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Supervisors: Frédéric Brunner

Identification of RXLR effectors from Phytophthora infestans that suppress PAMP-triggered immunity in Arabidopsis and tomato Liu Xiaoyu ZMBP, Universität Tübingen. Supervisors: Frédéric Brunner. Phytophthora infestans.

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Supervisors: Frédéric Brunner

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  1. Identification of RXLR effectors from Phytophthora infestans that suppress PAMP-triggered immunity in Arabidopsis and tomatoLiu XiaoyuZMBP, Universität Tübingen Supervisors: Frédéric Brunner

  2. Phytophthora infestans • Phytophthora infestans is an oomycete that causes the serious potato disease known as late blight or potato blight • In 2009 scientists completed the sequencing of the genome of P. infestans. The genome is much larger (240 Mb) compared to other Phytophthora species Symptom of Phytophthora infestans on the potato leaf Potato infected with Phytophthora infestans

  3. Pathogen-Associated Molecular Patterns (PAMPs) as triggers of innate immune responses in Arabidopsis thaliana

  4. Oomycete RXLR effectors as virulence factors that contribute to plant susceptibility N signal peptide for secretion into extrahaustorial space oomycete haustorium RXLR motif for targeting into plant cell extrahaustorial matrix effector domain for host-manipulating effector function plant cell C modified from Schornack et al., 2009

  5. w/wo GFP effector p35S flg22 luciferase pFRK1 FLS2 MAPKKK WRKY WRKY effector MAPKK luciferase FRK1 MAPK MAPK MAPK FRK1 pFRK1 luciferase luminescence measurement pFRK1 Screening for RXLR effectors repressing PAMP-Triggered innate immunity (PTI) in Arabidopsis Arabidopsis leaf protoplast method adapted from Yoo et al., 2007

  6. Phytophthora infestans RXLR effectors repressing PTI in Arabidopsis • About 100 effectors are screened for repression of flg22-induced pFRK1-Luc activity • ~ 20 repress pFRK1-Luc activity • Best candidates: piRXLR15125, piRXLR16737, piRXLR04145, piRXLR14833, piRXLR21388, piRXLR20303, piRXLR00821, piRXLR05750, piRXLR04097 priority list from Paul Birch’ s lab

  7. RXLR effector candidates block flg22-induced pFRK1-Luc activity pFRK1::Luc reporter gene assay 4,89 5,43 Promoter activity (pFRK1::Luc/GUS) 1,11 1,11 Promoter activity (pFRK1::Luc/GUS) 1.5 1.4 0.6

  8. piRXLR04145, 20303, (14833) repress endogenous FRK1 activation upon flg22 treatment FRK1 relative expression level

  9. piRXLR04097 repress endogenous FRK1 activation upon flg22 treatment FRK1 relative expression level

  10. RXLR effector candidates do not block flg22-induced MAP kinase activation in Arabidopsis protoplasts ev AvrPto PiRXL16737 PiRXLR14833 Post-translational MAP kinase activation +Flg22 0 15 30 0 15 30 0 15 30 0 15 30 @-phosphop42/p44 Ponceau S @-phosphop42/p44 ev AvrPto PiRXLR04145 PiRXLR20303 +Flg22 0 15 30 0 15 30 0 15 30 0 15 30 ev AvrPto PiRXLR00821 PiRXLR151525 +Flg22 0 15 30 0 15 30 0 15 30 0 15 30 Ponceau S @-phosphop42/p44 Ponceau S ev PiRXLR04097 PiRXLR05750 PiRXLR21388 +Flg22 15 0 15 30 0 15 30 0 15 30 @-phosphop42/p44 Ponceau S

  11. GFP sub-cellular localisation of piRXLR20303, 21388 and 14833 in Arabidopsis protoplasts GFP-piRXLR20303 (cytoplasm and nucleus) GFP-piRXLR21388 (nucleus) GFP-piRXLR14833 (nucleus)

  12. sub-cellular localisation of piRXLR15125, 04145 and 16737 in Arabidopsis protoplasts GFP-piRXLR15125 (cytoplasm) GFP-piRXLR04145 (nucleus) GFP-piRXLR16737 (plasma membrane)

  13. sub-cellular localisation of piRXLR00821, 05750 and 04097 in Arabidopsis protoplasts GFP-piRXLR00821 (cytoplasm and PM?) GFP-piRXLR05750 (cytoplasm and PM?) GFP-piRXLR04097 (cytoplasm and PM?)

  14. Conclusions • ~100 P.infestans RXLR effectors have been screened for PTI repression in Arabidopsis and several interesting candidates were identified. • piRXLR15125, piRXLR16737, piRXLR04145, piRXLR14833, piRXLR21388, piRXLR20303 piRXLR00821, piRXLR04097, piRXLR05750 repress flg22-induced pFRK1-Luc activity but do not blockMAP kinase activation. • piRXLR04145,20303,04097,(14833) abort the up-regulation of FRK1 expression after PAMP treatment. • GFP fusion of piRXLR15125, piRXLR16737, piRXLR04145, piRXLR14833, piRXLR21388, piRXLR20303 piRXLR00821, piRXLR04097, piRXLR05750 reveal different sub-cellular localisations.

  15. Outlook • Test sub-cellular localisation, repression of MAP kinase activation and of reporter gene activation in tomato protoplasts. • Identify and characterise plant interactors for the RXLRs via approaches like immunoprecipitation, BiFC/FACS in protoplasts... • What is the effect of RXLRs in planta? - Generate Dex-inducible RXLR-transgenic Arabidopsis and tomato plants (w/wo tag) - Analyse immune response with PTI bioassays and test susceptibility to phytopathogens - Perform microarray study to detect gene expression changes

  16. Thank you! Questions?

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