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?