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We dedicate this volume to Jeff Schell, friend, colleague, and sometimes competitor. Jeff was a pioneer in studies on Agrobacterium and his many seminal contributions through basic and applied research form a cornerstone of this anthology. His scientific legacy will continue not only through his own writings but also in the publications of the numerous individuals whose careers he influenced.


CONTENTS Dedication .................................................................................................................. iii Van Montagu, M. 2003. Jeff Schell (1935–2003). Nature 423:934. ................................. iv

Preface ......................................................................................................................... xi Sources ...................................................................................................................... xiii Biology of Agrobacterium tumefaciens A CENTURY OF PIONEERING WORK BEGINS Paul D. Peterson ........................................................................................................... Smith, E. F., and Townsend, C. O. 1907. A plant-tumor of bacterial origin. Science, New Series, 25:671–673. ...................................................................

1

4

ARMIN C. BRAUN AND THE DISCOVERY OF AGROBACTERIUMMEDIATED TRANSFORMATION OF PLANT CELLS Andrew N. Binns ..........................................................................................................

7

White, P. R., and Braun, A. C. 1941. Crown gall production by bacteria-free tumor tissues. Science 94:239–241. ........................................................................ 11 Braun, A. C., and Mandle, R. J. 1948. Studies on the inactivation of the tumorinducing principle in crown gall. Growth 12:255–269. .......................................... 14

TREASURE THE UNEXPECTED

Allen Kerr ............................................... 29

Kerr, A. 1969. Transfer of virulence between isolates of Agrobacterium. Nature 223:1175–1176. ........................................................................................... 31

GEORGE MICHEL MOREL (1916–1973): CHEMIST, BOTANIST, PLANT TISSUE CULTURE PIONEER Jacques Tempé ............................... 33 Petit, A., Delhaye, S., Tempé, J., and Morel, G. 1970. Recherches sur les guanidines des tissus de crown gall: Mise en évidence d’une relation biochimique spécifique entre les souches d’Agrobacterium tumefaciens et les tumeurs qu’elles induisent. Physiol. Vég. 8:205–213. ............................................ 36

LOSS OF TUMOR-INDUCING ABILITY

Robert H. Hamilton .................. 45

Hamilton, R. H., and Fall, M. Z. 1971. The loss of tumor-initiating ability in Agrobacterium tumefaciens by incubation at high temperature. Experientia 27:229–230. ............................................................................................................ 47

DISCOVERY OF THE Ti PLASMID

Marc Van Montagu ............................ 49

Van Larebeke, N., Genetello, C., Schell, J., Schilperoort, R. A., Hermans, A. K., Hernalsteens, J.-P., and Van Montagu, M. 1975. Acquisition of tumourinducing ability by non-oncogenic agrobacteria as a result of plasmid transfer. Nature 255:742–743. ....................................................................................... 51 v


Agrobacterium tumefaciens: From Plant Pathology to Biotechnology

A PLASMID WAS PRESENT AFTER ALL Bruce Watson and Eugene W. Nester ......................................................................... 53 Watson, B., Currier, T. C., Gordon, M. P., Chilton, M.-D., and Nester, E. W. 1975. Plasmid required for virulence of Agrobacterium tumefaciens. J. Bacteriol. 123: 255–264. ........................................................................................................ 56

MY LIFE WITH PHYTOHORMONES

Milton P. Gordon ........................... 66

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. ............................................................................................ 68

I’M CERTAINLY GLAD I WAS THERE

Martin Drummond ..................... 77

Drummond, M. H., Gordon, M. P., Nester, E. W., and Chilton, M.-D. 1977. Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumors. Nature 269:535–536. ....................................................................................... 79

AN ADVENTURE IN AGROBACTERIOLOGY

Marcelle Holsters ........... 81

Holsters, M., Silva, B., Van Vliet, F., Genetello, C., De Block, M., Dhaese, P., Depicker, A., Inzé, D., Engler, G., Villarroel, R., Van Montagu, M., and Schell, J. 1980. The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid 3:212–230. ........................................................................... 83

WOW!!! THE T-DNA IS INTEGRATED

Michael F. Thomashow ............. 102

Thomashow, M. F., Nutter, R., Montoya, A. L., Gordon, M. P., and Nester, E. W. 1980. Integration and organization of Ti plasmid sequences in crown gall tumors. Cell 19:729–739. .......................................................................................... 105

WHEN TOOTHPICKS MATTERED: USING MICROBIAL GENETICS AND TRANSPOSON TAGGING TO MAP THE T-DNA David J. Garfinkel ...................................................................................................... 116 Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M. P., and Nester, E. W. 1981. Genetic analysis of crown gall: Fine structure map of the T-DNA by site-directed mutagenesis. Cell 27:143–153. .............................. 119

CROSS, COUNT AND CALCULATE! T-DNA SATISFIES MENDELIAN RULES

Léon Otten ............................. 130

Otten, L., De Greve, H., Hernalsteens, J.-P., Van Montagu, M., Schieder, O., Straub, J., and Schell, J. 1981. Mendelian transmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens. Mol. Gen. Genet. 183:209–213. ............................................................................................... 133

HOW DOES AGROBACTERIUM “KNOW” WHAT IS T-DNA ON THE Ti PLASMID? Mary-Dell Chilton .................................................... 138 Yadav, N. S., Vanderleyden, J., Bennett, D. R., Barnes, W. M., and Chilton, M.-D. 1982. Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc. Natl. Acad. Sci. U.S.A. 79:6322–6326. .................................................. 140 vi


Agrobacterium tumefaciens: From Plant Pathology to Biotechnology

SUCH A CLEVER BUG

Scott E. Stachel ........................................................ 145

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. ............... 150 Stachel, S. E., Timmerman, B., and Zambryski, P. 1986. Generation of singlestranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322:706–712. ....................... 156

EARLY EVENTS IN THE T-DNA TRANSFER PROCESS Martin Yanofsky ......................................................................................................... 162 Yanofsky, M. F., Porter, S. G., Young, C., Albright, L. M., Gordon, M. P., and Nester, E. W. 1986. The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell 47:471–477. .................................................. 165

VirD TARGETS T-DNA INTO THE PLANT NUCLEUS Alfredo Herrera-Estrella ........................................................................................... 172 Herrera-Estrella, A., Van Montagu, M., and Wang, K. 1990. A bacterial peptide acting as a plant nuclear targeting signal: The amino-terminal portion of Agrobacterium VirD2 protein directs a β-galactosidase fusion protein into tobacco nuclei. Proc. Natl. Acad. Sci. U.S.A. 87:9534–9537. ....................................... 175

FROM PLANT PATHOGEN TO VECTOR FOR PLANTS, YEASTS, AND FUNGI Paul J. J. Hooykaas ................................................... 179 Bundock, P., den Dulk-Ras, A., Beijersbergen, A., and Hooykaas, P. J. J. 1995. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 14:3206–3214. .................................................... 182

Application of A. tumefaciens in Genetic Engineering A RECKLESS DECISION?

Jean-Pierre Hernalsteens ................................. 191

Hernalsteens, J.-P., Van Vliet, F., De Beuckeleer, M., Depicker, A., Engler, G., Lemmers, M., Holsters, M., Van Montagu, M., and Schell, J. 1980. The Agrobacterium tumefaciens Ti plasmid as a host vector system for introducing foreign DNA in plant cells. Nature 287:654–656. ............................................ 194

SHRINKING THE Ti PLASMID

Mary-Dell Chilton ................................... 197

de Framond, A. J., Barton, K. A., and Chilton, M.-D. 1983. Mini-Ti: A new vector strategy for plant genetic engineering. Bio/Technology 1:262–269. ........ 199 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. ......................... 207 vii


Agrobacterium tumefaciens: From Plant Pathology to Biotechnology

THE FIRST TUMORLESS TRANSFORMED PLANT WAS TREATED WITH UTMOST CARE Patricia C. Zambryski ............. 209 Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M., and Schell, J. 1983. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J. 2:2143–2150. .......... 212

FOREIGN GENES CAN BE EXPRESSED IN PLANT CELLS Luis Herrera-Estrella ................................................................................................ 220 Herrera-Estrella, L., Depicker, A., Van Montagu, M., and Schell, J. 1983. Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature 303:209–213. ................................................................................. 223 Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P. R., Flick, J. S., Adams, S. P., Bittner, M. L., Brand, L. A., Fink, C. L., Fry, J. S., Galluppi, G. R., Goldberg, S. B., Hoffmann, N. L., and Woo, S. C. 1983. Expression of bacterial genes in plant cells. Proc. Natl. Acad. Sci. U.S.A. 80:4803–4807. .................... 228 Bevan, M. W., Flavell, R. B., and Chilton, M.-D. 1983. A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304:184–187. ............................................................................................... 233

THERE WERE MULTIPLE TRANSFORMANTS ON EACH PLATE Kenneth A. Feldmann ................................................................................................ 237 Feldmann, K. A., and Marks, M. D. 1987. Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach. Mol. Gen. Genet. 208:1–9. ............................................................. 240

AGROBACTERIUM INFECTS MAIZE, AFTER ALL! Barbara Hohn ............................................................................................................ 249 Grimsley, N., Hohn, T., Davies, J. W., and Hohn, B. 1987. Agrobacteriummediated delivery of infectious maize streak virus into maize plants. Nature 325:177–179. ............................................................................................... 252

THE UPS AND DOWNS OF Bt GENE EXPRESSION IN PLANTS David Fischhoff .......................................................................................................... 255 Vaeck, M., Reynaerts, A., Höfte, H., Jansens, S., De Beuckeleer, M., Dean, C., Zabeau, M., Van Montagu, M., and Leemans, J. 1987. Transgenic plants protected from insect attack. Nature 328:33–37. .................................................. 258 Perlak, F. J., Fuchs, R. L., Dean, D. A., McPherson, S. L., and Fischhoff, D. A. 1991. Modification of the coding sequence enhances plant expression of insect control protein genes. Proc. Natl. Acad. Sci. U.S.A. 88:3324–3328. ................. 263

TRANSFORMATION OF CEREALS BY AGROBACTERIUM Toshihiko Komari ...................................................................................................... 268 Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6:271–282. ........................................... 271 viii


Agrobacterium tumefaciens: From Plant Pathology to Biotechnology

Quorum Sensing TraR AND AAI: AUTOINDUCTION GOES MAINSTREAM Stephen K. Farrand and Lian-Hui Zhang ............................................................... 283 Zhang, L., Murphy, P. J., Kerr, A., and Tate, M. E. 1993. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362:446–447. ............................................................................................... 287 Piper, K. R., Beck von Bodman, S., and Farrand, S. K. 1993. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362:448–450. ..................................................................... 289

DNA Sequencing GOING AFTER THE WHOLE GENOME FROM TWO DIFFERENT ANGLES Brad Goodner and Derek Wood ........................................................ 292 Wood, D. W., Setubal, J. C., Kaul, R., Monks, D. E., Kitajima, J. P., Okura, V. K., Zhou, Y., Chen, L., Wood, G. E., Almeida, N. F., Jr., Woo, L., Chen, Y., Paulsen, I. T., Eisen, J. A., Karp, P. D., Bovee, D., Sr., Chapman, P., Clendenning, J., Deatherage, G., Gillet, W., Grant, C., Kutyavin, T., Levy, R., Li, M.-J., McClelland, E., Palmieri, A., Raymond, C., Rouse, G., Saenphimmachak, C., Wu, Z., Romero, P., Gordon, D., Zhang, S., Yoo, H., Tao, Y., Biddle, P., Jung, M., Krespan, W., Perry, M., Gordon-Kamm, B., Liao, L., Kim, S., Hendrick, C., Zhao, Z.-Y., Dolan, M., Chumley, F., Tingey, S. V., Tomb, J.-F., Gordon, M. P., Olson, M. V., and Nester, E. W. 2001. The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294:2317–2323. ................. 297 Goodner, B., Hinkle, G., Gattung, S., Miller, N., Blanchard, M., Qurollo, B., Goldman, B. S., Cao, Y., Askenazi, M., Halling, C., Mullin, L., Houmiel, K., Gordon, J., Vaudin, M., Iartchouk, O., Epp, A., Liu, F., Wollam, C., Allinger, M., Doughty, D., Scott, C., Lappas, C., Markelz, B., Flanagan, C., Crowell, C., Gurson, J., Lomo, C., Sear, C., Strub, G., Cielo, C., and Slater, S. 2001. Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 294:2323–2328. ..................................... 304

Biological Control of Crown Gall WE HAVE A STRANGE NEW BACTERIOCIN THAT CONTROLS CROWN GALL Allen Kerr and Max Tate ....................................................... 310 Htay, K., and Kerr, A. 1974. Biological control of crown gall: Seed and root inoculation. J. Appl. Bacteriol. 37:525–530. ................................................... 313 Roberts, W. P., Tate, M. E., and Kerr, A. 1977. Agrocin 84 is a 6-N-phosphoramidate of an adenine nucleotide analogue. Nature 265:379–380. .................... 319

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PREFACE The journey from plant pathology to biotechnology began in 1904, one hundred years ago, when Erwin F. Smith started detailed work on crown gall, a disease that affects a wide range of plants. The disease is so called because affected plants have large swellings or galls, usually at the crown of the plant, at or just below soil level. Smith showed that crown gall was caused by a soil-inhabiting bacterium that he called Bacterium tumefaciens, later renamed Agrobacterium tumefaciens. At that time, crown gall was entirely a plant pathological problem but many years later, plant physiologists became interested in the disease, largely due to the work of Armin C. Braun. He made the remarkable discovery that crown gall was really a plant cancer by showing that the causal bacteria could be eliminated from galled tissue but the tissue continued to grow and divide out of control. Also, sterile galls could be grown in culture in the absence of the plant hormones, auxin and cytokinin, which all normal plant tissues require. Braun coined the phrase “tumor inducing principle,” which implied that the bacteria transmitted something to the plant. This work was done in the 1940s. Gradually, evidence built up that the tumor inducing principle had to be nucleic acid, either RNA or DNA. A new phase of understanding began when it was observed that the ability to induce tumors could be transmitted from a pathogenic strain of A. tumefaciens to a non-pathogenic strain. Final proof of the nature of the tumor inducing principle had to wait for the development of techniques in molecular biology. In 1977, a small piece of DNA, called transferred DNA, or T-DNA, located on a large plasmid in A. tumefaciens was shown to be the elusive tumor-inducing principle. But how did it work and even more intriguing, how did it get from bacterium to plant cell? Details of the mechanism are still under investigation. By the mid-1980s, we understood how T-DNA caused plant tumors and we had a fair idea of how the T-DNA was transferred from the bacterium to the plant cell. Could the system be manipulated? Could foreign DNA be transferred by the same system? The answer was a resounding “YES.” Vectors were developed to simplify the transfer process and make it more efficient. Useful genes were located, cloned and transferred to plants. This was the start of the genetic engineering of crops, now popularly (or unpopularly) known as GM crops. Last year, 167.2 million acres (67.7 million hectares) of genetically engineered crops were cultivated in 18 countries, but the cultivation of GM crops is still very controversial. Thus far,

insect and herbicide resistance are the principle traits incorporated into plants, although genes coding for many other useful characters, such as nutrition and flavor, and cold and drought-resistance are waiting in the wings. What started out as an investigation of a plant disease has been developed into a revolutionary tool for agriculture. In this book, we trace the change from plant pathology to biotechnology. We chose what we considered to be the seminal scientific papers in the biology and application of Agrobacterium. This was not an easy task and others would undoubtedly have made a different selection. It is certainly true that many excellent papers are not included because of space limitations. Once we agreed on the papers, we asked several people, who, as far as possible, had been directly involved in the original work, to write a commentary on one or more paper(s). The commentators were asked to give a background to the papers, explain the problems faced and the techniques used. The papers chosen are reprinted here along with the commentaries. We believe they provide an interesting insight into the way fundamental research progresses, which is not readily gleaned from the inherently formal styles required for scientific papers. We hope you find the journey interesting. However, the journey is not over. As the commentary by Paul Hooykaas indicates, it looks as though TDNA will insert into any cell, be it plant, fungal or even mammalian. Is there a possibility of using Agrobacterium in gene therapy? Will Agrobacterium prove to be as useful a tool in fungal genetics as it has been in plant genetics? Its potential is mind-boggling. As is inevitable with the progress of science, important minor themes developed out of the studies on crown gall. Three are represented here, each by two papers and one commentary. First, we have the intriguing phenomenon known as “quorum sensing” in bacteria, a term coined by Stephen Winans to describe population density dependence. Although first discovered in another organism, Vibrio fischeri, it was only when quorum sensing was discovered in Agrobacterium that the widespread nature of the phenomenon was realized. Second, the complete DNA sequencing of one strain of A. tumefaciens by two independent groups was a major achievement, which we thought deserved to be included. The third minor theme in this book is the biological control of crown gall. This control is probably the best known and most efficient biological control system for any plant disease. The control organism, strain K84, had to be genetically engixi


Agrobacterium tumefaciens: From Plant Pathology to Biotechnology

neered to prevent loss of effectiveness and the resulting organism, strain K1026, was the first genetically engineered organism to be released for commercial use in 1988. The crown gall saga is a classic case of curiositydriven research performed by small groups of widely dispersed scientists that ultimately led to a major revolution in agriculture. We suggest that Agrobacterium still contains mysteries that remain to be explored and exploited by future investigators. The assembling of this anthology was greatly aided by the dedicated efforts of numerous individuals who contributed enormously to all stages of the project. Doug Vollgraff, Donna Stewart and Gail Rawson were responsible for typing emails to commentators, locating addresses, requesting permission from journals and commentators and maintaining order to the inflow and outflow of countless email messages from editors, commentators and journal representatives. Gail Rawson deserves special thanks for her yeoman efforts in getting the last version of the manuscript into its final camera-ready form. We also extend our gratitude to

Karen Scholthof for carefully reading two drafts of the commentaries, clarifying unclear statements and catching innumerable trivial mistakes in the format. Karen also identified two anonymous reviewers who provided helpful critiques. Martha Hawes provided the photomicrograph reproduced on the front and back covers. The project would not have been initiated without an invitation from Randy Ploetz and Karen Scholthof representing APS Press. It could not have been completed without the willing participation of all of the commentators who took the time to recall and then write down their own personal significant experience with Agrobacterium tumefaciens. We hope that the finished product will make everyone’s efforts seem worthwhile. Eugene Nester Milton P. Gordon Allen Kerr June 2004

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SOURCES maize plants. Reprinted by permission from Nature 325:177–179. Copyright 1987 Macmillan Publishers Ltd.

Bevan, M. W., Flavell, R. B., and Chilton, M.-D. 1983. A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Reprinted by permission from Nature 304: 184–187. Copyright 1983 Macmillan Publishers Ltd.

Hamilton, R. H., and Fall, M. Z. 1971. The loss of tumor-initiating ability in Agrobacterium tumefaciens by incubation at high temperature. Reprinted by permission from Experientia 27:229–230.

Braun, A. C., and Mandle, R. J. 1948. Studies on the inactivation of the tumor-inducing principle in crown gall. Reprinted by permission from Growth 12:255–269. Copyright 1948 Growth Publishing Co., Inc.

Hernalsteens, J.-P., Van Vliet, F., De Beuckeleer, M., Depicker, A., Engler, G., Lemmers, M., Holsters, M., Van Montagu, M., and Schell, J. 1980. The Agrobacterium tumefaciens Ti plasmid as a host vector system for introducing foreign DNA in plant cells. Reprinted by permission from Nature 287:654–656. Copyright 1980 Macmillan Publishers Ltd.

Bundock, P., den Dulk-Ras, A., Beijersbergen, A., and Hooykaas, P. J. J. 1995. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. Reprinted by permission from EMBO Journal 14:3206–3214.

Herrera-Estrella, A., Van Montagu, M., and Wang, K. 1990. A bacterial peptide acting as a plant nuclear targeting signal: The amino-terminal portion of Agrobacterium VirD2 protein directs a β-galactosidase fusion protein into tobacco nuclei. Reprinted by permission from Proceedings of the National Academy of Sciences of the United States of America 87:9534–9537.

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. Reprinted from Cell 11:263–271. Copyright 1977, with permission from Elsevier. de Framond, A. J., Barton, K. A., and Chilton, M.-D. 1983. MiniTi: A new vector strategy for plant genetic engineering. Reprinted by permission from Bio/Technology 1:262–269.

Herrera-Estrella, L., Depicker, A., Van Montagu, M., and Schell, J. 1983. Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Reprinted by permission from Nature 303:209–213. Copyright 1983 Macmillan Publishers Ltd.

Drummond, M. H., Gordon, M. P., Nester, E. W., and Chilton, M.-D. 1977. Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumors. Reprinted by permission from Nature 269:535–536. Copyright 1977 Macmillan Publishers Ltd.

Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Reprinted from The Plant Journal 6:271–282, by permission from Blackwell Publishing Ltd.

Feldmann, K. A., and Marks, M. D. 1987. Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach. Reprinted by permission from Molecular and General Genetics 208:1–9. Copyright 1987 SpringerVerlag.

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. Reprinted by permission from Nature 303:179–180. Copyright 1983 Macmillan Publishers Ltd.

Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P. R., Flick, J. S., Adams, S. P., Bittner, M. L., Brand, L. A., Fink, C. L., Fry, J. S., Galluppi, G. R., Goldberg, S. B., Hoffmann, N. L., and Woo, S. C. 1983. Expression of bacterial genes in plant cells. Reprinted by permission from Proceedings of the National Academy of Sciences of the United States of America 80:4803–4807.

Holsters, M., Silva, B., Van Vliet, F., Genetello, C., De Block, M., Dhaese, P., Depicker, A., Inzé, D., Engler, G., Villarroel, R., Van Montagu, M., and Schell, J. 1980. The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Reprinted from Plasmid 3:212–230. Copyright 1980, with permission from Elsevier.

Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M. P., and Nester, E. W. 1981. Genetic analysis of crown gall: Fine structure map of the T-DNA by site-directed mutagenesis. Reprinted from Cell 27:143–153. Copyright 1981, with permission from Elsevier.

Htay, K., and Kerr, A. 1974. Biological control of crown gall: Seed and root inoculation. Reprinted from Journal of Applied Bacteriology 37:525–530, by permission from Blackwell Publishing Ltd.

Goodner, B., Hinkle, G., Gattung, S., Miller, N., Blanchard, M., Qurollo, B., Goldman, B. S., Cao, Y., Askenazi, M., Halling, C., Mullin, L., Houmiel, K., Gordon, J., Vaudin, M., Iartchouk, O., Epp, A., Liu, F., Wollam, C., Allinger, M., Doughty, D., Scott, C., Lappas, C., Markelz, B., Flanagan, C., Crowell, C., Gurson, J., Lomo, C., Sear, C., Strub, G., Cielo, C., and Slater, S. 2001. Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Reprinted by permission from Science 294:2323–2328. Copyright 2001 American Association for the Advancement of Science.

Kerr, A. 1969. Transfer of virulence between isolates of Agrobacterium. Reprinted by permission from Nature 223:1175–1176. Copyright 1969 Macmillan Publishers Ltd.

Grimsley, N., Hohn, T., Davies, J. W., and Hohn, B. 1987. Agrobacterium-mediated delivery of infectious maize streak virus into

Perlak, F. J., Fuchs, R. L., Dean, D. A., McPherson, S. L., and Fischhoff, D. A. 1991. Modification of the coding sequence en-

Otten, L., De Greve, H., Hernalsteens, J.-P., Van Montagu, M., Schieder, O., Straub, J., and Schell, J. 1981. Mendelian transmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens. Reprinted by permission from Molecular and General Genetics 183:209–213. Copyright 1981 SpringerVerlag.

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Agrobacterium tumefaciens: From Plant Pathology to Biotechnology hances plant expression of insect control protein genes. Reprinted by permission from Proceedings of the National Academy of Sciences of the United States of America 88:3324–3328.

Van Montagu, M. 2003. Jeff Schell (1935–2003). Reprinted by permission from Nature 423:934. Copyright 2003 Macmillan Publishers Ltd. Photograph of Jeff Schell reprinted by permission of Betsy Schell.

Petit, A., Delhaye, S., Tempé, J., and Morel, G. 1970. Recherches sur les guanidines des tissus de crown gall: Mise en évidence d’une relation biochimique spécifique entre les souches d’Agrobacterium tumefaciens et les tumeurs qu’elles induisent. Reprinted from Physiologie Végétale 8:205–213.

Watson, B., Currier, T. C., Gordon, M. P., Chilton, M.-D., and Nester, E. W. 1975. Plasmid required for virulence of Agrobacterium tumefaciens. Reprinted by permission from Journal of Bacteriology 123:255–264.

Piper, K. R., Beck von Bodman, S., and Farrand, S. K. 1993. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Reprinted by permission from Nature 362:448–450. Copyright 1993 Macmillan Publishers Ltd.

White, P. R., and Braun, A. C. 1941. Crown gall production by bacteria-free tumor tissues. Reprinted by permission from Science 94:239–241. Copyright 1941 American Association for the Advancement of Science.

Roberts, W. P., Tate, M. E., and Kerr, A. 1977. Agrocin 84 is a 6-Nphosphoramidate of an adenine nucleotide analogue. Reprinted by permission from Nature 265:379–380. Copyright 1977 Macmillan Publishers Ltd.

Wood, D. W., Setubal, J. C., Kaul, R., Monks, D. E., Kitajima, J. P., Okura, V. K., Zhou, Y., Chen, L., Wood, G. E., Almeida, N. F., Jr., Woo, L., Chen, Y., Paulsen, I. T., Eisen, J. A., Karp, P. D., Bovee, D., Sr., Chapman, P., Clendenning, J., Deatherage, G., Gillet, W., Grant, C., Kutyavin, T., Levy, R., Li, M.-J., McClelland, E., Palmieri, A., Raymond, C., Rouse, G., Saenphimmachak, C., Wu, Z., Romero, P., Gordon, D., Zhang, S., Yoo, H., Tao, Y., Biddle, P., Jung, M., Krespan, W., Perry, M., Gordon-Kamm, B., Liao, L., Kim, S., Hendrick, C., Zhao, Z.-Y., Dolan, M., Chumley, F., Tingey, S. V., Tomb, J.-F., Gordon, M. P., Olson, M. V., and Nester, E. W. 2001. The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Reprinted by permission from Science 294: 2317–2323. Copyright 2001 American Association for the Advancement of Science.

Smith, E. F., and Townsend, C. O. 1907. A plant-tumor of bacterial origin. Reprinted from Science, New Series, 25:671–673. 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. Reprinted by permission from Nature 318:624–629. Copyright 1985 Macmillan Publishers Ltd. Stachel, S. E., Timmerman, B., and Zambryski, P. 1986. Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells. Reprinted by permission from Nature 322:706–712. Copyright 1986 Macmillan Publishers Ltd.

Yadav, N. S., Vanderleyden, J., Bennett, D. R., Barnes, W. M., and Chilton, M.-D. 1982. Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Reprinted by permission from Proceedings of the National Academy of Sciences of the United States of America 79:6322–6326.

Thomashow, M. F., Nutter, R., Montoya, A. L., Gordon, M. P., and Nester, E. W. 1980. Integration and organization of Ti plasmid sequences in crown gall tumors. Reprinted from Cell 19:729–739. Copyright 1980, with permission from Elsevier.

Yanofsky, M. F., Porter, S. G., Young, C., Albright, L. M., Gordon, M. P., and Nester, E. W. 1986. The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Reprinted from Cell 47:471–477. Copyright 1986, with permission from Elsevier.

Vaeck, M., Reynaerts, A., Höfte, H., Jansens, S., De Beuckeleer, M., Dean, C., Zabeau, M., Van Montagu, M., and Leemans, J. 1987. Transgenic plants protected from insect attack. Reprinted by permission from Nature 328:33–37. Copyright 1987 Macmillan Publishers Ltd.

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A CENTURY OF PIONEERING WORK BEGINS Paul D. Peterson Pee Dee Research & Education Center, Clemson University Florence, South Carolina, United States E-mail: ppeters@clemson.edu Commentary on the paper: Smith, E. F., and Townsend, C. O. 1907. A plant-tumor of bacterial origin. Science, New Series, 25:671–673. In 1902 the United States Department of Agriculture reported that crown gall was “one of the most serious enemies to fruit growers . . . from California to the Alleghenies” (Galloway 1902). This announcement may have been news to some readers, but judging from the amount and frequency of references to the disease on fruit trees and many other economic plants in the late nineteenth and early twentieth century both in America and Europe, crown gall hardly went unnoticed. Various theories had been advanced as to its cause, particularly frost damage, mechanical injury, and fungi. Some researchers suspected that bacteria might be the culprit, but their studies had been largely disregarded due to questionable methodology and to the limited acceptance of bacteria as plant pathogens (Smith and Townsend 1911). Circumstances changed, however, when Erwin Frink Smith, the foremost authority on phytobacteriology, with the assistance of fellow USDA Plant Pathologist Charles Orrin Townsend, began a series of critical investigations in 1904. Three years later Smith and Townsend published the preliminary results from these studies providing wellestablished evidence for the bacterial origin of crown gall. Much had changed in the career of E. F. Smith during the twelve years between 1892 when he first examined tumor-like growths on the roots of peach trees and 1904 when he decided to study a similar gall of Paris daisy (Chrysanthemum frutescens). It was this research that would lead directly to his classic paper establishing the etiology of crown gall. Smith, in 1892, had been a fairly unknown American plant pathologist trying desperately to find his scientific footing in a recently created division of the USDA devoted to solving plant disease problems. The late nineteenth century was an exciting time for advances in plant pathology at the USDA. Departmental researchers had rapid success solving fungal life cycles, and efforts to control several major plant diseases with fungicides were fundamentally altering agricultural practices. Smith had joined the USDA in 1886

with a B.S. in biology from the University of Michigan and spent the first year or so as an assistant in what would later be regarded as seminal work to understand and control grape diseases caused by fungi (Campbell et al. 1999). But while Erwin F. Smith shared in the exciting advances in agricultural science at the USDA, he also suffered his share of frustrating disappointments during his early career. In 1887, he received a new assignment to study two mysterious peach diseases, yellows and rosette. Thus began one of the most arduous periods of his long career. The discovery of the causal agents and controls for these two maladies resisted even Smith’s most thorough observations and meticulous research over the next six years. Smith’s fortune began to change, however, with a reawakened interest in bacteriology. His connection to bacteriology actually extended back to the mid-1880s when he briefly worked in the field of human hygiene and sanitation in Michigan. During this time, Smith took a passionate interest in reviewing the exciting advances in bacteriology coming from the European laboratories of Louis Pasteur, Robert Koch, and Joseph Lister. There also was important work being done in the United States during the period, particularly in the areas of animal and plant bacteriology, and Smith had followed it closely after joining the USDA. He especially was impressed with the landmark study of hog cholera published in 1889 by Theobald Smith and Veranus A. Moore of the USDA’s Bureau of Animal Industry. This paper appears to have inspired his immutable commitment to bacteriological technique. Smith also paid close attention to the groundbreaking research on fire blight of pome fruits by the Americans Thomas J. Burrill and Joseph C. Arthur in the 1880s that had led to the first discovery of plant pathogenic bacteria (Campbell et al. 1999, Griffith et al. 2003). By focusing on bacteriology, Smith discovered his forté. He devoted considerable time to mastering, improving, and communicating information on bacterio1


Agrobacterium tumefaciens: From Plant Pathology to Biotechnology

logical methodology and technique, before applying his increasing expertise to plant diseases. In 1893, he began a series of seminal studies that would lead to the demonstration of the bacterial origins of a number of infamous diseases such as cucumber wilt and southern bacterial wilt (Campbell et al. 1999). Eight years later he had given Alfred Fischer, a distinguished professor of botany at the University of Leipzig, an intellectual thrashing during the well-known FischerSmith controversy between 1898–1901. Fischer, along with other prominent European scientists, refused to accept either the existing proof that myriad plant diseases were caused by bacteria and thus, that plant bacteriology was a credible field of study, or that American scientists had attained the scientific maturity to be leading the movement. In a series of articles in the Centralblatt für Bakteriologie, Smith championed the scientific evidence based on experimental proof using the tools of bacteriology. His characteristic attention to detail and brilliant summations of the state of knowledge led to a victory that was complete and conpicuous (Campbell 1981). He had come a long way from those frustrating days with peach yellows. It was as the acknowledged leader of the field of phytobacteriology that Smith undertook research on the mysterious disease known as crown gall. He had first examined crown gall on the roots of peach trees in 1892, but more pressing research on peach yellows and rosette prevented anything beyond cursory observations. During the 1890s James W. Toumey, a botanist and entomologist at the University of Arizona Agricultural Experiment Station had investigated crown gall of almond and speculated that it might be caused by a myxomycete similar to Plasmodiophora brassicae, known to be the causal agent of club root of cabbage. Toumey and other researchers had demonstrated that crown gall was an infectious disease by obtaining symptomatic tumors on healthy plants either through close contact with diseased tissue or by grafting. Around the turn of the twentieth century, other scientists in the United States and Europe continued to study the disease, but its cause remained baffling (Braun 1982). It was Smith, his colleague C. O. Townsend (an expert on sugar-beet diseases), and their laboratory assistants, Alice Haskins and Nellie A. Brown, who finally discovered the cause of crown gall. Smith’s interest in the disease was aroused when a shipment of tumor-bearing Paris daisy plants arrived at the USDA Bureau of Plant Industry in 1904 from a commercial grower in New Jersey. Unlike his brief observations in 1892, this time Smith was prepared for sustained investigations into the nature of the crown gall disease. Characteristic of his scientific practice, he began

with an intensive review of the literature available on the subject. He paid particular attention, for example, to the research that had been done in France and Italy on gall-forming diseases, especially olive knot caused by Bacillus oleaetuberculosis (Pseudomonas savastanoi), a disease with symptoms similar in appearance to crown gall. After familiarizing himself with other work, he moved on to a series of thorough investigations in his USDA laboratory (Braun 1982, Campbell et al. 1999). Smith and his colleagues found their work painstakingly slow over the next two years. It did not take long for them to eliminate insects and fungi from the list of possible causal agents, but initial attempts to isolate suspect bacteria detected in the interior of undecayed galls went nowhere. Only after it was discovered that bacteria were not “uniformly distributed” in gall tissues and that isolations worked “from that part of the stem where the tumor joins the healthy tissues,” did their investigations produce results. With the ability “to isolate the organism and to demonstrate it unmistakenly in stained sections,” Smith and his coworkers proceeded to produce “subcultures from poured plate colonies” and to inoculate healthy Paris daisy plants with needle punctures, pricking some plants without inoculation as checks. From subsequent galls produced, the bacterium was “reisolated in pure culture,” its identity confirmed, and the experiments repeated. In April, 1907, Smith and Townsend announced their success in Science with “A Plant-Tumor of Bacterial Origin.” They proposed that “the organism causing these tumors” be named Bacterium tumefaciens, while they carefully described the causal agent as “a schizomycete causing rapid multiplication of the young tissues of Chrysanthemum frutescens, Prunus persica, etc.” The authors were anxious to present their discovery of a new bacterial disease, yet these were only the early experiments on the host range of crown gall that Smith and Townsend soon broadened to include a wide variety of cultivated plants (Smith and Townsend 1911). As a matter of note, Smith later credited Fridiano Cavara of Italy with isolating the crown gall bacterium from grape galls a decade before and successfully reinoculating the organism into healthy plants reproducing the disease symptoms (Smith and Townsend 1911). Cavara’s limited studies, however, were less than convincing and seem generally to have been ignored. It appears almost certain that Smith was unaware of this work before publishing his paper in 1907 (Braun 1982, Campbell et al. 1999). This does not detract from the importance of the Smith and Townsend publication. Its merits firmly rest on careful 2


Agrobacterium tumefaciens: From Plant Pathology to Biotechnology

methodology and technique lacking in Cavara’s incomplete studies. Along with the paper in Science, Smith and Townsend also published a translated version of the information on their crown gall studies in the prestigious Centralblatt für Bakteriologie (20: 89–91, 1907). As it turned out, these were only Smith’s first forays into what became a fervid focus on plant galls and the histology of tumors over the rest of his career. Before long he was expanding his idea, hinted at in the 1907 paper, of comparisons between the growth and structure of plant tumors and human cancer. Yet, even with his long and celebrated career in plant bacteriology that followed, it is the classic 1907 paper on the etiology of crown gall that perhaps is best remembered. For with it, a century of pioneering work on Agrobacterium had begun.

Galloway, B. T. 1902. Report of the chief of the Bureau of Plant Industry. Pages 47–108 in: Annual Reports of the Department of Agriculture. GPO, Washington, D.C., U.S.A. Griffith, C. S., Sutton, T. B., and Peterson, P. D., eds. 2003. Fire Blight: The Foundation of Phytobacteriology. American Phytopathological Society, St. Paul, MN, U.S.A. Smith, E. F., and Townsend, C. O. 1911. Crown Gall of Plants; Its Cause and Remedy. USDA Bureau of Plant Industry Bulletin No. 213. GPO, Washington, D.C., U.S.A.

PAUL D. PETERSON: I graduated with a B.A. degree in history, an M.A. in public history/archival management, and a Ph.D. in plant pathology from North Carolina State University. I began my plant pathology studies under the direction of the late Dr. C. Lee Campbell, whose infectious passion for plant pathology and epidemiology cultivated my unique pursuit of merging a career in history and science. I am currently a postdoctoral fellow at Clemson University where I am working with Dr. Bruce Martin on a new disease of turfgrass referred to as Rapid Blight. Also, I am a research associate in the Department of Plant Pathology at North Carolina State University where I direct several history projects. I have written numerous journal articles and three books on the history of plant pathology. I also serve as the historian for the American Phytopathological Society.

References Braun, A. C. 1982. A history of the crown gall problem. Pages 155–210 in: Molecular Biology of Plant Tumors. G. Kahl and J. S. Schell, eds. Academic Press, New York, U.S.A. Campbell, C. L., ed. and trans. 1981. The Fischer– Smith Controversy: Are There Bacterial Diseases of Plants? Phytopathological Classics 13. American Phytopathological Society, St. Paul, MN, U.S.A. Campbell, C. L., Peterson, P. D., and Griffith, C. S. 1999. The Formative Years of Plant Pathology in the United States. American Phytopathological Society, St. Paul, MN, U.S.A.

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