Agrobacterium tumefaciens The Journey from Plant Pathology to

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

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

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.

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

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

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

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/winter 01. htm

Crown gall tumors on trees

Agrobacterium tumefaciens • Aerobic (requires oxygen), rodshaped, motile bacterium with 1 to 6 flagella Photo from Schaad et al. , 2001.

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

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

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

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

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)

Single cell of A. tumefaciens Ti = Tumor-inducing • Chromosome Photo - APS Ti plasmid

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

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/pr 01101. htm

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

Ti Plasmid http: //arabidopsis. info/students/paaras/ti_plasmid. jpg Vir genes endonuclease permeases,

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/Agrotrans. I. gif

Process of transferring T-DNA from the Tiplasmid 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

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

What genetic information is carried on the Ti plasmid?

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

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

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)

What genetic information on the Ti plasmid is actually transferred to the plant on TDNA? • Production of plant hormones (cytokinins and auxin) • Production and release of opines

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

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

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)

Control of crown gall • Biological • Use Agrocin K 1026 (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

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

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

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

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

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

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