Requirements for Ptr ToxA
Entry into Sensitive Wheat
Mesophyll Cells
Sara M. Hamilton
Viola A. Manning
Dr. Lynda M. Ciuffetti
Department of Botany and Plant Pathology
Pyrenophora tritici-repentis

Filamentous ascomycete

Causes the disease tan spot of wheat

Crop losses up to 50% worldwide
Host-selective Toxins
Pathogen

Ptr ToxA
• Ptr ToxB
• Ptr ToxC
• Ptr ToxD
HST
Ptr ToxA History

First host-selective toxin (HST)
isolated from P. tritici-repentis

First proteinaceous HST identified

Encoded by a single gene, ToxA

Required for disease
Ptr ToxA

Causes necrosis on sensitive wheat cultivars

Does not require pathogen to cause disease
symptoms (HST slide)

Reproduces disease symptoms in absence of
pathogen
Sensitive
Insensitive
Amino Acids Required for
Disease

Mutagenesis



RGD cell attachment
site within additional
essential amino acids
CKII phosphorylation
site
Crystal Structure
Ganapathy Sarma and Dr. Andy Karplus
Biochemistry & Biophysics Dept. OSU
ToxA Internalization
Green Fluorescent Protein
Sensitive
ToxA
GFP-ToxA
Ptr ToxA
Insensitive
Summary of Preliminary Data

Amino acids required for disease:
 RGD cell attachment site and
surrounding amino acids
 CKII site

GFP-ToxA and confocal microscopy:
 Internalization into mesophyll cells
 Association with chloroplasts

First protein shown to be internalized into plant cells
from the apoplastic space
What amino acids are involved
in ToxA internalization?
GFP-ToxA Mutant Proteins


Mutagenize parent GFP-ToxA plasmid:

Site-directed mutagenesis

Subclone from previously mutagenized ToxA
constructs
PCR site-directed mutagenesis was more
efficient
GFP-ToxA Mutant Proteins
Mutation
to Alanine
Method of
Mutagenesis
Motif
Mutagenized
Activity
t66
site-directed
PKC
Active
n76
site-directed
Essential A.A.
Not active
v78
site-directed
Essential A.A.
Not active
t79
site-directed
CKII
Not active
r80
site-directed
RGD
Not active
g81
site-directed
RGD
Not active
d82
subcloned
RGD
Not active
v83
site-directed
Essential A.A.
Not active
GFP-ToxA Mutant Proteins

Expressed in
E.coli
n76


Purified on a
column
SDS-PAGE

1 µg
v78 t79 r80 g81 d82 v83 t66
GFP-ToxA mutant proteins = 55kDa
Proteinase K Assay

Infiltrate ToxA


Incubate for 2hrs
Infiltrate PK

Incubate for 1hr

Extract total protein

Isolate ToxA

Western Blot
Proteinase K Assay
PK
TA
TA
TA
TA
TA
TA
TA
TA
TA
TA
Sensitive
TA
PK
TA
TA
TA
TA
TA
TA
Insensitive
ToxA Internalization into Cells of
Sensitive Leaves ONLY
Western blot with anti-ToxA antibody
insensitive sensitive
ToxA
Proteinase K Assay
GFP-
PK
TA
TA
GFPTA
TA
TA
GFPTA
GFPTA
TA
GFPTA
GFPTA
GFPTA
TA
GFPTA
GFPTA
TA
TA
GFP-ToxA Internalized
PK
GFPTA
GFPTA
GFPTA
GFPTA
GFPTA
GFPTA
GFPTA
GFP-ToxA Not Internalized
Proteins with Mutations in the
RGD Loop are NOT Internalized
ToxA
GFP-ToxA
A
A
A = Active protein
I = Inactive protein
n76
I
t77
v78
I
I
t79
I
r80
g81
d82
I
I
I
v83
I
t66
A
Conclusions

GFP-ToxA acts like ToxA

Thus, it is a great tool
Active ToxA is internalized
 ToxA internalization is required for
disease
 Amino acids required for cell entry:

RGD and essential aa’s
 CKII phosphorylation site

Acknowledgements

Dr. Lynda M. Ciuffetti









HHMI
Dr. Kevin Ahern
Viola A. Manning
Dr. Iovanna Pandelova
Rachael Andrie
Kristin Skinner
Alex Babinin
Dr. Andy Karplus
Ganapathy Sarma
Questions?