Requirements for Ptr ToxA Requirements for Ptr ToxA Entry into Sensitive Wheat Entry into Sensitive Wheat

Mesophyll CellsMesophyll Cells

Sara M. HamiltonSara M. Hamilton

Viola A. ManningViola A. Manning

Dr. Lynda M. CiuffettiDr. Lynda M. Ciuffetti

Department of Botany and Plant PathologyDepartment of Botany and Plant Pathology

PyrenophoraPyrenophora triticitritici--repentisrepentis

Filamentous ascomyceteFilamentous ascomycete

Causes the disease tan spot of wheatCauses the disease tan spot of wheat

Crop losses up to 50% worldwideCrop losses up to 50% worldwide

Host-selective ToxinsHost-selective Toxins

• Ptr ToxC

• Ptr ToxD

•Ptr ToxB

Pathogen HST

PtrPtr ToxAToxA

Ptr ToxA HistoryPtr ToxA History

First host-selective toxin (HST) First host-selective toxin (HST) isolated from isolated from P. tritici-repentisP. tritici-repentis

First proteinaceous HST identifiedFirst proteinaceous HST identified

Encoded by a single gene, Encoded by a single gene, ToxAToxA

Required for diseaseRequired for disease

Ptr ToxAPtr ToxA Causes necrosis on sensitive wheat cultivarsCauses necrosis on sensitive wheat cultivars

Does not require pathogen to cause disease Does not require pathogen to cause disease symptoms (HST slide)symptoms (HST slide)

Reproduces disease symptoms in absence of Reproduces disease symptoms in absence of pathogen pathogen

Sensitive

Insensitive

MutagenesisMutagenesis RGD cell attachment RGD cell attachment

site within additional site within additional essential amino acidsessential amino acids

CKII phosphorylation CKII phosphorylation sitesite

Crystal StructureCrystal Structure

Ganapathy Sarma and Dr. Andy Karplus Biochemistry & Biophysics Dept. OSU

Amino Acids Required for Amino Acids Required for DiseaseDisease

ToxA

GFP-ToxA

ToxA Internalization ToxA Internalization

Green Fluorescent Protein Ptr ToxA

InsensitiveSensitive

Summary of Preliminary DataSummary of Preliminary Data Amino acids required for disease:Amino acids required for disease:

RGD cell attachment site and RGD cell attachment site and surrounding amino acidssurrounding amino acids

CKII siteCKII site

GFP-ToxA and confocal microscopy:GFP-ToxA and confocal microscopy: Internalization into mesophyll cells Internalization into mesophyll cells Association with chloroplastsAssociation with chloroplasts

First protein shown to be internalized into plant cells First protein shown to be internalized into plant cells from the apoplastic spacefrom the apoplastic space

What amino acids are involved What amino acids are involved

in ToxA internalization?in ToxA internalization?

GFP-ToxA Mutant ProteinsGFP-ToxA Mutant Proteins

Mutagenize parent GFP-ToxA plasmid:Mutagenize parent GFP-ToxA plasmid:

Site-directed mutagenesisSite-directed mutagenesisSubclone from previously mutagenized ToxA Subclone from previously mutagenized ToxA

constructsconstructs

PCR site-directed mutagenesis was more PCR site-directed mutagenesis was more efficientefficient

MutationMutationto Alanineto Alanine

Method of Method of MutagenesisMutagenesis

Motif Motif MutagenizedMutagenized

ActivityActivity

t66t66 site-directedsite-directed PKCPKC ActiveActive

n76n76 site-directedsite-directed Essential A.A.Essential A.A. Not activeNot active

v78v78 site-directedsite-directed Essential A.A.Essential A.A. Not activeNot active

t79t79 site-directedsite-directed CKIICKII Not activeNot active

r80r80 site-directedsite-directed RGDRGD Not activeNot active

g81g81 site-directedsite-directed RGDRGD Not activeNot active

d82d82 subclonedsubcloned RGDRGD Not activeNot active

v83v83 site-directedsite-directed Essential A.A.Essential A.A. Not activeNot active

GFP-ToxA Mutant ProteinsGFP-ToxA Mutant Proteins

GFP-ToxA Mutant ProteinsGFP-ToxA Mutant Proteins

Expressed in Expressed in E.coliE.coli

Purified on a Purified on a columncolumn

SDS-PAGESDS-PAGE1 µg1 µg

n76 v78 t79 r80 g81 d82 v83 t66n76 v78 t79 r80 g81 d82 v83 t66

GFP-ToxA mutant proteins = 55kDa

Proteinase K AssayProteinase K Assay

Infiltrate ToxAInfiltrate ToxA Incubate for 2hrsIncubate for 2hrs

Infiltrate PKInfiltrate PK Incubate for 1hrIncubate for 1hr

Extract total proteinExtract total protein

Isolate ToxA Isolate ToxA

Western BlotWestern Blot

Proteinase K AssayProteinase K Assay

TA

TATA TA TA

TA

TA

PK

Insensitive

TA TA TA

TATATATA

TA

TATA

TA TA

TATAPK

Sensitive

TATA

TA

ToxA Internalization into Cells of ToxA Internalization into Cells of Sensitive Leaves ONLYSensitive Leaves ONLY

insensitive sensitive

ToxA

Western blot with anti-ToxA antibody

Proteinase K AssayProteinase K Assay

PK

TA TA TA

TATATATAPK

GFP-ToxA Internalized

GFP-TA

GFP-ToxA Not Internalized

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

GFP-TA

Proteins with Mutations in the Proteins with Mutations in the RGD Loop are NOT InternalizedRGD Loop are NOT Internalized

ToxA

A

GFP-ToxA

A

t66

A

n76 t77 v78 t79

I

r80 g81 d82 v83

I I I I II I

A = Active proteinI = Inactive protein

ConclusionsConclusionsGFP-ToxA acts like ToxAGFP-ToxA acts like ToxA

Thus, it is a great toolThus, it is a great tool

Active ToxA is internalized Active ToxA is internalized ToxA internalization is required for ToxA internalization is required for

diseasediseaseAmino acids required for cell entry: Amino acids required for cell entry:

RGD and essential aa’sRGD and essential aa’sCKII phosphorylation siteCKII phosphorylation site

AcknowledgementsAcknowledgements

Dr. Lynda M. CiuffettiDr. Lynda M. Ciuffetti

HHMIHHMI Dr. Kevin AhernDr. Kevin Ahern

Viola A. ManningViola A. Manning Dr. Iovanna PandelovaDr. Iovanna Pandelova Rachael AndrieRachael Andrie Kristin SkinnerKristin Skinner Alex Babinin Alex Babinin

Dr. Andy KarplusDr. Andy Karplus Ganapathy SarmaGanapathy Sarma

Questions?Questions?