Transcript Does Plant Cell Death Require Toxin Entry?

Does Plant Cell Death
Induced by Ptr ToxA
Require Toxin Entry?
Sara M. Hamilton
Viola A. Manning
Dr. Lynda M. Ciuffetti
Department of Botany and Plant Pathology
Pyrenophora tritici-repentis


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Filamentous fungus-ascomycete
Plant pathogen causing the disease tan spot of
sensitive wheat species
Crop losses estimated up to 50% in susceptible
varieties worldwide
Races of Pyrenophora tritici-repentis
Race 1
Race 2
Race 3
Race 4
Race 5
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
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Glenlea
Katepwa
6B662
6B365
Salamouni
N (ToxA)
N (ToxA)
R
R
R
N (ToxA)
N (ToxA)
R
R
C (ToxB)
R
R
R
R
C (ToxB)
C (ToxC)
R
C (ToxC)
R
R
R
R
R
R
R
N = causes necrosis
C = causes chlorosis
R = resistant to pathogen
Ptr ToxA
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First host selective toxin (HST) isolated from P.
tritici-repentis
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First proteinaceous HST
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Encoded by a single gene, the ToxA gene
Ptr ToxA
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Causes necrosis on sensitive wheat cultivars
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Does not require pathogen to cause disease
symptoms
Sensitive
Insensitive
We want to know:
1. What part of the ToxA protein is
necessary for disease symptoms?
2. Where does the protein exert activity
(i.e. where is the site-of-action)?
Question #1

What part of the ToxA protein is
necessary for disease?
Conserved ToxA Motifs
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“RGD” cell attachment site
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RGD sites mediate interaction of cell matrix
proteins with a family of membrane-bound
receptors called integrins.
Casein kinase II (CKII) and Protein kinase C
(PKC) phosphorylation sites
ToxA Protein Sequence
QGSCMSITINPSRPSVNNIGQVDIDSVILG
RPGAIGSWELNNFITIGLNRVNADTVRVNI
RNTGRTNRLIITQWDNTVTRGDVYELFGDY
ALIQGRGSFCLNIRSDTGRENWRMQLEN
Both the RGD and casein kinase II phosphorylation motifs are
required for ToxA activity
Question #2

Where does the protein exert activity
(i.e. where is the site-of-action)?
ToxA Localization
 ToxA is imported into mesophyll cells of
sensitive wheat genotypes and localizes to the
chloroplasts of these cells.
 ToxA localization can be visualized in vivo by
treatment of wheat with a green flourescent
protein (GFP) fused to ToxA (GFP-ToxA).
GFP-ToxA:
Localization to Chloroplasts
Sensitive
ToxA
GFP-ToxA
Insensitive
Hypothesis
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ToxA entry into mesophyll cells is
required to cause cell death.
Current Study
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Produce GFP-ToxA proteins harboring
mutations and determine their localization in
planta
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Mutations include amino acids in the RGD cell
attachment site and phosphorylation motifs.
GFP-ToxA:
Construction of Fusion Protein Vector
Green Fluorescent Protein
Ptr ToxA
GFP-ToxA Mutants
 Mutagenize parent GFP-ToxA plasmid:
 Site-directed mutagenesis
 Subcloning from previously mutagenized ToxA
constructs
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PCR site-directed mutagenesis proved to be
more efficient than subcloning.
Mutations of GFP-ToxA
Mutation
to Alanine
Method of Mutagenesis Motif Mutagenized
t63
subcloned
PKC
t66
site-directed
PKC
n76
site-directed
*Essential A.A.
t77
subcloned
*Essential A.A.
v78
site-directed
*Essential A.A.
t79
site-directed
CKII
r80
site-directed
RGD
g81
site-directed
RGD
d82
subcloned
RGD
v83
site-directed
*Essential A.A.
e85
site-directed
*Essential A.A.
* Essential amino acids surround the RGD motif
Expression of GFP-ToxA
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Transformation of E. coli with vector
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Expression of GFP-ToxA in E. coli
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Purification of GFP-ToxA
Protein Purification
pCVM77 fusion protein gel
kDa
72
55
40
33
24
To Be Completed:
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Infiltration of GFP-ToxA mutant proteins into
sensitive/insensitive wheat leaves:
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Assay activity
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Determine localization via fluorescent
microscopy
Dissecting the ToxA Pathway
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This information will allow us to determine if the
mutant proteins synthesized will:

Cross the cell wall
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Cross the plasma membrane
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Localize to an organelle (ex. chloroplast)
Acknowledgements

Howard Hughes Medical
Institution
Ernest and Pauline Jaworski
USDA

Dr. Kevin Ahern

Dr. Lynda M. Ciuffetti
Viola A. Manning
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Dr. Pat Martinez
Dr. Iovanna Pandelova
Kristin Skinner
Rachael Andrie
Rebecca Tippner- Hedges
Alex Babinin