2. About Pseudomonas syringae
Pseudomonas syringae is an agriculturally important
pathogen – more than 50 identified pathovars
cause disease on various plants.
It is rod-shaped, Gram negative, with polar
flagella.
http://centennial.plantpath.wisc.edu/seminars/lindow/
3. About Pseudomonas syringae
It used as a model system for the study of bacterial
plant pathogenesis. It has a similar mode of action as
the human pathogen Yersinia pestis.
http://www.avrdc.org/LC/tomato/tomato_diseases/index.html, http://www.caf.wvu.edu/kearneysville/disease_descriptions/omblist.html
4. Mode of infection
Successful infection by P. syringae depends upon
bacterial effector proteins injected into plant cells via
type III secretion system (T3SS).
Many Gram negative plant pathogens use type-III
secretion systems to infect plants.
T3SS proteins can be grouped into three categories:
Structural proteins
Effector proteins
Chaperones
5. Harpins - similar proteins
Structurally unrelated “Harpin” or “Harpin-like”
proteins share biochemical features.
Harpins have been reported to associate with
membranes and form ion conducting pores; this
suggests a role in nutrient release or effector
delivery.
In Pseudomonas syringae the protein HrpZ1 has a
similar role – pore formation
6. HrpZ1
Essential for type-III secretion effector delivery
hrpJ mutants are impaired in HrpZ1 secretion and
T3SS effector delivery; but not in T3SS effector
secretion. Therefore, HrpZ1 is probably involved in
effector delivery during bacterial infection.
HrpZ1 is similar to Yersinia’s YopB, part of the pore
complex for effector translocation. It’s essential for
the translocation of Yop effector proteins and
displays a contact-dependent membrane disrupting
activity
7. Microbial genome analysis: insights into virulence, host adaptation and evolution
(http://www.ncbi.nlm.nih.gov/pubmed/11262871)
8. hrp/hrc genes
The genes for the type III secretion system are on a
pathogenicity island that has an hrp operon
(hypersensitivity response and pathogenicity).
The Pseudomonas syringae hrp pathogenicity island
is composed of a cluster of type III secretion genes
bounded by exchangeable effector and conserved
effector loci that contribute to parasitic fitness and
pathogenicity in plants
hrp/hrc genes are probably universal among
necrosis-causing Gram-negative plant pathogens
10. Journal of General Plant Pathology; Feb2006, Vol. 72 Issue 1, p26-33, DOI:10.1007/s10327-005-0240-1
11. The hypersensitive response
In nonhost plants or in host plants with race-specific
resistance, the bacteria elicit the hypersensitive
response (HR), a rapid, defense-associated
programmed death of plant cells in contact with the
pathogen.
Cell death creates a physical barrier to movement
of the pathogen and in some plants dead cells can
release compounds toxic to the invading pathogen.
12.
13. Harpins
Research had indicated that pathogenic bacteria
were likely to have a single factor that was
responsible for triggering the HR
The target protein was encoded in the hrp gene
cluster.
This protein was given the name Harpin (encoded
by hrpN).
14. More about harpins
Harpin acts by eliciting a complex natural defense
mechanism in plants, analogous to a broad
spectrum immune response in animals.
Harpin elicits a protective response in the plant that
makes it resistant to a wide range of fungal,
bacterial, and viral diseases.
Harpin protein has the potential to substantially
reduce use of more toxic pesticides, especially
fungicides and certain soil fumigants, such as methyl
bromide.
15. “Harpin”, patented
This meant that Harpin Protein triggered a Systemic
Acquired Resistance (SAR), a plant defense
mechanism that provides resistance to a variety of
viral, bacterial, and fungal pathogens.
Sprayed topical application of Harpin in small
quantities would effectively activate plant defense
responses. Without eliciting any visible HR. The
effects of Harpin on disease resistance and growth,
together with the simple means of application,
provided the basis for commercializing Harpin
Proteins.
16.
17. Harpin Protein, the active ingredient of Messenger, acts as a
pathogen attacking the plant when sprayed. This stimulates growth
within the plant and increases its natural self defense system. The
benefits of Messenger treated plants are better disease control, less
viruses, increased yield, better quality crop and longer shelf life.
http://www.insectscience.co.za/index.cfm?Cid=1817838152
18. In Summary
Harpins are heat-stable, glycine-rich type III-
secreted proteins produced by plant pathogenic
bacteria, which cause a hypersensitive response
(HR)
HrpZ1 and related proteins elicit innate immune
responses in non-cultivar specific manner in various
plants; therefore these harpins are proposed to
resemble pathogen associated molecular patterns
(PAMPS), activating PAMP-triggered immunity (PTI).
19. Focus on HrpZ1
HrpZ1 has a N-term harpin-like domain.
HrpZ1 associates with hrp pili, possibly serving as
stabilizers or has pilus-tip associated functions
during effector delivery.
HrpZ1 can trigger MAPK activation, production of
antimicrobial ROS and phytoalexins, trigger
hypersensitive response (HR), and mount systemic
acquired resistance (SAR) responses in various
plants.
20. The questions
Is the pore forming activity of HrpZ1 functionally
linked to the immunity stimulating activities of the
protein?
What is the mode of recognition of HrpZ1 at the
plant cell surface?
21. Pore formation experiments
HrpZ1 proteins can integrate into planar lipid
bilayers and form cation conducting pores.
They added a sodium sensitive fluorescent dye,
Sodium Green into synthetic liposomes.
22. Pore formation experiments
They added NaCl to dye filled liposomes, tried to
excite the dye with a 530 nm wavelength, and
nothing happened.
When they added recombinant HrpZ1with NaCl
and excited the dye, fluorescence was detected.
This suggests that HrpZ1 facilitated the entry of
sodium and thus, excitation of the dye.
23. Pore formation experiments
They used complete destruction of liposomes using the
detergent Triton X-100 to determine the maximum
fluorescence, and from there calculate relative fluorescence.
24. Pore formation experiments
They found that fluorescence was dependent on
sodium concentration used – they went from 2.5 mM
to 15 to 25 mM.
Based on this, they decided to use a concentration
of HrpZ1 of .5 to 2 microMolar and 25 miliMolar
NaCl for rapid detection of pore forming activity.
25. Experiments on parsley cells
They used a parsley cell suspension. Parsley’s
reponse to microbial molecules include the activation
of two MAPK: MPK3 and MPK6.
When they treated the parsley cells with HrpZ1
they saw the MAPK within 10 minutes. They found
this by immunoprecipitation, using 2 monospecific
antibodies.
26.
27. Experiments on parsley cells
They also saw that recognition of microbial molecular
patterns results in rapid alteration of gene expression.
They did a cDNA-AFLP experiment (complementary DNA-
amplified fragment length polymorphism)
They used RNA samples from parsley cells, and treated
with either HrpZ1 or a different PAMP, Pep-13, for 1 or 4
hours. The Venn diagrams show the overlap of genes that
are expressed as a result of the 2 treatments.
They verified several of these genes with semi-
quantitative RT-PCR
28. Experiments on parsley cells
Another reason they chose to use parsley cells for
these experiments is that they produce an
antimicrobial “furanocoumarin phytoalexin”
The addition of increasing concentrations of HrpZ1
resulted in increased concentration of phytoalexins.
29. Experiments on parsley cells
Based on this data, they declared that:
Different, unrelated microbial patterns trigger
conserved, generic but complex transcriptome response
HrpZ1 triggers immunity-associated responses in
parsley cells.
30. Binding experiments
For their next experiments, they used radio-
iodinated HrpZ1 to characterize the HrpZ1 binding
site on parsley membranes.
They add the radio-ligand and it’s shown that
maximum binding is achieved 20-30 min after
addition. They added 100-fold molar excess
HrpZ1, unlabeled, and saw an almost complete
replacement of the radioligand; this told them that
binding of HrpZ1 is reversible.
31.
32. Binding experiments
Researchers did binding experiments with increasing
concentrations of radio-iodinated HrpZ1 and
discovered that the binding site was saturated at
concentrations higher than 200 nM.
In competition experiments with increasing
concentrations of unlabeled HrpZ1 in the presence
of radioiodinated HrpZ1, the inhibitor concentration
required to block 50% of binding sites is revealed.
All this data told them that there is a single binding
site for HrpZ1 on parsley membranes.
33. Binding experiments
What they also wanted to know is the molecular nature of this
binding site. To find out, the treated the parsley membranes
with either trypsin, the nonspecific proteinase E, or heated for
10 min at 95 C., and observed the proteolysis by SDS-PAGE.
This is in contrast to the binding of the PAMP Pep3 to its
receptor, which is heat and protease sensitive.
34.
35. Comparison
These graphs compare :
The pore-formation ability of HrpZ1 to other glycine-
rich heat stable proteins that trigger plant defenses.
The triggering of phytoalexin production by HrpZ1 vs
other proteins.
Only HrpZ1, but not the other related proteins,
triggers phytoleaxin production. Then, pore
formation doesn’t really explain the ability of
HrpZ1 to trigger plant immunity associated
defenses in parsley.
36. Deletion mutants
To see if individual regions within HrpZ1 were
important for both activities of the protein, they
produced and tested a library of recombinant
HrpZ1 deletion mutants, for both pore formation
and stimulation of plant immune response.
They measured stimulation of plant immune
response based on MAPK activation, pathogenesis
related gene expression, and phytoalexin
production.
37. Deletion mutants
They used:
A N-term fragment aa 1 to 80
A central fragment aa 100 to 200
A C-term fragment aa 201-345
All constructs were expressed as His-tagged fusion
proteins in E. Coli and purified in Ni-NTA affinity
chromatography.
38.
39. Deletion mutants
Only full length HrpZ1 can form pores in the liposome
assay.
Both full length HrpZ1 and the C-term fragment can
elicit MAPK activity, phytoalexin production, and PR
gene expression.
When ligand binding experiments were performed
with HrpZ1 fragments as competitors, only the C-term
fragment of HrpZ1 was as effective as intact HrpZ1.
This indicates that the binding of HrpZ1 to the binding
site mediates HrpZ1-induced plant defense.
40. Insertional mutagenesis
To get some info about the motif within the HrpZ1
C-term that is sufficient for plant defense activation,
they used a series of HrpZ1 mutants with single
insertions of 15 nts.
Mutants were tested for their abilities to trigger
Na+ dependent fluorescence in the liposome assay,
and phytoalexin production in parsley cells.
41.
42. Insertional mutagenesis
The same mutants were tested as elicitors of
phytoalexin production and the researchers saw a
lot of differences .
They found that insertions in the C-term part of
HrpZ1 negatively affected the elicitor activity of
the protein.
43. Conclusion
Pore formation and plant-immunity stimulating
activities of HrpZ1 are structurally separable.
HrpZ1 can bind membranes in a ligand-receptor
like manner but the binding site appears to not be
a protein.
44. Major findings that support this conclusion:
HrpZ1 related proteins from various phytopathogenic
bacteria possess pore-forming abilities, but fail to
trigger defense responses in parsley.
A C-terminal fragment of HrpZ1 is sufficient to trigger
immunity associated responses in parsley and tobacco,
but is insufficient to form ion-conducting pores.
Insertional mutagenesis revealed a number of structural
alterations within the protein without significantly
altering its biochemical activity.
45. Sources
Separable roles of the Pseudomonas syringae pv. phaseolicola accessory protein
HrpZ1 in ion-conducting pore formation and activation of plant immunity. Engelhardt
S, Lee J, Gäbler Y, Kemmerling B, Haapalainen ML, Li CM, Wei Z, Keller H, Joosten
M, Taira S, Nürnberger T. Plant J. 2009 Feb;57(4):706-17. Epub 2008 Oct 16.
Microbial genome analysis: insights into virulence, host adaptation and evolution. B
W Wren. Nat Rev Genet. 2000 October; 1(1): 30–39. doi: 10.1038/35049551.
Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are
functionally similar to HrpK1 in promoting translocation of type III secretion system
effectors. Brian H. Kvitko, Adela R. Ramos, Joanne E. Morello, Hye-Sook Oh, and
Alan Collmer. MPMI Vol. 22, No. 9, 2009, pp. 1069–1080.
The majority of the type III effector inventory of Pseudomonas syringae pv. tomato
DC3000 can suppress plant immunity. Guo M, Tian F, Wamboldt Y, Alfano JR. Mol
Plant Microbe Interact. 2009 Sep;22(9):1069-80
