Crown Gall of Higher Plants and Its Biocontrol

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Crown Gall of Higher Plants and Its Biocontrol

• Fure-Chyi Chen
• ???
• Department of Plant Industry
• National Pingtung University of Science
  Technology

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Animal and Plant Protection and Agriculture

• Fall Semester 2005 (Sep. 22, 29)
• Classroom CM108
• Instructor Prof. Fure-Chyi Chen
• E-mail fchen_at_mail.npust.edu.tw
• Laboratory HO210, Phone 6337
• Office Hour 10-12 Saturday or by appointment

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Crown Gall Disease
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Small, pimple-like galls of crown gall
(Agrobacterium vitis) extending up the trunk of a
grapevine
Crown gall of Mark apple rootstock
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Crown gall (arrows) on common bean plant
(Phaseolus vulgaris cv. Pinto)
Crown gall on a 4-year-old alfalfa plant
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Symptoms and signs

• Crown gall is identified by overgrowths appearing
  as galls on roots and at the base or "crown" of
  woody plants such as pome (e.g., apple, pear) and
  stone (e.g., cherry, apricot) fruit and nut
  (e.g., almond, walnut) trees
• Crown galls are also formed on ornamental woody
  crops such as roses, Marguerite daisies, and
  Chrysanthemum spp. as well as on vines and canes
  such as grapevines and raspberries

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Symptoms and signs

• Marguerite daisies, chrysanthemums and grapevines
  can become infected systemically
• Crown gall is caused by Agrobacterium
  tumefaciens, a Gram-negative, bacilliform
  bacterium that is normally associated with the
  roots of many different plants in the field.
• This bacterium can survive in the free-living
  state in many soils with good aeration such as
  sandy loams where crown gall diseased plants have
  grown. The bacterium can also survive on the
  surface of roots (rhizoplane) of many orchard
  weeds

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Assay Hosts

• Plants representing over 93 plant families are
  susceptible to crown gall as judged by
  experimental inoculations.
• Owing to their high susceptibility to crown gall,
  plants such as Jimson weed (Datura stramonium)
  and sunflower (Helianthus annuus) are used as
  assay hosts for testing the degree of virulence
  of A. tumefaciens. Also, Kalanchoë daigmontiana
  (also known as Bryophyllum) is used for assaying
  A. tumefaciens, but the plant is less sensitive
  than Datura

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Representation of the bacilliform Agrobacterium
tumefaciens with circumthecal flagellation,
common pili and the T pilus (produced in induced
cells).
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Pathogen Biology

• Agrobacterium tumefaciens is a rhizoplane
  bacterium whose characteristics are
  Gram-negative, strictly aerobic, bacilliform rods
  measuring 1 x 3 µm, and whose nutritional
  requirements are non-fastidious. The rods bear
  flagella that are arranged subpolarly around the
  cylindrical circumference of the cell, referred
  as circumthecal flagellation
• When A. tumefaciens cells perceive plant phenolic
  compounds, the virulence genes that are located
  in the resident Ti (tumor-inducing) plasmid are
  expressed, resulting in the formation of a long
  flexuous filament called the T pilus

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• The activation of VirA also shuts off motility of
  the circumthecal flagella, presumably when A.
  tumefaciens cells attach to plant cells.
  Attachment to the plant cells is a prerequisite
  for initiating the transfer of the T-DNA into the
  plant cell. Both the circumthecal flagella and
  the T pilus play an essential role in virulence,
  presumably by bringing the bacterial cell to its
  target followed by attachment to the plant host,
  respectively

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T-DNA transfer animation
http//www.biology.ualberta.ca/facilities/multimed
ia/uploads/microbiology/agrobacteriumIII.swf
http//oak.ppws.vt.edu/sforza/agro/agro.html
http//www.ppws.vt.edu/sforza/prokaryote.html
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T-DNA transfer process

• Chemotaxis
• Phenolics
• Sugars
• Amino acids
• Attachment
• chvA
• chvB
• pscA
• AttR
• Virulence-region induction

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Journey of T-DNA transfer

• T-DNA processing
• T-DNA transfer
• Nuclear import of T-DNA
• T-DNA integration

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VirA/VirG

• Two genes, the VirA/VirG two-component
  sensor-transducer system, regulate the production
  of both the transferred oncogenic DNA and the DNA
  transfer machinery from the tumor-inducing Ti
  plasmid
• VirA, a membrane-localized histidine sensor
  kinase, is autophosphorylated upon perceiving
  signals characteristic of host wound sites and
  transfers the phosphoryl group to VirG
• Phosphorylated VirG acts as a transcription
  activator that induces the expression of the
  remaining genes of the vir regulon

J. Bacteriology (2005) 187 2182-2189
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Agrobacterium tumefaciens biotypes and biovars

• Based on some distinct phenotypic differences, A.
  tumefaciens isolates were originally classified
  into three biotypes or biovars (biotype I, II and
  III biovar 1, 2, and 3). Biotype I or biovar 1
  strains produce 3-ketosugars and usually have
  wide host ranges Biotype II or biovar 2 strains
  mainly classify as the hairy root-forming
  organism, A. rhizogenes

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• Biotype III or biovar 3 isolates are mainly
  confined to grapevines, prefer L-tartaric acid
  over glucose and produce polygalacturonase.
  Because grapevine isolates formed a distinct
  group verified by DNA homology studies and were
  frequently limited in host-range to grapevines,
  biovar 3 strains have been reclassified into one
  species, A. vitis. Agrobacterium rubi strains
  infect canes of the genus Rubus, representing
  blackberry and raspberry

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Ti plasmid and virulence genes

• Experimental inoculation of an assay host plant
  such as Jimson weed (Datura stramonium) results
  in tumor formation within two weeks (Figure 6).
  Virulence and the host-range of A. tumefaciens
  are conferred by a large extrachromosomal DNA
  element designated as the Ti plasmid (for
  tumor-inducing) that resides in all virulent
  strains of this pathogen. The Ti plasmid is
  approximately 200 kilobases in length and is
  comprised of a covalently closed, double-stranded
  DNA circular molecule

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Experimental inoculation on the stem of a potted
Datura stramonium (Jimson weed) plant with A.
tumefaciens (electron micrograph). After 2-3
weeks, a crown gall tumor is generated. The
integration of the T-DNA originating from the Ti
plasmid harbored in A. tumefaciens is visualized
by in situ T-DNA-DNA hybridization of the crown
gall chromosome within gall tissue. The T-DNA was
labeled with tritium and the integrated T-DNA
hybridization is seen as a dark band (white
arrow) as detected by x-ray emulsion film layered
on the chromosomes from cells in crown gall.
(Courtesy C. Kado)
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Crown gall causes stunting of growth

• Both loss of yield and stunting of growth may
  occur when seedlings or young cuttings are
  infected in the early stages of plant growth. The
  lack of vigor, reduction in foliage, and water
  stress are associated with chronically diseased
  root systems. When more mature tree crops become
  infected, secondary growths will appear from the
  root systems near the trunk. These "suckers" are
  a good sign that the root system is infected

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Bio-control

• Infection of plants by pathogenic strains of
  Agrobacterium tumefaciens causes crown gall
  tumors with devastating economic consequences
• The most successful bacterial biocontrol agent,
  nonpathogenic A. radiobacter strain K84, prevents
  disease by production of the "Trojan horse" toxin
  agrocin 84

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• Because it imitates a tumor-derived substrate
  agrocinopine A (fig. S1), agrocin 84 is
  specifically imported into A. tumefaciens strains
  that harbor certain types of tumor-inducing (Ti)
  plasmids
• A toxic moiety is released from agrocin 84 that
  inhibits the pathogen by an unknown mechanism

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• Plasmid pAgK84 in strain K84 contains the genes
  for agrocin 84 production and two immunity
  elements
• The translation product of one of these immunity
  genes, agnB2, showed gt40 sequence identity
  between its coding sequence and many leucyl-tRNA
  synthetases (LeuRSs).
• LeuRSs catalyze attachment of leucine to its
  cognate tRNAs in the first step of protein
  synthesis (aminoacylation)
• Aminoacylation assays showed the recombinant
  AgnB2 protein exhibits robust LeuRS activity

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• The structure of the toxic moiety of agrocin 84
  is similar to that of leucyl-adenylate (Leu-AMP),
  a critical enzyme-bound reaction intermediate
  (Fig. 1A), having a relatively stable
  5'-phosphoramidate bond instead of the labile
  phosphoanhydride linkage
• Plausibly, the stable toxic moiety of agrocin 84
  could impart its antibiotic effect on the
  bacteria by binding to the catalytic domain of
  the A. tumefaciens genomic-encoded LeuRS
  (LeuRSAt) as a Leu-AMP mimic

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Science 309 1533 (2005)
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Disease incidence of crown gall
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Disease incidence of crown gall

• Inoculation of Mazzard cherry rootstocks with A.
  tumefaciens B49c or B49c Rfr resulted in a
  significant increase (P 0.05) in the incidence
  of crown gall in all four field sites compared
  with that in the water-treated control (Table 1).

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• Biocontrol of crown gall tumors by agrocin 84
  thus targets a tRNA synthetase in the pathogen
• In turn, strain K84 carries a second,
  self-protective copy of the synthetase
• In principle, this strategy from nature could be
  applied to other crop diseases by delivering
  pathogen-specific toxins with agents that protect
  the delivery vehicle

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Crown gall Management

• Preplanting Management Options
• Management in Established Fields

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Preplanting Management Options

• Planting stocks
• Site selection
• Crop rotation
• Chemical eradicants
• Biological control
• Genetically engineering

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Planting stocks

• Visual examination for crown gall tumors has been
  the conventional primary screen for diseased
  material. The method is limited for complete
  disease control because A. tumefaciens can reside
  on the rhizoplane and systemically in certain
  host plants such as grapevines, chrysanthemums,
  and marguerite daisy

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Site selection

• Fields that have grown cereal crops for a long
  period are favored as crown gall-free sites.
  Fields previously used for growing fruit and nut
  crops can remain infested with A. tumefaciens.
  Certain weeds such as morning glory (Ipomoea
  leptphylla) can serve as natural hosts of A.
  tumefaciens and therefore perpetuate the survival
  of this pathogen in field soils

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Crop rotation

• A crop rotation program employing cereal crops
  followed by green manuring helps reduce the
  population size of A. tumefaciens

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Chemical eradicants

• Eradication of crown gall using creosote-based
  compounds, copper-based solutions, and strong
  oxidants such as sodium hypochlorite are
  transiently effective. The chemical eradicant
  application procedure is labor intensive and
  therefore costly both monetarily and to the
  environment. The superficial treatments are
  ineffective against systemically infected plants.
  Generally, chemicals are rarely used for control
  of crown gall

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Biological control

• Certain strains of A. tumefaciens are sensitive
  to the antibiotic agrocin produced by A.
  radiobacter, a closely related soil-borne
  bacterium that does not infect plants. An example
  of an antibiotic produced is Agrocin-84, which is
  an analog of the opine agrocinopine A.
  Agrocinopine A is produced in crown gall tumors
  induced by A. tumefaciens strains whose Ti
  plasmid encodes for nopaline and agrocinopine A.
  Agrocin-84 mimics agrocinopine A and therefore is
  taken up by the same transport system used by A.
  tumefaciens to utilize agrocinopine A. Inside the
  A. tumefaciens cell, the antibiotic Agrocin-84
  inhibits DNA replication and cellular growth

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Biological control

• Plants are protected against Agrocin-84 sensitive
  strains of the pathogen by dipping the root
  system into a suspension of A. radiobacter K84
  before planting in infested fields. Biological
  control of crown gall has been a very effective
  method to control crown gall in several
  locations. In many other regions where
  agrocin-insensitive strains of A. tumefaciens
  (strains that do not acquire agrocinopine A)
  reside, this biological control strategy is
  ineffective

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Genetically engineering

• Transgenic crop plants harboring one or more
  unique genes tailored to protect the plant from
  crown gall have been developed. Genes encoding
  products that degrade or inactivate the T-DNA
  strand complex when it enters the host cell, that
  prevent the expression of T-DNA genes encoding
  indoleacetic acid and cytokinin biosynthesis, and
  that prevent A. tumefaciens attachment to its
  target are some examples currently being tested.
  Biotechnology companies, such as DNA Plant
  Technology (Oakland, CA), are applying sense
  strand messenger RNA or small-interfering RNAs to
  develop crown gall resistant fruit and nut crops

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Management in Established Fields

• Trees in fruit and nut orchards can be maintained
  over long periods if the trees became infected at
  maturity. Diseased trees will bear crop, but with
  age the trees will become unthrifty and suffer
  dehydration as their root system becomes
  progressively infected. The removal of infected
  trees and vines is costly in loss in time and in
  money. Annual row crops such as sugar beets and
  cotton occasionally will have a few plants with
  crown gall, but the disease is considered of low
  economic importance. Perennial field crops such
  as alfalfa will occasionally become infected with
  A. tumefaciens where the organism is spread by
  the mowing equipment. Usually, rogueing of the
  diseased plants is sufficient to minimize further
  spread of crown gall