METU-Plant Mol Biol & Gen
Effector-triggered immunity
Catanzariti et al. 2007
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
Predicted secreted effectors of cDNA library of Pst haustoria (Yin et al., 2009)
METU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
Pstha2a5 gene motif region
MEME Suite GLAM2 of putative and predicted effector candidates
METU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
Pstha2a5 gene synthesis and pJL48-TRBO cloning
PCR
METU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
METU-Plant Mol Biol & Gen
Interacting host proteins
Tagged protein co-immunoprecipitationMETU-Plant Mol Biol & Gen
PstHa2a5 interacts with Ta-GloxMETU-Plant Mol Biol & Gen
PstHa2a5 facilitates ETS
Pieterse et al. 2009
METU-Plant Mol Biol & Gen
METU-Plant Mol Biol & Gen
Subcellular localization
PCR
Flag-Tag Linker CACCATGGACTACAAGGACGACGATGACAAAGTCAAGCTTCTCGAGAATTCCttcaagtgtcccggtttgcatggaacgccaagccaaacacatggttattgcaccagatcaatcaccgatgaagaacgaaaggcaaaaaagattggcaaggagttcaccatgtggaaggaagaaatcaagacagtcgacgggaaattctcgtgtgataaagtggacttgaatgggtcggttgccacagatagcttctgttgtgacgttgcaggtagaattggtgaagttgagaaaagtaaacaagctatgtggacaaacaactgctccaaagcatcttag
METU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
METU-Plant Mol Biol & Gen
Gateway cloningMETU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
METU-Plant Mol Biol & Gen
GFP & RFP expression
GFP and FLAG-RFP expression in Nicotiana benthamiana, 3-day post infiltration. In picture A and B, leaves infiltrated with Agrobacterium containing pJL48-TRBO-GFP were shot under GFP fluorescence filter at 40X and 10X magnifications, respectively. Pictures D and E were taken using RFP fluorescence filter, and leaf infiltrated with Agrobacterium with pJL48-TRBO-FLAG-RFP was shot under 40X and 10X magnifications, respectively. In controls, non-infiltrated (normal) leaf was shot in picture C and F under GFP and RFP fluorescence filter, respectively, at 40 X magnifications.
METU-Plant Mol Biol & Gen
Subcellular localizations
Subcellular localization studies can assist in determining the classification of a particular protein
and it can give more information about its possible function.
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
Predicted secreted effectors of cDNA library of Pst haustoria (Yin et al., 2009)
METU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
METU-Plant Mol Biol & Gen
Subcellular localization of PstHa12h2
METU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
METU-Plant Mol Biol & Gen
pEDV6: Effector detector vector
pEDV6 (Effector Detector Vector) is a Gateway destination vector which is designed for delivering bacterial effectors to the wheat by using TTSS of Pseudomonas to activate plant defense system.
METU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
METU-Plant Mol Biol & Gen
Avr?
PstHa15N21 / Siete Cerros
METU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
METU-Plant Mol Biol & Gen
Pst-78:Determination of candidate effector proteins
• PST-78 (gene, protein and transcript) sequences
downloaded from Broad Institute
• 20482 protein and 19542 gene in total are investigated
• Filtration of big proteins ( >150 a.a)
• SignalP analysis to select secreted proteins
• Cysteine count to first-rate Cyc rich proteins
• Multiple motif search
• The proteins are proved not to share any sequence
similarity to known proteins through BLAST
• Besides the motifs, high sequence similarity between the
proteins are found
METU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
METU-Plant Mol Biol & Gen
22 are identified as a specific type of effectorsMETU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
FunctionMETU-Plant Mol Biol & GenMETU-Plant Mol Biol & Gen
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014
• Bayantes Dagvadorj• Ahmet Çağlar Özketen• Ayşe Andaç• Kübra Narcı• Burak Demıralay
• Osman Tolga BOZKURT (former PhD student)Sainsbury Lab
• Hasan OgulBaskent University, Computer Science
• Tolga CanMETU, Computer Science
• Dr. Xianming CHEN• Lesley BOYD• Mogens Hovmoller
METU-Plant Mol Biol & Gen
ACKNOWLEDGEMENT
Supported by
•DPT
•ICGEB
•TUBITAK
2nd International Stripe Rust Symposium Izmir, 28 April-1 May 2014