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



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



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

First host selective toxin (HST) isolated from P.
tritici-repentis

First proteinaceous HST

Encoded by a single gene, the ToxA gene
Ptr ToxA

Causes necrosis on sensitive wheat cultivars

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

“RGD” cell attachment site


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

ToxA entry into mesophyll cells is
required to cause cell death.
Current Study

Produce GFP-ToxA proteins harboring
mutations and determine their localization in
planta

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

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

Transformation of E. coli with vector

Expression of GFP-ToxA in E. coli

Purification of GFP-ToxA
Protein Purification
pCVM77 fusion protein gel
kDa
72
55
40
33
24
To Be Completed:

Infiltration of GFP-ToxA mutant proteins into
sensitive/insensitive wheat leaves:

Assay activity

Determine localization via fluorescent
microscopy
Dissecting the ToxA Pathway

This information will allow us to determine if the
mutant proteins synthesized will:

Cross the cell wall

Cross the plasma membrane

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



Dr. Pat Martinez
Dr. Iovanna Pandelova
Kristin Skinner
Rachael Andrie
Rebecca Tippner- Hedges
Alex Babinin