does plant cell death induced by ptr toxa require toxin entry? sara m. hamilton sara m. hamilton...
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Does Plant Cell Death Does Plant Cell Death Induced by Ptr ToxA Induced by Ptr ToxA RequireRequire Toxin Entry? Toxin Entry?
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 fungus-ascomyceteFilamentous fungus-ascomycete Plant pathogen causing the disease Plant pathogen causing the disease tantan spotspot of of
sensitive wheat speciessensitive wheat species Crop losses estimated up to 50% in susceptible Crop losses estimated up to 50% in susceptible
varieties worldwidevarieties worldwide
Races of Races of Pyrenophora tritici-repentisPyrenophora tritici-repentis
N = causes necrosisN = causes necrosis C = causes chlorosisC = causes chlorosis R = resistant to pathogenR = resistant to pathogen
Race 1Race 2
Race 3
Race 4
Race 5
SalamouniGlenlea
N (ToxA)
N (ToxA)
R
R
R
R
R
R
R
R
Katepwa
N (ToxA)
N (ToxA)
R
R
C (ToxB)
6B365
C (ToxC)
R
C (ToxC)
R
R
6B662
R
R
R
R
C (ToxB)
Ptr ToxAPtr ToxA
First host selective toxin (HST) isolated from First host selective toxin (HST) isolated from P. P. tritici-repentistritici-repentis
First proteinaceous HSTFirst proteinaceous HST
Encoded by a single gene, the Encoded by a single gene, the ToxAToxA gene gene
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
symptomssymptoms
Sensitive
Insensitive
We want to know:We want to know:
1. What part of the ToxA protein is 1. What part of the ToxA protein is necessary for disease symptoms?necessary for disease symptoms?
2. Where does the protein exert activity 2. Where does the protein exert activity (i.e. where is the site-of-action)? (i.e. where is the site-of-action)?
Question #1Question #1
What part of the ToxA protein is What part of the ToxA protein is necessary for disease?necessary for disease?
Conserved ToxA Motifs Conserved ToxA Motifs
““RGD” cell attachment site RGD” cell attachment site RGD sites mediate interaction of cell matrix RGD sites mediate interaction of cell matrix
proteins with a family of membrane-bound proteins with a family of membrane-bound receptors called integrins.receptors called integrins.
Casein kinase II (CKII) and Protein kinase C Casein kinase II (CKII) and Protein kinase C (PKC) phosphorylation sites(PKC) phosphorylation sites
QGSCMSITINPSRPSVNNIGQVDIDSVILGRPGAIGSWELNNFITIGLNRVNADTVRVNIRNTGRTNRLIITQWDNTVTRGDVYELFGDYALIQGRGSFCLNIRSDTGRENWRMQLEN
ToxA Protein SequenceToxA Protein Sequence
Both the RGD and casein kinase II phosphorylation motifs are required for ToxA activity
Question #2Question #2
Where does the protein exert activity Where does the protein exert activity (i.e. where is the site-of-action)? (i.e. where is the site-of-action)?
ToxA LocalizationToxA Localization
ToxA is imported into mesophyll cells of ToxA is imported into mesophyll cells of sensitive wheat genotypes and localizes to the sensitive wheat genotypes and localizes to the chloroplasts of these cells.chloroplasts of these cells.
ToxA localization can be visualized ToxA localization can be visualized in vivoin vivo by by treatment of wheat with a green flourescent treatment of wheat with a green flourescent protein (GFP) fused to ToxA (GFP-ToxA).protein (GFP) fused to ToxA (GFP-ToxA).
GFP-ToxA: GFP-ToxA: Localization to ChloroplastsLocalization to Chloroplasts
Sensitive Insensitive
ToxA
GFP-ToxA
HypothesisHypothesis
ToxA entry into mesophyll cells is ToxA entry into mesophyll cells is required to cause cell death.required to cause cell death.
Current StudyCurrent Study
Produce GFP-ToxA proteins harboring Produce GFP-ToxA proteins harboring mutations and determine their localization mutations and determine their localization inin plantaplanta
Mutations include amino acids in the RGD cell Mutations include amino acids in the RGD cell attachment site and phosphorylation motifs.attachment site and phosphorylation motifs.
GFP-ToxA: GFP-ToxA: Construction of Fusion Protein VectorConstruction of Fusion Protein Vector
Green Fluorescent Protein
Ptr ToxA
GFP-ToxA MutantsGFP-ToxA Mutants
Mutagenize parent GFP-ToxA plasmid:Mutagenize parent GFP-ToxA plasmid:
Site-directed mutagenesisSite-directed mutagenesis Subcloning from previously mutagenized ToxA Subcloning from previously mutagenized ToxA
constructsconstructs
PCR site-directed mutagenesis proved to be PCR site-directed mutagenesis proved to be more efficient than subcloning.more efficient than subcloning.
Mutationto Alanine
Method of Mutagenesis Motif Mutagenized
t63 subcloned PKC
t66 site-directed PKC
n76 site-directed *Essential A.A.
t77 subcloned *Essential A.A.
v78 site-directed *Essential A.A.
t79 site-directed CKII
r80 site-directed RGD
g81 site-directed RGD
d82 subcloned RGD
v83 site-directed *Essential A.A.
e85 site-directed *Essential A.A.
Mutations of GFP-ToxAMutations of GFP-ToxA
* Essential amino acids surround the RGD motif
Expression of GFP-ToxAExpression of GFP-ToxA
Transformation of Transformation of E. coliE. coli with vector with vector
Expression of GFP-ToxA in Expression of GFP-ToxA in E. coliE. coli
Purification of GFP-ToxAPurification of GFP-ToxA
Protein PurificationProtein Purification
kDa
24
72
33
40
55
pCVM77 fusion protein gelpCVM77 fusion protein gel
To Be Completed:To Be Completed:
Infiltration of GFP-ToxA mutant proteins into Infiltration of GFP-ToxA mutant proteins into sensitive/insensitive wheat leaves:sensitive/insensitive wheat leaves:
Assay activityAssay activity
Determine localization via fluorescent Determine localization via fluorescent microscopymicroscopy
Dissecting the ToxA PathwayDissecting the ToxA Pathway
This information will allow us to determine if the This information will allow us to determine if the mutant proteins synthesized will:mutant proteins synthesized will:
Cross the cell wallCross the cell wall
Cross the plasma membraneCross the plasma membrane
Localize to an organelle (ex. chloroplast)Localize to an organelle (ex. chloroplast)
AcknowledgementsAcknowledgements
Howard Hughes Medical Howard Hughes Medical InstitutionInstitution
Ernest and Pauline JaworskiErnest and Pauline Jaworski USDAUSDA
Dr. Kevin AhernDr. Kevin Ahern
Dr. Lynda M. CiuffettiDr. Lynda M. Ciuffetti Viola A. ManningViola A. Manning
Dr. Pat MartinezDr. Pat MartinezDr. Iovanna PandelovaDr. Iovanna PandelovaKristin SkinnerKristin SkinnerRachael AndrieRachael AndrieRebecca Tippner-Rebecca Tippner- HedgesHedgesAlex BabininAlex Babinin