Analysis of the Site-of-Action and
Evolution of the Host-Selective
Toxin Ptr ToxB
Wade Holman
Dr. Lynda Ciuffetti’s Lab
Department of Botany and Plant Pathology
Oregon State University
Pyrenophora tritici-repentis (Ptr)
Disease: Tan Spot of Wheat
Host-Selective Toxins (HST):
• Only produced by fungi
•Primary determinants of
pathogenicity
•Reproduce symptoms of
disease
Host-Selective Toxins (HST)s of Ptr
• Protein Toxins
– ToxA
• Necrosis
– ToxB
• Chlorosis
ToxB characteristics
• Multiple-copy gene
• 261 bp Open Reading Frame (ORF)
S
S
87 aa (8.9 kDa)
ToxB preprotein
23 aa- Signal sequence
Mature Ptr ToxB
64 aa (6.5 kDa)
ToxB Mode-of-action?
• It’s still unknown
• Chlorophyll degradation
• Activity requires light
• ToxB sequence does not provide insights into the
toxin’s mode or site-of-action.
Objectives
• To determine whether Ptr ToxB is
internalized into the toxin sensitive cell
• To determine if Ptr ToxB homologs are
present in different ascomycete species
Experimental approach
Expression and Purification
of ToxB and His ToxB
Test activity of toxins
Proteinase K Protection Assay (PK assay)
Purification of Pichia pastoris
expressed ToxB and His-ToxB
QMA column
Pichia pastoris
culture
concentration/dialysis
of collected proteins
KD
250
50
37
BCA assay/Adjust
ToxB concentration
to 15 µM
20
15
10
QMA fractions containing contaminantfree ToxB collected during purification
process.
Activity of toxins
Proteinase K
Protection assay
PK
TB
TB
TB
TB
TB
TB
TB
TB
TB
TB
Internalized
PK=Proteinase K
TB=ToxB
Mesophyll Cells
TB
PK
TB
TB
TB
TB
TB
TB
Not Internalized
Toxin-sensitive wheat leaf
His-ToxB
infiltration
PK
infiltration
2h
Tissue grinding
and protein extraction
Symptom observation
•Chlorosis was monitored
over 5 days
Ni-NTA Protein
Purification System
Protein Gels
For silver stain
and western blot
•Compared to the extracted
protein results
Proteinase K Protection Assay using
Pichia pastoris ToxB
ToxB
PK
PK Time Points
Assay shows a time dependent PK degradation effect on ToxB
Proteinase K Protection Assay using
Pichia pastoris His-ToxB
His-ToxB
extraction
Symptom Observation
20 kD
Water Control
15 kD
His-ToxB Only
His-ToxB+24h PK
10 kD
His-ToxB
PK Only
PK:
-
+
24 h
Assay shows there is not PK degradation effect on His-ToxB
PK Assay Troubleshooting
• PK not working?
•High concentration of His-ToxB?
In vitro digestion of His-ToxB
PMSF:
+
+
-
-
His-ToxB:
+
+
+
+
PK:
-
+
+
-
• PMSF is a protease inhibitor
• Four tubes for in vitro
digestion
•In vitro digestions went for 30
mins. at room temperature
In vitro digestion of His-ToxB
20kD
15 kD
His-ToxB
10 kD
+ PMSF
No PK
+ PMSF
PK
- PMSF
PK
- PMSF
No PK
PK is active, although His-ToxB shows
resistance to degradation.
Effect of PK concentrations on
symptoms caused by His-ToxB
• PK
causes chlorosis when infiltrated
• Exacerbates His-ToxB symptoms on leaves
•Difficult to determine if yellowing is due to toxin or PK
His-ToxB
+
+
+
+
+
PK
-
+
+
+
+
150 ug/ml
200 ug/ml
300 ug/ml
400 ug/ml
Water
His-ToxB
PK 150 ug/ml+His-ToxB
PK 200 ug/ml+His-ToxB
PK 300 ug/ml+His-ToxB
PK 400 ug/ml+His-ToxB
• Ideal concentration is between 150 and 200
ug/ml
• 300 and 400 ug/ml develop extensive
chlorosis
• PK adjusted to 150 µg/ml in subsequent
experiments
Screening for the presence of Ptr ToxB
homologous sequences in different
Ascomycete species
• Ptr ToxB homologs have been found in Pyrenophora
bromi, a sister taxon to Ptr
Pb Bf-1lg
Pb TAM115
Pb Bf-1sm
Pb SM20Alg
Pb SM20Asm
Pb SM101sm
Pb SM106sm
Pb SM106lg
Pb TW123
Pb MPKlg
Pb MPKsm
Pb SM101lg
Ptr toxb
Ptr ToxB
Amino acid alignments of Ptr ToxB, Ptr toxb, and ToxB
homologs from different P. bromi isolates (Pb)
(Andrie et.al., 2007)
Residue Key
Global
Identical
Similar
Normal
• Identification of ToxB homologs will be carried out
by PCR.
• Screening of several ascomycete isolates
• Primers have been designed for the Internal
Transcribed Spacer (ITS) sequence and the Ptr
ToxB sequence within the ORF
• The ITS regions will serve as positive controls
PCR Results
1 kB
500 bp
200 bp
Fig. 1:PCR for ToxB gene with positive
ToxB homolog in lane 6
1 kB
500 bp
Fig. 4: PCR for ITS regions
• Some isolates were too low in concentration for PCR amplification
• Two possible ToxB homologs were identified in this experiment
= Pyrenophora tritici-repentis
= Pleospora herbarum
= Hysterium pulicare
• 29 isolates total were
screened
• Of those, 2 possible homologs
were identified
• One homolog was closely
related to Ptr and one was
distally related
Future Research
• To try PK protection assay with a more specific protease
•To screen the remaining isolates for ToxB
•To perform nested primer PCR on isolates I have already
screened
• To locate possible ToxA homologs in ascomycetes
Acknowledgements
•
•
•
•
•
•
•
•
Dr. Lynda Ciuffetti
Dr. Melania Betts
M.Sc. Viola Manning
Dr. Iovanna Pandelova
Dr. Tom Wolpert’s Lab
Ernest and Pauline Jaworski Fund
HHMI and Kevin Ahern
OSU Department of Botany and
Plant Pathology
Questions?