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Pathogencity and Virulence factors
Agrobacterium tumefaciens as for
model for PPB
Artificially induced galls in young tobacco plant by injecting crown gall bacteria
into the stem.
Agrobacterium tumefaciens
• A.tumefaciens is a soil-borne, non-sporulating, motile,
rod-shaped
• phytopathogenic bacterium that elicits neoplastic growth
at the site of infection
• in many dicotyledonous plants causing the “crown-gall”
disease
• This disease by far represents a unique event, involving
the transfer of DNA from a prokaryote into the
chromosomes of plants
• The Agrobacterium genus consists three biovars
Agrobacterium tumefaciens
A. tumefaciens= the causative agents for crown gall disease and A. rubi =
cane gall belong to biovar I group;
A. rhizogenes that causes hairy root cultures belongs to biovar II;
and
A. vitis that causes the galls on grapevines belong to the third group,
biovar III.
Stages of Agrobacterium tumefaciens infection
• There are several stages in the process of infection by A.
tumefaciens
• coordinated responses between an individual viable
bacterium and the host cell
• the crown gall, is imprinted on a large tumor-inducing (Ti)
plasmid residing in the bacterium Ti plasmids are in the
order of 200 to 800 kb
Some of the critical steps involved in the crown gall disease and
subsequent plant
transformation by A. tumefaciens are:
1) Chemotaxis and activation of bacterial virulence;
2) Bacterial attachment;
3) T-DNA processing and transport into plant cell; and
4) Nuclear import, integration of the T-DNA into the plant
genome and xpression of oncogenes.
Chemotaxis and activation of bacterial virulence
• The phenolics and sugar compounds, extruded at the
plant wounded site, is perceived as the signal for
infection by Agrobacterium and serve as inducers (or co-
inducers) for the bacterial virulence (vir) genes located
on the Ti plasmid.
• Agrobacterium uses a two component sensory systems
for both sensing and responding to the presence of
susceptible host cells.
• The VirA sensory system perceives the presence of the
phenolics at the wounded site either directly or indirectly
that is mediated by two chromosomally encoded proteins
P10 and P21
•
Chemotaxis and activation of bacterial virulence
• This results in autophosphorylation of the VirA protein and the
subsequent ransphosphorylation of the VirG protein cytoplasmic
response regulator protein.
• These interactions within the bacterial cell result in the
transcriptional activation of other vir genes.
• Among the various phenolic compounds (e.g., flavonoid precursors)
known to induce vir gene expression the best categorized inducer is
a monocyclic phenolic compound, acetosyringone
Chemotaxis and activation of bacterial virulence
• This protein in turn phosphorylates a second constitutive protein VirG,
followed by a cascade of reactions resulting in the activation of many
other vir genes.
• This cascade of events within the bacterium culminates in the nicking
and transference of a single stranded DNA molecule from the
bacterium into the plant cell.
• The single-stranded DNA molecule, the T-strand resides within the
tumor inducing (Ti) plasmid called T-DNA.
• Most wild-type T-DNAs encode enzymes for the synthesis of low
molecular weight compounds called opines as well as enzymes
involved in the synthesis of plant growth regulators auxin and
cytokinins.
Chemotaxis and activation of bacterial virulence
• The uncontrolled proliferation of crown gall cells results
from the production of auxins and cytokinins by the
bacterium at the wounded sites.
• The wild-type strains of Agrobacterium contain
• T-DNA strands that carry genes involved in the synthesis
of plant growth hormones and opines, while the Ti
plasmid carry genes that encode proteins that can
catabolize opines
Chemotaxis and activation of bacterial virulence
• Opines are low molecular weight carbon compounds (amino
acid and sugars phosphate derivatives) that serve as carbon
and/or nitrogen sources for the tumor-inducing bacterium.
• More than 20 different opines have been identified in crown
galls and hairy roots, but only a few subset of them are
encoded by the T-DNA of any one Agrobacterium strain.
• The basis of different types of opine(s) metabolized in the
infected cells, has been also used for classification of the
infecting Agrobacterium strains (e.g. octopine, nopaline and
agropine-type strains
Chemotaxis and activation of bacterial virulence
• Octopine-type T-DNAs posses four opine synthesis
genes that catalyze the production of octopine (ocs),
agropine (ags), and mannopine (mas1, mas2) and
octopine-type Ti plasmid.
• Octopine-type Ti plasmid encodes nearly 40 genes
related to octopine, agropine and mannopine uptake and
use.
• Opines not only provide a growth substrate but probably
are also involved in conjugal Ti plasmid exchange and
chemotaxsis (
Chemotaxis and activation of bacterial virulence
• The bacterial recognition and attachment is an important step in the infection process.
• In nature, Agrobacterium normally infects wounded plant tissues that are rich in exudates
containing phenolic compounds and sugars that trigger chemotaxis of the bacterium for
attachment
• The phenolics and sugar compounds, extruded at the plant wounded site, is perceived as
the signal for infection by Agrobacterium and serve as inducers (or co-inducers) for the
bacterial virulence (vir) genes located on the Ti plasmid.
• Agrobacterium uses a two component sensory systemsfor both sensing and responding to
the presence of susceptible host cells
• The VirA sensory system perceives the presence of the phenolics at the wounded site either
directly or indirectly that is mediated by two chromosomally encoded proteins P10 and P21
• This results in autophosphorylation of the VirA protein and the subsequent
transphosphorylation of the VirG proteincytoplasmic response regulator protein
(2) Bacterial attachment;
• Following the loose binding to the host, the bacterium synthesizes
cellulose microfilaments, which helps in anchoring the bacterium to
• the plant cell surface
• The binding of the bacterium to the host cell-surface involves a cell-to-
cell recognition step involving a cascade of bacterial attachment proteins
that is encoded by chromosomal genes; chvA, chvB, pscA and att.
• Agrobacterium infection most likely occurs through the formation of an
active biofilm and the subsequent attachment of the bacterium to the
susceptible host cell.
• The process of infection is mediated by certain cell wall receptors, which
could be
• protease-sensitive molecules.
(2) Bacterial attachment;
• These interactions within the bacterial cell result in the transcriptional
activation of other vir genes.
• Among the various phenolic compounds (e.g.,flavonoid precursors)
known to induce vir gene expression the best categorized inducer is a
monocyclic phenolic compound, acetosyringone
• The bacterial recognition and attachment is an important step
in the infection process. In nature, Agrobacterium normally
infects wounded plant tissues that are rich in exudates
containing phenolic compounds and sugars that trigger
chemotaxis of the bacterium for attachment
T-DNA processing and transmembrane transfer of the T-DNA into the
plant
cytoplasm
• The activation of the vir operon leads to the production of a single-
stranded (ss) molecules of T-DNA, termed T-strand.
• All T-DNA elements are delimited by a conserved 25-bp homologous
sequence that defines their left and right borders
Nuclear import of the T-strand, T-DNA integration and expression of the
oncogenes
• The final step in the process of successful transformation of the host
cell depends on the T-strand targeting to the nucleus by effectively
crossing the nuclear membrane barrier.
• The T-strand coated with the protein complex when synthesized has
a very large size (≈12.6 nm); that by far exceeds the size exclusion
limits of the nuclear pore (9 nm),
• the VirD2 and VirE2 proteins are very vital for the nuclear import of
the T-DNA into the nucleus of the plant cell.
• Finally once inserted into the plant genome the crown gall tumors
result from overproduction of auxin and cytokinin in plant cells
transformed by A. tumefaciens
• These abnormally high phytohormone levels result from
• expression of three genes transferred stably into the plant genome
from the A. tumefaciens tumor-inducing (Ti) plasmid
Nuclear import of the T-strand, T-DNA integration and expression
of the
oncogenes
• Once inside the plant cell, the T-DNA is targeted to the plant nucleus
wherein it randomly integrates into the host genomic DNA through
an unknown process, which most likely is by non-homologous
recombination
GENOME ORGANIZATION OF AGROBACTERIUM TUMEFACIENS
• The completion of the genome sequencing and annotation
of the different genomes of A. tumefaciens C58 strain,
reaffirmed the presence of four replicons.
• a circular chromosome, a linear chromosome and plasmids
pAtC58 and pTiC58
• The current annotation of the genome of A. tumefaciens
contains 5,482 predicted protein coding genes.
• Proteins encoded by 5,415 genes have been assigned a
putative function or classified as hypothetical protein,
whose functions are unknown in other related genomes.
A.tumefaciens
• The host range is wide and includes dicotyledons of more than 643
species from 331 general of 93 families.
• Monocotyledons are generally much less susceptible.
• . However, autonoumous growth has been observed in tumor cells
of some conifer species
The resistance of monocots to crown gall disease is a result of some of
the following reasons.
1. bacterial attachment to the surface host plant cell is
insufficient.
2. Transfer of T – DNA is incomplete because wound
healing reactions do not accompany the hypertrophy
and/or hyperplasia,
3. T – DNA cannot be incorporated in plant genome.
4. host plant cells do not respond to plant hormones that
are produced by the transformed tumor cells.

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Chapter_ 7 Pathogencity Agrobacterium.ppt

  • 1. Pathogencity and Virulence factors Agrobacterium tumefaciens as for model for PPB
  • 2. Artificially induced galls in young tobacco plant by injecting crown gall bacteria into the stem.
  • 3. Agrobacterium tumefaciens • A.tumefaciens is a soil-borne, non-sporulating, motile, rod-shaped • phytopathogenic bacterium that elicits neoplastic growth at the site of infection • in many dicotyledonous plants causing the “crown-gall” disease • This disease by far represents a unique event, involving the transfer of DNA from a prokaryote into the chromosomes of plants • The Agrobacterium genus consists three biovars
  • 4. Agrobacterium tumefaciens A. tumefaciens= the causative agents for crown gall disease and A. rubi = cane gall belong to biovar I group; A. rhizogenes that causes hairy root cultures belongs to biovar II; and A. vitis that causes the galls on grapevines belong to the third group, biovar III.
  • 5. Stages of Agrobacterium tumefaciens infection • There are several stages in the process of infection by A. tumefaciens • coordinated responses between an individual viable bacterium and the host cell • the crown gall, is imprinted on a large tumor-inducing (Ti) plasmid residing in the bacterium Ti plasmids are in the order of 200 to 800 kb
  • 6. Some of the critical steps involved in the crown gall disease and subsequent plant transformation by A. tumefaciens are: 1) Chemotaxis and activation of bacterial virulence; 2) Bacterial attachment; 3) T-DNA processing and transport into plant cell; and 4) Nuclear import, integration of the T-DNA into the plant genome and xpression of oncogenes.
  • 7.
  • 8. Chemotaxis and activation of bacterial virulence • The phenolics and sugar compounds, extruded at the plant wounded site, is perceived as the signal for infection by Agrobacterium and serve as inducers (or co- inducers) for the bacterial virulence (vir) genes located on the Ti plasmid. • Agrobacterium uses a two component sensory systems for both sensing and responding to the presence of susceptible host cells. • The VirA sensory system perceives the presence of the phenolics at the wounded site either directly or indirectly that is mediated by two chromosomally encoded proteins P10 and P21 •
  • 9. Chemotaxis and activation of bacterial virulence • This results in autophosphorylation of the VirA protein and the subsequent ransphosphorylation of the VirG protein cytoplasmic response regulator protein. • These interactions within the bacterial cell result in the transcriptional activation of other vir genes. • Among the various phenolic compounds (e.g., flavonoid precursors) known to induce vir gene expression the best categorized inducer is a monocyclic phenolic compound, acetosyringone
  • 10. Chemotaxis and activation of bacterial virulence • This protein in turn phosphorylates a second constitutive protein VirG, followed by a cascade of reactions resulting in the activation of many other vir genes. • This cascade of events within the bacterium culminates in the nicking and transference of a single stranded DNA molecule from the bacterium into the plant cell. • The single-stranded DNA molecule, the T-strand resides within the tumor inducing (Ti) plasmid called T-DNA. • Most wild-type T-DNAs encode enzymes for the synthesis of low molecular weight compounds called opines as well as enzymes involved in the synthesis of plant growth regulators auxin and cytokinins.
  • 11. Chemotaxis and activation of bacterial virulence • The uncontrolled proliferation of crown gall cells results from the production of auxins and cytokinins by the bacterium at the wounded sites. • The wild-type strains of Agrobacterium contain • T-DNA strands that carry genes involved in the synthesis of plant growth hormones and opines, while the Ti plasmid carry genes that encode proteins that can catabolize opines
  • 12. Chemotaxis and activation of bacterial virulence • Opines are low molecular weight carbon compounds (amino acid and sugars phosphate derivatives) that serve as carbon and/or nitrogen sources for the tumor-inducing bacterium. • More than 20 different opines have been identified in crown galls and hairy roots, but only a few subset of them are encoded by the T-DNA of any one Agrobacterium strain. • The basis of different types of opine(s) metabolized in the infected cells, has been also used for classification of the infecting Agrobacterium strains (e.g. octopine, nopaline and agropine-type strains
  • 13. Chemotaxis and activation of bacterial virulence • Octopine-type T-DNAs posses four opine synthesis genes that catalyze the production of octopine (ocs), agropine (ags), and mannopine (mas1, mas2) and octopine-type Ti plasmid. • Octopine-type Ti plasmid encodes nearly 40 genes related to octopine, agropine and mannopine uptake and use. • Opines not only provide a growth substrate but probably are also involved in conjugal Ti plasmid exchange and chemotaxsis (
  • 14. Chemotaxis and activation of bacterial virulence • The bacterial recognition and attachment is an important step in the infection process. • In nature, Agrobacterium normally infects wounded plant tissues that are rich in exudates containing phenolic compounds and sugars that trigger chemotaxis of the bacterium for attachment • The phenolics and sugar compounds, extruded at the plant wounded site, is perceived as the signal for infection by Agrobacterium and serve as inducers (or co-inducers) for the bacterial virulence (vir) genes located on the Ti plasmid. • Agrobacterium uses a two component sensory systemsfor both sensing and responding to the presence of susceptible host cells • The VirA sensory system perceives the presence of the phenolics at the wounded site either directly or indirectly that is mediated by two chromosomally encoded proteins P10 and P21 • This results in autophosphorylation of the VirA protein and the subsequent transphosphorylation of the VirG proteincytoplasmic response regulator protein
  • 15. (2) Bacterial attachment; • Following the loose binding to the host, the bacterium synthesizes cellulose microfilaments, which helps in anchoring the bacterium to • the plant cell surface • The binding of the bacterium to the host cell-surface involves a cell-to- cell recognition step involving a cascade of bacterial attachment proteins that is encoded by chromosomal genes; chvA, chvB, pscA and att. • Agrobacterium infection most likely occurs through the formation of an active biofilm and the subsequent attachment of the bacterium to the susceptible host cell. • The process of infection is mediated by certain cell wall receptors, which could be • protease-sensitive molecules.
  • 16. (2) Bacterial attachment; • These interactions within the bacterial cell result in the transcriptional activation of other vir genes. • Among the various phenolic compounds (e.g.,flavonoid precursors) known to induce vir gene expression the best categorized inducer is a monocyclic phenolic compound, acetosyringone • The bacterial recognition and attachment is an important step in the infection process. In nature, Agrobacterium normally infects wounded plant tissues that are rich in exudates containing phenolic compounds and sugars that trigger chemotaxis of the bacterium for attachment
  • 17. T-DNA processing and transmembrane transfer of the T-DNA into the plant cytoplasm • The activation of the vir operon leads to the production of a single- stranded (ss) molecules of T-DNA, termed T-strand. • All T-DNA elements are delimited by a conserved 25-bp homologous sequence that defines their left and right borders
  • 18. Nuclear import of the T-strand, T-DNA integration and expression of the oncogenes • The final step in the process of successful transformation of the host cell depends on the T-strand targeting to the nucleus by effectively crossing the nuclear membrane barrier. • The T-strand coated with the protein complex when synthesized has a very large size (≈12.6 nm); that by far exceeds the size exclusion limits of the nuclear pore (9 nm), • the VirD2 and VirE2 proteins are very vital for the nuclear import of the T-DNA into the nucleus of the plant cell. • Finally once inserted into the plant genome the crown gall tumors result from overproduction of auxin and cytokinin in plant cells transformed by A. tumefaciens • These abnormally high phytohormone levels result from • expression of three genes transferred stably into the plant genome from the A. tumefaciens tumor-inducing (Ti) plasmid
  • 19. Nuclear import of the T-strand, T-DNA integration and expression of the oncogenes • Once inside the plant cell, the T-DNA is targeted to the plant nucleus wherein it randomly integrates into the host genomic DNA through an unknown process, which most likely is by non-homologous recombination
  • 20. GENOME ORGANIZATION OF AGROBACTERIUM TUMEFACIENS • The completion of the genome sequencing and annotation of the different genomes of A. tumefaciens C58 strain, reaffirmed the presence of four replicons. • a circular chromosome, a linear chromosome and plasmids pAtC58 and pTiC58 • The current annotation of the genome of A. tumefaciens contains 5,482 predicted protein coding genes. • Proteins encoded by 5,415 genes have been assigned a putative function or classified as hypothetical protein, whose functions are unknown in other related genomes.
  • 21. A.tumefaciens • The host range is wide and includes dicotyledons of more than 643 species from 331 general of 93 families. • Monocotyledons are generally much less susceptible. • . However, autonoumous growth has been observed in tumor cells of some conifer species
  • 22. The resistance of monocots to crown gall disease is a result of some of the following reasons. 1. bacterial attachment to the surface host plant cell is insufficient. 2. Transfer of T – DNA is incomplete because wound healing reactions do not accompany the hypertrophy and/or hyperplasia, 3. T – DNA cannot be incorporated in plant genome. 4. host plant cells do not respond to plant hormones that are produced by the transformed tumor cells.