Requirements for Ptr Requirements for Ptr ToxA Entry into ToxA Entry into Sensitive Wheat Sensitive Wheat Mesophyll Cells Mesophyll Cells Sara M. Hamilton Sara M. Hamilton Viola A. Manning Viola A. Manning Dr. Lynda M. Ciuffetti Dr. Lynda M. Ciuffetti Department of Botany and Plant Pathology Department of Botany and Plant Pathology
20
Embed
Requirements for Ptr ToxA Entry into Sensitive Wheat Mesophyll Cells Sara M. Hamilton Viola A. Manning Dr. Lynda M. Ciuffetti Department of Botany and.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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
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
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