Pto system literature review

1
Pto system literature review

• Pto isolation
• Pto function in other species
• Function of other Pto family members
• avrPto isolation
• Pto over expression
• Identification of other components of
  recognition/signaling
• Pto recognition of avrPto
• avrPto function as an effector

2
Pto functions in related species
Southern blot hybridization of Pto to DNA of
related species
Rommens et al. 1995 Plt. Cell. 7 1537-
3
Pto functions in related species
avrPto-P.s. tabaci N. benthamiana
avrPto-P.s. tabaci Pto-N. benthamiana
avrPto-P.s. tabaci N. benthamiana
avrPto-P.s. tabaci Pto-N. benthamiana
Pto doesnt work in more distantly related
species Why not?
Rommens et al. 1995 Plt. Cell. 7 1537-
4
Some other genes in the Pto gene cluster have
phenotypes
Representation of gene cluster in line introduced
from L. pimpinellifolium
Line carrying this gene cluster (haplotype) get
small necrotic spots when sprayed with the
organophosphate insecticide fenthion. The
phenotype mapped to this locus. Segregation in
1200 progeny showed it mapped perfectly to the
area of the gene family. Expression of a cDNA
from one family member found it did not confer
Pto resistance, but made the plant sensitive to
fenthion the gene was called Fen (Martin et al.
1994 Plant Cell 61543-)
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Effect of the Fen gene in transgenic tomato
plants after fenthion application
Martin et al. 1994 Plant Cell 61543-
6
Several related compounds elicit the Fen-induced
necrosis
Fen Fen-
Do some R genes detect small compounds?
Soybean-P. s. pv glycinea interaction avrD is
associated with the production of elicitors
consisting of two acyl glycosides called
syringolides (Keen et al. 1990 MPMI 3, 112-121)
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Isolation of AvrPto from Pseudomonas syringae pv.
tomato
Ronald et al., (1992) J Bact. 1741604-1611
P. s. tomato race 0 (avrPto)
Chromosome avrPto
What next?
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Isolation of AvrPto, Ronald et al. cont
Examination of 381 clones identified one that
caused HR
Subcloning and mutagenesis was used to identify
the gene on the cosmid
Insertions
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Isolation of AvrPto, cont
When avrPto from P.s. tomato strain was replaced
with a mutant version, it was still avirulent on
Pto tomato lines (but not pto tomato). Explanatio
n?
Another 681 cosmids-transformed strains were
examined, but none identified a second Avr gene!
Does that mean there isnt one?
994 cosmids x 27.5 Kb 27,335 Kb, or 27
Mb This is 4 genome equivalents In theory, you
should have a 95 chance of carrying a sequence
with 3 genome equivalents and a 99 chance with 4
equivalents. In reality, it maybe more
difficult to clone some sequences.
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Would this approach be good for isolating Avr
genes from fungi?

• Problems
• Need very efficient transformation
• Genomes size is a limitation
• O.K. for bacteria, 5 Mb (5000 kb) 1 genome
  equiv. 150 Cosmids
• Yeast, 15 Mb genome 450 Cosmids
• Filamentous fungi, 30-100 Mb genome

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Dissection of defense signaling pathways
How does one find other (besides R Avr gene)
components of specific recognition and signaling
pathways?

• Genetic approaches?
• Biochemical approaches?

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Identifying proteins that interact with R/Avr
gene products

• Yeast two hybrid system for identifying
  interacting proteins
• Demonstrating the components interact in vivo
• Purification of intact protein complexes
• Demonstrating that the interactors are
  biologically relevant

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Yeast 2 hybrid approach example finding
interactors for R protein

• Make fusion protein construct with R protein
  (bait) and ½ of 2-domain protein with functional
  assay (e.g. two domain transcription factor)
• Make cDNA library in a vector that fuses these
  transcripts (prey) to the other half of the
  2-domain protein.
• Transform the R gene fusion construct and the
  cDNA library into yeast.
• Look for cells where with the reporter gene
  phenotype indicating the 2 domains of the protein
  are working together.

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Overview of two-hybrid assay, checking for
interactions between two proteins
                                                
               
A. Gal4 transcription factor gene produces two
domain protein (BD and AD) which is essential for
transcription of the reporter gene (LacZ). B,C.
Two fusion proteins are prepared Gal4BDBait and
Gal4ADPrey. None of them is usually sufficient
to initiate the transcription (of the reporter
gene) alone. D. When both fusion proteins are
produced and Bait part of the first interact with
Prey part of the second, transcription of the
reporter gene occurs.
http//en.wikipedia.org/wiki/Two-hybrid_screening
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Interaction of Pto with avrPto
Tang et al 1996 Science 2742060-
Bait construct none Pto Pto pto
Pto(K69Q) Fen Prey construct avrPto
none avrPto avrPto avrPto avrPto
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Interaction of Pto with avrPto
Do they have to interact to trigger resistance?
Can you tell what part of the protein is critical
to distinguish Fen from Pto?
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Isolation of Pto interactors Pti1
Zhou et al. 1995 Cell 83925-
Bait vector Pto Prey vector Pti1
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Pto interactors Pti1, continued
RNA blot
DNA blots
Pti1 is a member of a conserved gene family
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Pto interactors Pti1, continued
Pti1 is a functional protein kinase.
Autophosphorylation assay Pti1 and a mutant were
purified as GST fusions, incubated with labeled
ATP and run on gel.
Pti1 was then hydrolyzed to see which amino acids
were phosphorylated.
20
Pto interactors Pti1, continued

Phosphorylation assay (autoradiograph) after
incubation with Pti1 and labeled ATP PAGE gel
of purified proteins

Conclusion Pti1 phosphorylates itself, but not
Pto or Fen
21
Pto interactors Pti1, continued

Phosphorylation assay PAGE gel of purified
proteins

Pto phosphorylates Pti1, but Fen doesnt
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Pto interactors Pti1, continued
Bait Prey

• Pto that is not phosphorylation competent doesnt
  interact with Pti1
• Because Pto needs to be phosphorylated?
• Pti1 doesnt need to be a competent kinase to
  interact with Pto.

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Isolation of Pto interactors Pti1, continued
Tobacco (with functional Pto homolog) transformed
with Pti1 showed a stronger resistance reaction
to P.s. tabaci carrying avrPto (Reactions 13 hr
after infiltration)

• Pti1 is not just a protein that Pto
  phosphorylates, it is relevant to the defense
  signaling!
• Also indicates its signaling is involved in
  inducing HR!

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Isolation of Pto interactors Pti1, continued
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Isolation of Pto interactors Pti4, 5 and 6
Zhou et al. 1997 EMBO 1632073218
Pti4, 5 and 6 interact with Pto, but not pto
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Pto interactors Pti4, 5 and 6, continued
DNA gel blots with probes from the cDNA clones
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Pto interactors Pti4, 5 and 6, continued
Alignment of Pti4,5 and 6 genes with proteins
from database reveals they are related to the
ethylene response element binding proteins EREBPs
of tobacco.
EREBPs bind a cis element in promoters of PR
genes that has the core sequence GCCGCC,
sometimes called PR box
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Pto interactors Pti4, 5 and 6, continued
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Pto interactors Pti4, 5 and 6, continued
90 bp
Gel mobility shift assays with labeled PR box
(with 4 GCCGCC) or mutant PR box (with 4 TCCTCC)
run on gel after incubation with various purified
proteins
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Pto interactors Pti4, 5 and 6, continued
31
Pto interactors Pti4, 5 and 6, continued
Tobacco with Pto challenged with Pseudomonas
strains carrying avrPto induces expression of
proteins with PR boxes
Why would the plant need three transcription
factors to induce the PR genes?
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Pto interactors Pti4, 5 and 6, continued
33
(No Transcript)
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Mutagenesis vrs protein interactions for
identifying receptor signaling components
Mutagenesis Problems with gene redundancy Shows
biological significance Protein
interactions No problems with redundancy Biologica
l significance must be determined Interaction may
be irrelevant and may not even occur in vivo.