1. Agrobacterium tumefaciens – pathogen and useful tool
Cherry
Agrobacterium tumefaciens is a plant pathogen that induces tumors on about 60% of dicotyledonous angiosperms and gymnosperms
Its tumor-inducing property also makes it a valuable tool for introducing genes into plants for research and agricultural purposes
Casimiro, I., Marchant, A., Bhalerao, R.P., Beeckman, T., Dhooge, S., Swarup, R., Graham, N., Inzé, D., Sandberg, G., Casero, P.J. and Bennett, M. (2001). Auxin Transport Promotes Arabidopsis Lateral Root Initiation. Plant Cell. 13: Herb Pilcher

2. Crown gall disease
The first written record of crown gall disease, on grape, dates from 1853
Fridiano Cavara (1897) found that a bacterium causes crown gall in grape
Crown gall induces growths at wound sites and severely limits crop yields and growth vigor
Edward L. Barnard, Florida Department of Agriculture and Consumer Services, Bugwood.org; Mike Ellis, Ohio State University; University of Georgia Plant Pathology Archive, University of Georgia, Bugwood.org; Wikimedia commons

3. “A plant tumor of bacterial origin”
1907: Crown gall is caused by a bacterium
Erwin Smith and C.O. Townsend isolated a bacterium from galls on daisy. When inoculated onto other plants, galls were produced
gall
There is a nice photograph of a crown gall on daisy here:
Smith, E.F. and Townsend, C.O. (1907). A plant-tumor of bacterial origin. Science. 25:

4. Unusual compounds called opines are found in many crown galls
Questions raised:
What are these compounds?
Do they cause the tumors?
How and why do the bacteria cause the plants to make opines?
Octopine
Octopine-utilizing strain
Nopaline
Nopaline-utilizing strain
1960s – 1970s, numerous studies
The type of opine is determined by the bacterium, not the plant

5. Agrobacterium-induced galls do not require bacterial persistence
Armin C. Braun
Gall tissues without any bacteria can persist indefinitely in culture, in contrast with most other pathogen-induced neoplastic growths that require the presence of the pathogen
Braun made fundamental discoveries about how Agrobacterium transforms plant cells
White, P.R. and Braun, A.C. (1941). Crown gall production by bacteria-free tumor tissues. Science. 94: ; Photo from Wood, H.N., and Kelman, A. (1987) Phytopathology 77: 991.

6. Gall tissues can grow indefinitely without exogenous phytohormones
1930s – 1950s, numerous studies
+ Auxin + CK
Normal plant tissue cannot live indefinitely in hormone-free medium
Normal plant tissue grows and survives when auxin and cytokinin (CK) are added to medium
Crown gall tissue grows well without added hormones
“It is possible for a cell to acquire the capacity for autonomous growth as a result of the permanent activation of growth-substance-synthesizing systems”
-AC Braun 1958
Auxin CK
High levels of auxin and cytokinin are found in gall tissues
Braun, A.C. (1958) A physiological basis for autonomous growth of the crown-gall tumor cell. Proc Natl Acad Sci U S A. 44: 344–349.

7. A few days after inoculation, tumors become independent of bacteria
Periwinkle (Catharanthus roseus) stems were inoculated with Agrobacterium tumefaciens, and then incubated at room temperature for various times, followed by 5 days at 47°C to kill the bacteria
When the tissue was incubated at room temperature for four days before heat-killing the bacteria, many tumors were formed
When the tissue was incubated at room temperature for one day before heat-killing the bacteria, no tumors were formed
Viable bacteria are no longer necessary beyond two days post-inoculation. After this period, tumors become independent of the bacteria, because the bacteria have altered the host cells, by transferring some factors into them.
Vinca was used because it can withstand the several days at elevated temperature needed to kill the bacteria
Braun, A.C. (1943) Studies on tumor inception in the crown-gall disease. Am. J. Bot. 30:

8. A large plasmid in gall-inducing Agrobacterium confers virulence
A very large plasmid was identified that is present in virulent but absent from avirulent strains
Heat treatment removes plasmid and makes bacteria non-pathogenic
Virulent
Avirulent
tumor
No tumor +
time
heat
A plasmid carrying a genetic marker (antibiotic resistance) was shown to be confer virulence
Zaenen, I., van Larebeke, N., Teuchy, H., van Montagu, M. and Schell, J. (1974). Supercoiled circular DNA in crown-gall inducing Agrobacterium strains. Journal of Molecular Biology. 86: Larebeke, N.V., Engler, G., Holsters, M., Den Elsacker, S.V., Zaenen, I., Schilperoort, R.A. and Schell, J. (1974). Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature. 252: Van Larebeke, N., Genetello, C.H., Schell, J., Schilperoort, R.A., Hermans, A.K., Hernalsteens, J.P. and Van Montagu, M. (1975). Acquisition of tumour-inducing ability by non-oncogenic agrobacteria as a result of plasmid transfer. Nature. 255:

9. Some DNA from the Ti plasmid is transferred into the plant cells (1977)
Restriction enzyme digestion
“Our results suggest that the tumor-inducing principle first proposed by Braun (1947) is indeed DNA, as many investigators have long suspected.”
Pos. controls (Ti plasmid)
Neg. control (untransformed plant DNA)
DNA from crown gall
Renaturation kinetics of labeled plasmid DNA fragments with various unlabeled DNA samples
Increasing amounts of labeled Ti plasmid DNA
The key finding was that Ti plasmid DNA anneals with DNA isolated from the crown gall, meaning that the gall contains Ti DNA
Reprinted from Chilton, M.-D., Drummond, M.H., Merlo, D.J., Sciaky, D., Montoya, A.L., Gordon, M.P. and Nester, E.W. (1977). Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell. 11: with permission from Elsevier. See also Yadav, N.S., Postle, K., Saiki, R.K., Thomashow, M.F. and Chilton, M.D. (1980). T-DNA of a crown gall teratoma is covalently joined to host plant DNA. Nature. 287:

10. Structure and function analysis of the Ti plasmid
vir genes
T-DNA
pTi
The virulence (vir) genes are required for T-DNA movement into the plant cell
Transfer DNA (T-DNA) moves into the plant cell nucleus. It is flanked by two direct 25 bp repeat border sequences, shown as yellow triangles
The organization of Ti plasmids varies between isolates, but all carry one or more T-DNA region and one vir region

11. The T-DNA region: tumor-inducing genes and opine synthesis genes
vir genes
T-DNA
pTi
Auxin synthesis
Cytokinin synthesis
Opine synthesis to “feed” Agrobacterium
Autonomous growth
Plant cell
T4SS
Not all T-DNAs are arranged like this and carry these genes; there is some variation amongst Ti plasmid structures
T4SS = Type IV Secretion System

12. The Ti plasmid can be used to introduce any gene into plants
The discovery that T-DNA was inserted into the plant genome raised the possibility that “any gene” could be transferred into plants
vir genes
T-DNA
pTi
Tumor-inducing and opine synthesis genes on T-DNA can be replaced by a “gene of interest” and selectable marker
Gene of interest
Selectable marker
The cloning of the smaller plasmid takes place in E. coli (not shown). Once prepared, this plasmid can be introduced into Agrobacterium by electroporation or conjugation.
Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. (1983). A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature. 303:

13. Applications of Agrobacterium-mediated transformation
Basic research – plant transformation allows in vivo study of plant genes
Expression pattern of an auxin-inducible promoter fused to GUS reporter gene
Population segregating for short-hypocotyl phenotype conferred by PHYB overexpression
Wild type
Overexpression
Lobed-leaf phenotype of plants overexpressing KNAT1 gene
Wagner, D., Tepperman, J.M. and Quail, P.H. (1991). Overexpression of phytochrome B induces a short hypocotyl phenotype in transgenic Arabidopsis. Plant Cell. 3: ; Chuck, G., Lincoln, C. and Hake, S. (1996). KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell. 8: Casimiro, I., Marchant, A., Bhalerao, R.P., Beeckman, T., Dhooge, S., Swarup, R., Graham, N., Inzé, D., Sandberg, G., Casero, P.J. and Bennett, M. (2001). Auxin transport promotes Arabidopsis lateral Root Initiation. Plant Cell. 13:

14. Production of genetically-modified (GM) plants
Photo credits: Herb Pilcher, Scott Bauer
Transgenic plants expressing insecticidal Bt gene
Wild-type peanut plant
Peanut plant expressing the Bt gene
Bacillus thuringiensis expressing Bt toxin
Plant cell expressing Bt toxin
Agrobacterium tumefaciens allows gene transfer into many crop plants, particularly dicots like soybean and peanut

15. Agrobacterium-mediated transformation has other uses
gene
Insertional mutagenesis
T-DNA
Mutated, tagged gene
Transient expression studies: Short-term expression of T-DNA genes gives results faster than generating transgenic plants
GFP expression in tobacco epidermal cells
Alonso, J.M. et al., and Ecker, J.R. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 301: ; Reprinted by permission from Macmillan Publishers Ltd Sparkes, I.A., Runions, J., Kearns, A. and Hawes, C. (2006). Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protocols. 1:

16. Summary – the development of a vector for plant transformation
Agrobacterium tumefaciens
vir genes
T-DNA
Introduce gene of interest into
T-DNA region, and introduce into Agrobacterium carrying vir genes
Inoculate plant with engineered Agrobacterium
Regenerate plant from transformed cells
Arabidopsis floral dip transformation method
Photo by Peggy Greb, USDA

17. Inside the black box – how Agrobacterium transfers DNA
5. Expression of T-DNA and pathogenicity
Agrobacterium
Plant cell
3. Movement of
T-DNA out of the bacterium
4. Nuclear import and integration of T-DNA
1. Chemoattraction and activation of virulence
2. T-DNA excision

18. Many of the genes needed for T-DNA transfer are found on the Ti plasmid
The Ti plasmid carries genes required for
T-DNA transfer, Ti plasmid conjugation and opine metabolism
T-DNA
Virulence genes
Opine catabolism
Conjugal Transfer

19. Perception of host signals induces expression of vir genes
Acetosyringone is likely perceived by the VirA protein encoded on the Ti plasmid
VirA and VirG induce other vir genes in response to plant signals
Plant-derived small molecules such as acetosyringone induce Agrobacterium vir genes
Stachel, S.E., Messens, E., Van Montagu, M. and Zambryski, P. (1985). Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature. 318: ; Stachel, S.E., Nester, E.W. and Zambryski, P.C. (1986). A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc. Natl. Acad. Sci. USA. 83:

20. T-DNA transfers through a multi-subunit type IV secretion system
Tilted outer membrane side
Tilted inner membrane side
Inner membrane
Outer membrane
VirB10
Side view
VirB9 and B7
Plant cell
Agrobacterium
Reprinted by permission from Macmillan Publishers Ltd. Fronzes, R., Christie, P.J. and Waksman, G. (2009). The structural biology of type IV secretion systems. Nat. Rev. Micro. 7:

21. Conjugation spreads the Ti plasmid throughout the population
Agrobacterium with Ti plasmid
Cell division
Agrobacterium without Ti plasmid
Conjugation – horizontal gene transfer
Replication of the large Ti plasmid is metabolically costly. When opines are present, the Ti plasmid is amplified in the population by conjugation. Thus, a small number of individuals carrying Ti can serve as a reservoir for the larger population
Opine

22. SUMMARY (Animated) Plant cell Agrobacterium vir genes induction T4SS.
Plant cell
Agrobacterium E3 F E2 D2
Transfer
T4SS
VirB/D4 F E3 E2
Integration
of T-DNA D2 D5
T-DNA processing D2 LB RB
Ti Plasmid
T-DNA
vir
genes
nucleus
vir genes induction
Expression of
T-DNA: auxin, cytokinin, opine
VirG p
VirA
Phenolics
VirG p
Signaling
in rhizosphere 22

23. Agrobacterium rhizogenes: inducer of roots
vir genes
T-DNA
pRi
The oncogenic genes on A. rhizogenes T-DNA are not as well understood as those on the Ti plasmid
Mulberry infected with A. rhizogenes
Infection by A. rhizogenes leads to production of a large root mass, rather than a tumor. Therefore, the large plasmid carried by A. rhizogenes is called a Root-inducing (Ri) plasmid
Image credits: William M. Brown Jr., Bugwood.org; Reprinted from Dhakulkar, S., Ganapathi, T.R., Bhargava, S. and Bapat, V.A. (2005). Induction of hairy roots in Gmelina arborea Roxb. and production of verbascoside in hairy roots. Plant Sci. 169: with permission from Elsevier.

24. Conclusions
Agrobacterium is an amazing organism, with a unique ability to transfer DNA into diverse host genomes, which has been exploited to facilitate research and breeding
Agrobacterium research and its application went far beyond what Smith and Townsend could foresee when they found crown gall was
caused by the bacterium in 1907
Edward L. Barnard, Florida Department of Agriculture and Consumer Services, Bugwood.org; Mike Ellis, Ohio State University;