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: 843-852. Herb Pilcher

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

“A plant tumor of bacterial origin” 1907: Crown gall is caused by a bacterium 1907 - 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: http://www.science.oregonstate.edu/bpp/Plant_Clinic/images/crown%20gall.htm Smith, E.F. and Townsend, C.O. (1907). A plant-tumor of bacterial origin. Science. 25: 671-673.

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

Agrobacterium-induced galls do not require bacterial persistence Armin C. Braun 1911 - 1986 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: 239-241; Photo from Wood, H.N., and Kelman, A. (1987) Phytopathology 77: 991.

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.

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: 674-677

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: 109-127. 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: 169-170. 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: 742-743.

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: 263-271. 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: 458-461.

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

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

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: 179-180.

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: 1275-1288; Chuck, G., Lincoln, C. and Hake, S. (1996). KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell. 8: 1277-1289. 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: 843-852.

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

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: 653-657; 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: 2019-2025.

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

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

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

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: 624-629; 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: 379-383.

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: 703-714.

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

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

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: 812-818 with permission from Elsevier.

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;