Purification, Crystallization, and Mutagenesis of ToxB, a HostSelective Toxin in Pyrenophora tritici-repentis Kara Miles-Rockenfield In collaboration with the labs of Drs.
Download ReportTranscript Purification, Crystallization, and Mutagenesis of ToxB, a HostSelective Toxin in Pyrenophora tritici-repentis Kara Miles-Rockenfield In collaboration with the labs of Drs.
Purification, Crystallization, and Mutagenesis of ToxB, a Host Selective Toxin in Pyrenophora tritici-repentis Kara Miles-Rockenfield In collaboration with the labs of Drs. Lynda Ciuffetti 1 and P. Andrew Karplus 2 1 Department of Botany and Plant Pathology 2 Department of Biochemistry and Biophysics
Tan Spot
One of the major diseases affecting wheat Wide geographical distribution Causes lightweight and shriveled grains Results in 3-50% crop loss
(Strelkov and Lamari 2003)
…and we know what causes it!
Pyrenophora tritici-repentis
Fungus that causes tan-spot in wheat Symptoms caused by Host-Selective Toxins Molecules secreted by the fungus
Host-Selective Toxins
Produced by fungi Small molecular weight molecules Proteins Reproduce symptoms of disease Pathogen not required Primary determinants of pathogenicity Toxic only to susceptible plants Non-host plants not affected
Healthy Leaf Chlorosis
Symptoms of HSTs
Necrosis Cell death Chlorosis Breakdown of chlorophyll Necrotic Lesions
HST of Ptr
ToxB Protein Causes chlorosis Multiple copies of the gene cause greater chlorosis Inactive form toxb Zone of Infiltration
Hypothesis
A specific region of the structure of ToxB is responsible for the toxicity of the protein.
Project Goals
Purify ToxB from Ptr Mutagenesis Important regions of the gene for toxicity
First Goal
Purification of
ToxB
Purification of
ToxB Protein Production in Crude Culture Filtrate
Plugs vs. Ground
Harvest Filtrate
Filters
Protein Precipitations
2-Step (NH 4 ) 2 SO 4
Chromatography
Cation Exchange
Fraction Analysis
Silver Stain, Western Blot, Protein Assay
Purification Results
Western Blot Silver Stain
25 20 15 10 kD P F1 F2 W1 W2 W3 M E1 E2 E3 E4 75 50 37
Protein Analysis Western Blot Silver Stain BCA Protein Assay Additional purification needed
ToxB
Second Goal
Mutagenesis of
ToxB
Sequence Comparison
I II III
toxb ToxB MAPIF E TAMLLAVAILPAALVSANC T ANILNINE V VIATGCVPAGG NLI I R VGS D HSYLI R ATVSCGLSL NPS QSFINGES LA SGGRC MAPIF K TTMLLAVAILPAALVSANC V ANILNINE A VIATGCVPAGG ELR I F VGS S HSYLI K ATSSCGLSL -TN QVFINGES VQ SGGRC
Differences between the sequences Deletion in
ToxB
Proline in
toxb
Restriction sites present in both Divides protein into three regions H 2 C N H 2 C C CH 2 COOH H H
Chimeras
Chimeric Proteins Swap Coding Regions
pCMR3 – BBb
I II
MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGCVPAGGELRIFVGSSHSYLIKATSSCG
III
LSLNPSQSFINGESLASGGRC pCMR4 – bbB MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGCVPAGGNLIIRVGSDHSYLIRATVSCG LSL-TNQVFINGESVQSGGRC pCMR5 – Bbb MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGC VPAGGNLIIRVGSDHSYLIRATVSCGLSLNPSQSFINGESLASGGRC pCMR6 – bBB MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGC VPAGGELRIFVGSSHSYLIKATSSCGLSL-TNQVFINGESVQSGGRC pCMR7 – BbB MAPIFKTTMLLAVAILPAALVSANCVANILNINEAVIATGC VPAGGNLIIRVGSDHSYLIRATVSCG LSL-TNQVFINGESVQSGGRC pCMR8 – bBb MAPIFETAMLLAVAILPAALVSANCTANILNINEVVIATGC VPAGGELRIFVGSSHSYLIKATSSCG LSLNPSQSFINGESLASGGRC
ToxB
/
toxb
Plasmids
ampicillin
Bsa I (2417)
pUC ori
Bse YI (1767) pCMR1-toxb 3270 bp II III I Eco RI (702) Bse YI (845)
toxb
Bsa I (924) Not I (1048)
ampicillin
Subcloned into pBSII One site each of BseYI and BsaI Bsa I (2414) pCMR2-ToxB 3267 bp II I III Eco RI (702) Bse YI (845)
ToxB
Bsa I (924) Not I (1045)
pUC ori
Bse YI (1764)
Expression of Chimeras
Subcloning into expression vector pPICZB Transformation into
Pichia pastoris
Expression of chimeras Secretes protein
Summary of Purification
Partial purification of ToxB
Next Steps of Purification
Finish purification Crystallization of ToxB 3-D Structure
Summary of Mutagenesis
Generation of 5 of 6 chimeras
BBb bbB Bbb bBB BbB bBb
Two chimeras transformed into
Pichia
BBb bbB Bbb bBB BbB bBb
Next Steps of Mutagenesis
Finish generation of chimeras Transform into
Pichia
Bioassay Additional chimeras?
Acknowledgements
HHMI URISC Dr. Lynda Ciuffetti Dr. P. Andrew Karplus Viola Manning Dr. Iovanna Pandelova Dr. Kevin Ahern