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Chapter 11
Hypersensitive reaction and pathogenicity
(Hrp)
Hypersensitive reaction and pathogenicity (Hrp)
 Pathogenic bacteria produce hypersensitive
responses on non-host plants but cause disease on
their respective host plants.
 Hrp genes are required for both the pathogenicity
and the elicitation of the HR.
 Hrp genes were first cloned by Tn5 mutagenesis by
Peter B. Lindgren in 1986.
Avirulence gene of bacteria
 The phytopathogenic bacteria seem to deposit
proteins within the host cytoplasm via a specialized
secretion mechanism known as a type-III secretion
system (TTSS). => Hrp
 The translocated proteins are known as “effectors”
by analogy to those of bacterial pathogens of
animals.
The Hrp secretory apparatus of P. syringae pv.
syringae 61
(conjugation)
Hrp pilus
 First found in P. syringae pv. tomato DC3000 (Pst DC3000) in
1997 by S. He.
 Also found in Ralstonia solanacearum, Erwinia amylovora,
Sinorhixobium fredii and Xanthomonas campestris
 Hrp pilus (8nm X 2mm) is encoded by hrp genes that involved
in the type III secretion system.
 The major subunits of the hrp-dependent pili are all small
proteins (6-11 kDa); hrpA in P. syringae and E. amylovora ,
hrpE in Xanthomonas campestris pv. vesicatoria and hrpY in
R. solanacearum.
 Their protein sequences are hypervariable, even within
pathovars of P. syringae.
 For example, the major subunit of the Hrp pilus of Pst
DC3000 is the 11kDa HrpA protein, which shares only 30%
identity with the HrpA protein of P. syringae pv syringae.
Hrp pilus
 The major subunit of the E. amylovora Hrp pilus is
a 6.5kDa protein that share only 30% identity
with the carboxy-terminus protein of the Pst
DC3000 HrpA protein
 The structure protein (7kDa HrpY) of the R.
solanacearum Hrp pilus share no detectable
similarity with the Hrp A protein of P. syringae or
E. amylovora.
Function of Hrp pilus
 The hrpA mutants of P. syringae and E. amylovora
and hrpY mutants of R. solanacearum do not cause
disease in susceptible plants, nor do they elicit the
defense-associated hypersensitive response in
resistant plants, suggesting a requirement of the Hrp
pilus for bacterial interactions with plants.
 Hrp pilus has been hypothesized to attach bacteria to
plant cells and to deliver type III secreted proteins.
 The immunogold labeling experiment showed that all
examined type III secreted proteins, including HrpZ,
HrpW, and AvrPto of P. syringae and HrpN and DspE
of E. amylovora, were localized along the entire length
of the Hrp pilus.
Function of Hrp pilus
 However, mutational analysis showed that the Hrp
pilus of R. solanacearum is not required for
adhering bacteria to infection sites in tomato.
(Mol. Microbiol. 2000. 36:249-260)
Assembly of the Hrp pilus
 Hrp pilus subunits (pilin) are assembled in the
periplasmic space by the assistance of the type III
secretion apparatus, which uses ATP.
 Unfolded effector proteins associate with the growing
Hrp pilus within the type III apparatus and are
transported like a cargo on a conveyor belt within the
lumen of the pilus.
 Secretion of effector proteins and release into plant
host cells might then occur by depolymerization of the
pilus tip, which might be closed by a putative cap
protein.
 Such cap protein could also function as an initiator of
pilus assembly and help to open the secretion channel
in the outer membrane (HrcC) for passage of the
growing pilus.
Model of the assembly of Hrp pili and
transport of effector proteins
Model for T-complex transfer through the transmembrane
VirB channel from A. tumefaciens to host plant cells
Type IV secretionassociated pilus
END
Proposed
biochemical
models of the
RRS1-R–PopP2
interaction
Impa, importin a
LRR, leucine-rich
repeat domain
NB, nucleotidebinding site
NLS, nuclear
localization signal
S, SUMO
TIR, homologous
to Toll/interleukin1-receptor
TTS, type-III
secretion system
WRKY, WRKY
DNA-binding
Structure of the plasmodesmatal channel and transport
complexes of the movement protein–viral RNA
Resistance
 Race-specific resistance
 Race-nonspecific resistance
Also called general resistance
 Nonhost resistance -> HR
 Systemic acquired resistance (SAR)
Quadratic check for gene-for-gene concept
R-
rr
A-
resistance
diseased
aa
diseased
diseased
A : avirulence gene (avr)
a : virulence gene
R : resistance gene (R)
r : susceptible
Oxidative burst
 Reactive oxygen species (ROS), such as superoxide
anion radical (O2-), hydrogen peroxide (H2O2),
hydroxyl radical (.OH), and singlet oxygen (1O2*)
are routinely generated at low levels by plant cells.
 ROS are generated from photosystem II activity in
chloroplast, electron transport in mitochondria, boxidation in glyoxysome, photorespiration in
peroxisome, and other oxidoreduction reaction in
cytosol.
 They are highly reactive and toxic to plant cells.
Reactive oxygen species (ROS)
 Higher plants posses various enzymes to detoxify
these ROS.), hydrogen peroxide (H2O2), hydroxyl
radical (.OH), and singlet oxygen (1*).
Superoxide dismutase
2O2- + 2H+
H2O2 + O2
Peroxidase
H2O2 + AH2
H2O2
H2O + A
Catalase
H2O + 1/2O2
Hypersensitive reaction (HR)
E. C. Stakman (1915) is generally credited
with the use of the term, hypersensitive
reaction (HR).
HR involves the extremely rapid death of
only a few host cells which limits the
progression of the infection.
Cf5 – avr5 interactions
(Race-specific
resistance)
Cladosporium fulfum
Avr5
Phosphatase
H+
NADPH
oxidase
G protein
H-ATPase H-ATPase
-Pi
CaMKII
PKC-like kianse
Superoxide Ascorbate
Peroxidase
dismutase peroxidase
Ferricyanide
reduction
H+
O2
O2 -
H2O2
O2
H2 O
O2
NAD+ NADH
O2-
Cf5
Characteristics of hypersensitive response
Cessation of cytoplasmic streaming
Formation of large vesicles in the cytoplasm
Membrane damage
Protoplast (vacuoles) collapse
Burst of reactive oxygen species
Release of second metabolites – fluorescent
compounds
Browning of cells
Hypersensitive response (HR)
The production of reactive oxygen species in
disease resistance response, a process
known as oxidative burst, was first observed
in potato discs inoculated with a race of P.
infestans that results in an HR.
Oxidative burst has been subsequently
found in several other host-parasite
interactions or elicited cells.
Hypersensitive response
 Proceded by the burst of reactive oxygen species, e.g.,
superoxide anion radical (O2-), hydrogen peroxide
(H2O2), hydroxyl radical (.OH), and singlet oxygen
(1O2*).
 An NADPH-dependent oxidase located on the plasma
membrane is thought to produce O2-, which in turn is
converted to .OH and H2O2.
 Hydroxyl radical (.OH) is the strongest oxidant and
can initiate radical chain reaction leading to lipid
peroxidation, enzyme inactivation, and nucleic acid
degradation.
 The active oxygen species may contribute to cell death
of plant cells or act to kill the pathogen directly.
Hypersensitive response
Exogenously supplied H2O2 stimulates plant
cells to accumulate phytoalexins.