Unraveling the senses of Phytophthora

Transcription

1 Unraveling the senses of Phytophthora Leads to novel control strategies? Francine Govers GILB Beijing April 2008

2 the senses of man hearing smell taste vision touch working together our SENSES tell us: What is out in the environment. How much is out there. Is there more or less of it than before. Where is it. Is it changing in time or place

3 from David Shaw the senses of Phytophthora hearing smell taste vision touch working together

4 Unraveling the senses of Phytophthora Leads to novel control strategies? What signals are perceived by Phytophthora? cyclic lipopeptides mating hormone.. How are signals perceived by Phytophthora? How are signals transmitted in Phytophthora?

5 perception of signals by receptors in the cell membrane

6 transmission of signals activation of downstream targets k

7 key players receptors / G-proteins / phospholipases k

8 G-protein α and β subunit k WD repeat

9 Gβ deficient mutants are fluffy, produce few or no sporangia, and sporangia are misshapen Gβ (wt) 6a (Pigpb1-silenced) Latijnhouwers & Govers Eukar. Cell, 2003

10 Gα deficient mutants: aberrent zoospore release and zoospores show higher turning frequency Gα wild type Pigpa1-silenced

11 Gα deficient mutants: can no longer infect potato Gα wild type gs3 R2 gs2 gs1 4 dpi Q1 Latijnhouwers et al. Molec. Microbiol 2004

12 k Phospholipase D

13 membranes contain phospholipids Phospholipid bilayer phospolipids outside of cell inside of cell

14 phospholipases hydrolyse phospholipids Head group lipid PLC OH HEAD GROUP PLD P -choline PtdCho PC -ethanolamine PtdEtn PE -glycerol PtdGro PG -inositol PtdIns PI GLYCEROL PLA 1 PLA 2 F A T T Y F A T T Y Phosphatidic Acid, PA Phosphatidyl moiety A C I D A C I D Why focus on phospholipase D? PLD PA PA induces zoospore encystment Phytophthora has novel PLDs

15 phospholipase D in eukaryotes C2-PLD C2 plant (At) 10 PXPH-PLD Catalytic site human 2 yeast 1 plant (At) 2 Pleckstrin homology domain Phox homology domain

16 PLD subfamilies in oomycetes P. sojae, P. ramorum, P. infestans, P. capsici, Hyaloperonospora parasitica AA oomycetes PXTM-PLD 1-1 PXPH-PLD Catalytic site TM-PLD 1-1 Transmembrane region oomycetes 1-1 Signal peptide Pleckstrin homology domain oomycetes spld-like (A) 1-2 Phox homology domain oomycetes spld-like (B) 11-3 Altered catalytic site oomycetes PLD-like 3-1

17 PLDs are out - and inside the cell PXTM-PLD PXPH-PLD TM-PLD PLD-like spld-like (A) spld-like (B) outside of cell inside of cell

18 Phytophthora secretes PLDs Is PXTM-PLD a receptor? PXTM-PLD wild type spld-like (A) spld-like (B) PXTM-PLD mutant outside of cell inside of cell

19 Late blight disease cycle cysts cyst germination zoospores appressoria infection infection cytoplasmic cleavage sporangium germination sporangia

20 PLD? PA zoospores cysts cyst germination appressoria Gα infection infection cytoplasmic cleavage sporangium germination sporangia Gβ

21 What signals does Phytophthora perceive? cysts cyst germination zoospores appressoria cyclic lipopeptides cytoplasmic cleavage Ile sporangia Leu Thr Glu Ile Leu Ser Leu Ser infection sporangium germination infection 10-C lipid tail 9 amino acids

22 What signals does Phytophthora perceive? cysts cyst germination zoospores appressoria cyclic lipopeptides cytoplasmic cleavage Ile sporangia Leu Thr Glu Ile Leu Ser Leu Ser infection sporangium germination infection 10-C lipid tail 9 amino acids

23 What signals does Phytophthora perceive? Cyclic Lipopeptides (CLPs) biosurfactants produced by bacteria including Pseudomonas - soil bacteria - disease suppressive soil role in natural biocontrol! - cyclic lipopeptides Leu Leu Ile Thr Ser Leu Glu Ile Ser 10-C lipid tail 9 amino acids vary in fatty acid, amino acids can reduce the surface tension function as chelators and virulence factors have antibiotic activities interfere with membranes Jos M. Raaijmakers

24 Pseudomonas fluorescens SS101 isolated from wheat roots in the Netherlands * * * * * O L-Leu D-Ser NH O NH O NH OH D-allo- Ile L-Leu O NH N H H O D-Ser O N O HO HN O O D-allo- Thr L-Ile D-Glu COOH N H O L-Leu H N O OH massetolide A Souza et al AEM the CLP massa : can lyse zoospores of oomycete pathogens like Phytophthora, Pythium, and Albugo inhibit hyphal growth of several fungi and oomycetes

25 Effects of SS101 on damping off SS101 mutant Hyacinth/Crocus - Pythium drop collapse assay Root weight (g) a c b c Pseudomonas C- C+ R1SS greenhouse experiment Control De Boer et al. 2006; Ficke et al. In prep. Field experiment (naturally infested soil) SS101 equally effective as metalaxyl

26 Effects of SS101 or massa on late blight Tran et al. New Phytologist 2007 SS101 mutant drop collapse assay prevents infection of tomato leaves? Disease Incidence (%) Cntrl R1SS µg/ml

27 Effects of SS101 or massa on late blight Tran et al. New Phytologist 2007 treated 3-4 days post inoculation Increase lesion area a b b Control 0 Control SS Increase lesion area a ab ab b b b 0µg/ml 5µg/ml 10µg/ml 25µg/ml 50µg/ml SS101

28 SS101 and massa > systemic resistance Tran et al. New Phytologist 2007 treated 24 h prior to inoculation Control Lesion area (mm2) Control SS101 MassA 10, MassA SS101

29 response of P. infestans to CLP treatment monitor changes in gene expression ~ 300 genes were up-regulated Major Facilitator Superfamily (MFS): 7x ABC transporters: 5x biosurfactant massetolide A treatment ug/ml Phytophthora microarray Affymetrix - GeneChip uni-genes P.infestans 3407 P.brassicae cdna-aflp analysis

30 What signals does Phytophthora perceive? September 2005 Extracellular medium from a Phytophthora nicotianae A1 mating type strain From 1830 liters > 1.2 mg of pure hormone α1 mating hormone α1

31 Chemical synthesis of mating hormone α1 four stereocenters sixteen putative stereoisomers methodology: catalytic enantioselective conjugate addition of Grignard reagents two of the sixteen stereoisomers were obtained. a) 1, hormone α1 HO HO * * * * O 1, hormone α1 OH TBDPSO OTES * * * * S S 2 OTBDPS b) Fe PCy 2 P CuBr Ph2 TBDPSO 3 * 3 S S + I * 7 * OTES * OTBDPS R' O SEt + RMgBr (R,S) mol% -78 o C, t BuOMe R O R' SEt ee up to 99% O OTBS EtS 3 15 EtS 5 6 O OTBDPS a % 2 b TBDPSO 78% OTES O 23 OTBDPS c 92% 1 PNAS 2008 accepted

32 Synthetic MH-α1 has biological activity changes in colony morphology of A2 strains but not A1 strains PNAS 2008 accepted

33 Synthetic MH-α1 has biological activity A1 x A2 P. capsici Induces oospore formation in A2 strains but not A1 strains P. capsici P. nicotianae P. infestans P. infestans 7 days at 800 ng P. capsici 7 days > 800 ng P. nicotianae > 7 days > 4000 ng PNAS 2008 accepted

34 Leads to novel controls strategies? GENOME Phytophthora specific features in signal perception and transmission cyclic lipopeptides NOVEL TARGETS mating hormone Reporter systems Screening of bioactive compounds

35 THANKS Klaas Bouwmeester Rob Weide *Rays Jiang > Broad *Hanspeter Versluis Harold Meijer Pieter van Poppel Peter Msimuko Wang Shutong *Zhao Zhijian > Yunnan *Peter van de Vondervoort *Maita Latijnhouwers > Norway *Guo Jun > CAAS Beijng / Yangling Laboratory of Phytopathology Oomycete Genome Initiatives Syuzanna R. Harutyunyan Tim den Hartog Adriaan J. Minnaard Ben L. Feringa Stratingh Institute, University of Groningen Jos Raaijmakers Ha Tran Lia Wagemakers Fengfeng Wang Molecular Ecology Group, Phytopathology Wageningen University Parapluplan Phytophthora