Download Photo - DC Gross

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
Crown gall of grape caused by
Agrobacterium vitis
Photo - D.C. Gross
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/winter01.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/pr01101.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
permeases, endonuclease
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
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 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
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-rise-charts
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