requirements for ptr toxa entry into sensitive wheat mesophyll cells sara m. hamilton viola a....
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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?
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