1. Agrobacterium tumefaciens The Journey from Plant Pathology to Biotechnology Bonnie Ownley Entomology and Plant Pathology University of Tennessee, Knoxville

2. Agrobacterium tumefaciens Causes crown gall disease of a wide range of mostly broad-leaf (dicots) plants

3. Disease Symptoms Young tumors are round, smooth, and white or lightly colored Older tumors are irregular, rough and dark brown Courtesy [http://biologi.uio.no/plfys/haa/gen/gmo.htm Halvor Aarnes. Gall caused by Agrobacterium tumefaciens.http://biologi.uio.no/plfys/haa/gen/gmo.htm

4. Symptoms of crown gall disease Tumors appear at the soil line of most plants In some plants (grape, blackberry, raspberry) tumors appear on trunk or twigs

5. A. tumefaciens Hosts include apple, pear, peach, cherry, almond, raspberry and roses (A) Agrobacterium vitis (related species) causes crown gall disease in grape (B) Photos – S. Von Broembsen

6. Crown gall of grape caused by Agrobacterium vitis Photo - D.C. Gross

7. Crown gall of grape caused by Agrobacterium vitis Photo - D.C. Gross

8. Disease Symptoms Agrobacterium tumefaciens does not usually cause serious damage to older plants Crown gall can be fatal on young trees if the tumor girdles the tree Crown gall on young olive tree www.australianolives.com.au/ TOP/winter01.htm www.australianolives.com.au/ TOP/winter01.htm

9. Crown gall tumors on trees

10. Agrobacterium tumefaciens Aerobic (requires oxygen), rod- shaped, motile bacterium with 1 to 6 flagella Photo from Schaad et al., 2001.

11. Agrobacterium tumefaciens Prokaryotic (lacks double membrane-bound organelles), Gram-negative cell wall structure G- G-G+ G+ Photo – B. Ownley

12. Agrobacterium tumefaciens Can survive in soil without a host plant for more than 20 years – very unusual! Challenging to control on some crops

13. Do all isolates of A. tumefaciens cause plant disease? No! Only about 10% of isolates are pathogenic

14. Do all isolates of A. tumefaciens cause plant disease? Only isolates of A. tumefaciens that carry the Ti (tumor-inducing) plasmid can cause tumors in plants

15. What is a plasmid?? Plasmid small, extra-chromosomal, hereditary, circular DNA replicates or multiplies independently of the bacterial chromosome can be transferred from one cell to another (by conjugation)

16. Single cell of A. tumefaciens ChromosomeTi plasmid Photo - APS Ti = Tumor-inducing

17. Disease Cycle of A. tumefaciens Wound parasite, plant injury is essential for infection

18. Disease Cycle of A. tumefaciens After entering a wound, the bacteria attach to the plant cell Source – http://www.nsf.gov/od/lpa/news/press/01/pr01101.htm

19. Disease Cycle of A. tumefaciens Plant produces phenolic compounds in response to wounding (these compounds are toxic to many plant pathogens) In response to plant phenolics (acetosyringone), A. tumefaciens begins processing T-DNA (Transfer-DNA) which is carried on the Ti (Tumor-inducing) plasmid

20. Ti Plasmid Vir genespermeases, endonuclease http://arabidopsis.info/students/paaras/ti_plasmid.jpg

21. In the process of causing crown gall disease, Agrobacterium tumefaciens inserts a portion of its Ti-plasmid (the T-DNA) into the chromosome of the host plant Source: http://boneslab.chembio.ntnu.no/Tore/Bilder/AgrotransI.gif

22. Process of transferring T-DNA from the Ti- plasmid in A. tumefaciens to the genomic DNA in the nucleus of the plant cell Source: http://bs.shinshu-u.ac.jp/ HTML/botany/Agro.htm bs.shinshu-u.ac.jp/ HTML/botany/Agro.htmbs.shinshu-u.ac.jp/ HTML/botany/Agro.htm

23. Disease cycle T-DNA is transferred to the plant cell and becomes integrated into the plant cell’s genetic material This permanent genetic change is called transformation

24. What genetic information is carried on the Ti plasmid?

25. 1. - Host range Many dicots and some gymnosperms can be infected by Agrobacterium, but individual isolates of A. tumefaciens are host specific Why? Host range is determined by genes on the Ti plasmid

26. 2. – Opine catabolism The transformed plant cell produces opines (amino acids linked to sugars) Why? A. tumefaciens uses opines as a food source. Opines cannot be used by the plant cell. Part of the plasmid left in the bacterium codes for uptake and catabolism of opines

27. 3. – Plant hormones The transformed plant cell enlarges (hypertrophy = abnormal cell enlargement) and divides (hyperplasia = increased cell division). A tumor results from hypertrophy + hyperplasia Why? The Ti plasmid codes for production of plant hormones (cytokinins and auxin)

28. What genetic information on the Ti plasmid is actually transferred to the plant on T- DNA? Production of plant hormones (cytokinins and auxin) Production and release of opines

29. Role of the Ti plasmid in nature Host range – determines which types of plants will be infected Opine catabolism – provides food for the bacterium Plant hormones – causes cell multiplication and enlargement in the plant

30. Disease Cycle Tumor development takes 5 days to several weeks Tumors enlarge on plants that are growing rapidly Tumors are inactive on plants that are dormant

31. Control of crown gall disease Cultural Practice sanitation Avoid wounding the plants Control root-chewing insects Plant disease-free plants Rotate to corn or grain in infested fields (may take several years)

32. Control of crown gall Biological Use Agrocin K1026 (nonpathogenic genetically engineered Agrobacterium, released in 1988) as a pre-plant root dip Apply before infection Produces an antibiotic (agrocin) and competes with pathogenic isolates for sites on the plant roots

33. Control of crown gall Chemical Bacticin (petroleum product) painted onto small galls or onto cut surface of large galls – limited success Fumigation of soil with chloropicrin

34. From plant disease....….to…….. a revolutionary tool for agriculture

35. From Plant Pathology to Biotechnology First report of genetic exchange between kingdoms Modified Agrobacterium system is used in plant biotechnology to transfer genetic material to plants

36. Significance beyond Plant Pathology Agrobacterium is co-evolving with hosts, away from pathogenicity, toward a more mutualistic relationship, like Rhizobium

37. Genetically modified (GM) Crops http://www.vox.com/2014/8/12/5995087/genetically-modified-crops-rise-charts

38. Genetically modified (GM) Crops Principle genetic traits: Insect resistance Bt toxin genes Herbicide resistance Glyphosate (Round-Up) Resistance to plant viruses Nutrition and flavor Cold and drought resistance