analysis of the site-of-action and evolution of the host-selective toxin ptr toxb wade holman dr....
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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
Host-Selective Toxins (HST):
• Only produced by fungi
•Primary determinants of pathogenicity
•Reproduce symptoms of disease
Pyrenophora tritici-repentis (Ptr)Disease: Tan Spot of Wheat
Host-Selective Toxins (HST)s of Ptr
• Protein Toxins
– ToxA• Necrosis
– ToxB• Chlorosis
ToxB characteristics
• 261 bp Open Reading Frame (ORF) 87 aa (8.9 kDa)
S 23 aa- Signal sequence
S
Mature Ptr ToxB
64 aa (6.5 kDa)
• Multiple-copy gene
ToxB preprotein
• It’s still unknown
• Chlorophyll degradation
• Activity requires light
• ToxB sequence does not provide insights into the toxin’s mode or site-of-action.
ToxB Mode-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
Proteinase K Protection Assay (PK assay)
Expression and Purification of ToxB and His ToxB
Test activity of toxins
Purification of Pichia pastoris expressed ToxB and His-ToxB
BCA assay/Adjust ToxB concentration to 15 µM
KD
QMA fractions containing contaminant-free ToxB collected during purification process.
10
15
20
37
250
50
Pichia pastoris culture
concentration/dialysis of collected proteins
QMA column
Activity of toxins
Proteinase K Protection assay
TB
TBTB TB TB
TB
TB
PK
Not Internalized
TB TB TB
TBTBTBTB
TB
TBTB
TB TB
TBTBPK
Internalized
TBTB
TB
PK=Proteinase KTB=ToxB
Mesophyll Cells
Toxin-sensitive wheat leaf
2 h
Tissue grindingand protein extraction
Ni-NTA Protein Purification System
Protein GelsFor silver stainand western blot
His-ToxB infiltration
PK infiltration
Symptom observation
•Chlorosis was monitored over 5 days
•Compared to the extracted protein results
Proteinase K Protection Assay using Pichia pastoris ToxB
Assay shows a time dependent PK degradation effect on ToxB
ToxB PK PK Time Points
Proteinase K Protection Assay using Pichia pastoris His-ToxB
24 h
His-ToxB extraction
Water Control
His-ToxB Only
His-ToxB+24h PK
PK Only
Symptom Observation
Assay shows there is not PK degradation effect on His-ToxB
PK: - +
10 kD
15 kD
20 kD
His-ToxB
• PK not working?
•High concentration of His-ToxB?
PK Assay Troubleshooting
In vitro digestion of His-ToxB
• PMSF is a protease inhibitor
• Four tubes for in vitro digestion
•In vitro digestions went for 30 mins. at room temperature
PMSF:
His-ToxB:
PK:
+ + - -
+ + + +
- + + -
+ PMSF No PK
+ PMSF PK
- PMSF PK
- PMSF No PK
20kD
15 kD
10 kD
PK is active, although His-ToxB shows resistance to degradation.
In vitro digestion of His-ToxB
His-ToxB
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 + + + + +
150 ug/ml 200 ug/ml 300 ug/ml 400 ug/ml
PK - + + + +
• 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
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
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-1lgPb TAM115Pb Bf-1sm
Pb SM20AlgPb SM20AsmPb SM101smPb SM106smPb SM106lg
Pb TW123Pb MPKlg
Pb MPKsmPb SM101lg
Ptr toxbPtr ToxB
Global
Identical
Similar
Normal
Residue KeyAmino acid alignments of Ptr ToxB, Ptr toxb, and ToxB homologs from different P. bromi isolates (Pb) (Andrie et.al., 2007)
• 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
Fig. 1:PCR for ToxB gene with positive ToxB homolog in lane 6
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
1 kB
500 bp200 bp
1 kB
500 bp
= 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?