-access = free }}</ref> on the basis of an unjustified type strain change in the Approved Lists of 1980, now reverted.<ref name=jo127/>
| synonyms_ref = <ref>{{cite journal | author = Buchanan RE | year = 1965 | title = Proposal for rejection of the generic name ''Polymonas'' Lieske 1928 | journal =[[International Bulletin of Bacteriological Nomenclature and Taxonomy]]| volume = 15 | issue = 1 | pages = 43–44 | doi = 10.1099/00207713-15-1-43| doi-access = free }}</ref><ref>{{cite journal |vauthors =  Sawada H, Ieki H, Oyaizu H, Matsumoto S | title = Proposal for rejection of ''Agrobacterium tumefaciens'' and revised descriptions for the genus ''Agrobacterium'' and for ''Agrobacterium radiobacter'' and ''Agrobacterium rhizogenes'' | journal =[[Int J Syst Bacteriol]]| year = 1993 | volume = 43 | issue = 4 | pages = 694–702| doi = 10.1099/00207713-43-4-694 | pmid = 8240952 | doi-access = free }}</ref><ref name=jo127/>
}}
'''''Agrobacterium tumefaciens'''''<ref name=ncbi>{{cite web|title=Taxonomy browser (''Agrobacterium tumefaciens'')|url=https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=358&lvl=3&lin=f&keep=1&srchmode=1&unlock |website=National Center for Biotechnology Information|access-date=7 January 2024}}</ref><ref name=jo127/> (also known as ''Rhizobium radiobacter'') is the causal agent of '''crown gall disease''' (the formation of [[tumour]]s) in over 140 species of [[eudicot]]s. It is a rod-shaped, [[Gram-negative]] soil [[bacterium]].<ref name=Smith1907/> Symptoms are caused by the insertion of a small segment of [[DNA]] (known as [[Transfer DNA|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,<ref>{{cite journal |last1=Chilton |first1=Mary-Dell |last2=Drummond |first2=Martin H. |last3=Merlo |first3=Donald J. |last4=Sciaky |first4=Daniela |last5=Montoya |first5=Alice L. |last6=Gordon |first6=Milton P. |last7=Nester |first7=Eugene W. | s2cid = 7533482 | title = Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis | journal =[[Cell (journal)|Cell]]| volume = 11 | issue = 2 | pages = 263–271 | date = June 1977 | pmid = 890735 |issn=0092-8674 | doi = 10.1016/0092-8674(77)90043-5 }}</ref> 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 [[Transfer DNA binary system|T-DNA binary vectors]].

''Agrobacterium tumefaciens'' is an [[Alphaproteobacterium]] of the family [[Rhizobiaceae]], which includes the [[nitrogen fixation|nitrogen-fixing]] [[legume]] [[symbionts]]. Unlike the nitrogen-fixing symbionts, tumor-producing ''Agrobacterium'' species are [[pathogen]]ic and do not benefit the plant. The wide variety of plants affected by ''Agrobacterium'' makes it of great concern to the agriculture industry.<ref>{{cite journal | vauthors = Moore LW, Chilton WS, Canfield ML | title = Diversity of opines and opine-catabolizing bacteria isolated from naturally occurring crown gall tumors | journal =[[Applied and Environmental Microbiology]]| volume = 63 | issue = 1 | pages = 201–7 | date = January 1997 | pmid = 16535484 | pmc = 1389099 | doi = 10.1128/AEM.63.1.201-207.1997 | bibcode = 1997ApEnM..63..201M }}</ref>

Economically, ''A. tumefaciens'' is a serious pathogen of [[walnuts]], [[grape vine]]s, [[stone fruit]]s, [[Nut (fruit)|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.<ref>{{Cite web |title=Crown Galls |url=https://www.missouribotanicalgarden.org/gardens-gardening/your-garden/help-for-the-home-gardener/advice-tips-resources/pests-and-problems/diseases/bacterial-galls/crown-galls.aspx |access-date=December 2, 2019 |website=[[Missouri Botanical Garden]]}}</ref>

''Agrobacterium tumefaciens'' grows optimally at {{ Convert |28|C}}. The doubling time can range from 2.5–4h depending on the media, culture format, and level of aeration.<ref name=Elise>{{cite journal | vauthors = Morton ER, Fuqua C | title = Laboratory maintenance of ''Agrobacterium'' | journal =[[Current Protocols in Microbiology]]| volume = Chapter 1 | pages = Unit3D.1 | date = February 2012 | pmid = 22307549 | pmc = 3350319 | doi = 10.1002/9780471729259.mc03d01s24 | isbn = 978-0471729259 }}</ref> At temperatures above {{ Convert |30|C}}, ''A. tumefaciens'' begins to experience heat shock which is likely to result in errors in cell division.<ref name=Elise/>

==Conjugation==

To be [[virulent]], the bacterium contains a [[Ti plasmid|tumour-inducing plasmid]] (Ti plasmid or pTi) 200 [[Base pair#Length measurements|kbp]] long, which contains the T-DNA and all the [[gene]]s necessary to transfer it to the plant cell.<ref>{{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 }}</ref> Many strains of ''A. tumefaciens'' do not contain a pTi.

Since the Ti plasmid is essential to cause disease, prepenetration events in the [[rhizosphere (ecology)|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.<ref>{{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}}</ref> This activates the [[transcription factor]] TraR, positively regulating the [[Transcription (genetics)|transcription]] of genes required for conjugation.<ref>{{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 }}</ref>

== Infection methods ==
''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 [[photoassimilate]]s that accumulate in the rhizosphere around roots. Some [[Strain (biology)|strains]] may [[chemotaxis|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.<ref name="stanton">{{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}}</ref>

At least 25 ''vir'' genes on the Ti plasmid are necessary for tumor induction.<ref>{{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}}</ref> In addition to their perception role, ''virA'' and ''chvE'' induce other ''vir'' genes. The VirA protein has auto[[kinase]] activity: it [[phosphorylation|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'' [[operon]]s. The ChvE protein regulates the second mechanism of the ''vir'' genes' activation. It increases VirA protein sensitivity to phenolic compounds.<ref name=stanton/>

Attachment is a two-step process. Following an initial weak and reversible attachment, the bacteria synthesize [[cellulose]] [[fibril]]s 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 in biology|calcium-dependent]] outer membrane [[protein]] called rhicadhesin is produced, which also aids in sticking the bacteria to the cell wall. [[Homology (biology)|Homologue]]s of this protein can be found in other rhizobia. Currently, there are several reports on standardisation of protocol for the [[genetic transformation of plants|''Agrobacterium''-mediated transformation]]. The effect of different parameters such as infection time, acetosyringone, DTT, and cysteine have been studied in soybean (''Glycine max'').<ref>{{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}}</ref>

Possible plant compounds that initiate ''Agrobacterium'' to infect plant cells:<ref>{{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)}}</ref>
* [[Acetosyringone]] and other phenolic compounds
* alpha-[[Hydroxyacetosyringone]]
* [[Catechol]]
* [[Ferulic acid]]
* [[Gallic acid]]
* [[4-Hydroxybenzoic acid|p-Hydroxybenzoic acid]]
* [[Protocatechuic acid]]
* [[Pyrogallic acid]]
* [[Resorcylic acid]]
* [[Sinapinic acid]]
* [[Syringic acid]]
* [[Vanillin]]

===Formation of the T-pilus===

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.<ref name=":1">{{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 }}</ref>

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 [[Code|encode]] components of the transporter. An [[ATPase]] for the [[active transport]] of the subunits would also be required.

===Transfer of T-DNA into the plant cell===
[[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.<ref>{{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}}</ref> 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 [[Sequence motif|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.<ref name=":1" />

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 signal]]s, 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 [[Cell nucleus|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.

== Genes in the T-DNA ==

=== Hormones ===
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 [[cytokinin]]s are also expressed. This stimulates cell proliferation and gall formation.

=== Opines ===
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]].<ref>{{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 }}</ref> [[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]].<ref>{{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 }}</ref>

Two nopaline type [[Ti plasmid]]s, 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.<ref>{{cite web |title=Species: Agrobacterium fabrum |url=https://lpsn.dsmz.de/species/agrobacterium-fabrum |website=lpsn.dsmz.de |language=en}}</ref>}}  the first fully sequenced [[pathovar]], was first isolated from a cherry tree crown gall. The genome was simultaneously sequenced by Goodner ''et al.''<ref name=Goodner>{{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 (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}}</ref> and Wood ''et al.''<ref name=Wood>{{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 (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}}</ref> in 2001. The genome of strain C58consists of a circular chromosome, two [[plasmid]]s, 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.<ref name=Goodner/><ref name=Wood/>

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.<ref>{{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 }}</ref>  If the Ti plasmid is removed, the tumor growth that is the means of classifying this species of bacteria does not occur.

== Biotechnological uses ==
{{See also|Agroinfiltration}}

[[Image:Transformation with Agrobacterium.JPG|thumb|Transformed [[plant tissue culture]]s]]
The [[Asilomar Conference on Recombinant DNA|Asilomar Conference]] in 1975 established widespread agreement that recombinant techniques were insufficiently understood and needed to be tightly controlled.<ref>{{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 }}</ref><ref name="GM-Pest-Prot">{{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 }}</ref> 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.<ref>{{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 }}</ref> The plasmid T-DNA that is transferred to the plant is an ideal vehicle for genetic engineering.<ref>{{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 }}</ref> This is done by cloning a desired gene sequence into [[Transfer DNA binary system|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.<ref name=":2" /> This [[luminescence]] has been a useful device in the study of plant chloroplast function and as a [[reporter gene]].<ref name=":2">{{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 }}</ref> 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.<ref>{{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 }}</ref>

The mechanism by which ''Agrobacterium'' inserts materials into the host cell is by a [[Secretion#In gram-negative bacteria|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 ==

[[Transformation (genetics)|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.<ref name="pmid11375171">{{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 }}</ref>

== Disease cycle ==
[[File:A tumefaciens disease cycle.jpg|alt=Disease cycle Agrobacterium tumefaciens|left|thumb|425px|[[Disease cycle]]]]
''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.<ref>{{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}}</ref> 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.<ref name=":0">{{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}}</ref>

== Disease management ==
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<ref>{{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}}</ref> 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.<ref name=":0" /> 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.<ref>{{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}}</ref>

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]]''<ref>{{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}}</ref> (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 (genetics)|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.<ref>{{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 }}</ref><ref>{{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 }}</ref>

== Environment ==
[[File:Crown Gall of Sunflower.jpg|alt=Crown gall of sunflower|thumb|upright|Crown gall of sunflower caused by ''A. tumefaciens'']]
Host, environment, and pathogen are extremely important concepts in regards to plant pathology. ''Agrobacteria'' have the widest host range of any plant pathogen,<ref>{{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}}</ref> 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.<ref>{{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}}</ref> 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.<ref>{{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 }}</ref> 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.<ref>{{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}}</ref>

== See also ==
* ''[[suhB]]''

== References ==
{{notelist}}
{{Reflist}}

== Further reading ==
{{Refbegin}}
* {{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}}
{{Refend}}

== External links ==
* {{Commons-inline}}
* [https://archive.today/20140223073629/http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=ntat01 "''Agrobacterium fabrum''" C58 Genome Page]&nbsp;— 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]]