2nd stripe rust izmir

METU-Plant Mol Biol & GenMiddle East Technical University

Mahinur S. [email protected]

u.tr

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)



Pstha2a5 gene motif region

MEME Suite GLAM2 of putative and predicted effector candidates



Pstha2a5 gene synthesis and pJL48-TRBO cloning

PCR




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



Subcellular localization

PCR

Flag-Tag Linker CACCATGGACTACAAGGACGACGATGACAAAGTCAAGCTTCTCGAGAATTCCttcaagtgtcccggtttgcatggaacgccaagccaaacacatggttattgcaccagatcaatcaccgatgaagaacgaaaggcaaaaaagattggcaaggagttcaccatgtggaaggaagaaatcaagacagtcgacgggaaattctcgtgtgataaagtggacttgaatgggtcggttgccacagatagcttctgttgtgacgttgcaggtagaattggtgaagttgagaaaagtaaacaagctatgtggacaaacaactgctccaaagcatcttag




Gateway cloningMETU-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.


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.


Predicted secreted effectors of cDNA library of Pst haustoria (Yin et al., 2009)




Subcellular localization of PstHa12h2




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.




Avr?

PstHa15N21 / Siete Cerros




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




22 are identified as a specific type of effectorsMETU-Plant Mol Biol & Gen


FunctionMETU-Plant Mol Biol & GenMETU-Plant Mol Biol & Gen


• 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


ACKNOWLEDGEMENT

Supported by

•DPT

•ICGEB

•TUBITAK


2nd stripe rust izmir

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plant defense system

gfp fluorescence filter

x magnifications

protein expression